11 Tissue and molecular diagnosis

ACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS
The authors are very grateful to the following contributors for assistance with previous versions of  the chapter and for providing images: Professor Mike Shea ff , London, UK; Dr Manuel Rodriguez-Justo, London, UK. ACKNOWLEDGEMENTS
The authors are very grateful to the following contributors for assistance with previous versions of  the chapter and for providing images: Professor Mike Shea ff , London, UK; Dr Manuel Rodriguez-Justo, London, UK. ACKNOWLEDGEMENTS
The authors are very grateful to the following contributors for assistance with previous versions of  the chapter and for providing images: Professor Mike Shea ff , London, UK; Dr Manuel Rodriguez-Justo, London, UK.

ASSESSMENT Light microscopy
ASSESSMENT Light microscopy
Most tissue assessment depends on conventional light micros - copy . Microscopes have several lenses with various powers of magnification, typically ranging from × 20 to × 400 or more. A low-power lens allows scanning of  a sample and assessment hile a higher power lens allows a of  overall architecture, w closer view with more detail ( Figure 11.21 ). Attachment of one or more teaching arms, a camera and other accessories are possible for many microscopes ( Figure 11.8 ). Polarisation assists with detection of  some types of  foreign material (e.g. sutures) or to assess a special stain (e.g. Congo red for amyloid deposition).
(b)
Figure 11.21
(a)
Low-power view of an umbilical nodule. Glands are
distributed irregularly through the tissue.
(b)
High-power view shows
benign columnar epithelium lining the glands (arrow), indicating endo
metriosis rather than carcinoma.
ASSESSMENT Light microscopy
Most tissue assessment depends on conventional light micros - copy . Microscopes have several lenses with various powers of magnification, typically ranging from × 20 to × 400 or more. A low-power lens allows scanning of  a sample and assessment hile a higher power lens allows a of  overall architecture, w closer view with more detail ( Figure 11.21 ). Attachment of one or more teaching arms, a camera and other accessories are possible for many microscopes ( Figure 11.8 ). Polarisation assists with detection of  some types of  foreign material (e.g. sutures) or to assess a special stain (e.g. Congo red for amyloid deposition).
(b)
Figure 11.21
(a)
Low-power view of an umbilical nodule. Glands are
distributed irregularly through the tissue.
(b)
High-power view shows
benign columnar epithelium lining the glands (arrow), indicating endo
metriosis rather than carcinoma.
ASSESSMENT Light microscopy
Most tissue assessment depends on conventional light micros - copy . Microscopes have several lenses with various powers of magnification, typically ranging from × 20 to × 400 or more. A low-power lens allows scanning of  a sample and assessment hile a higher power lens allows a of  overall architecture, w closer view with more detail ( Figure 11.21 ). Attachment of one or more teaching arms, a camera and other accessories are possible for many microscopes ( Figure 11.8 ). Polarisation assists with detection of  some types of  foreign material (e.g. sutures) or to assess a special stain (e.g. Congo red for amyloid deposition).
(b)
Figure 11.21
(a)
Low-power view of an umbilical nodule. Glands are
distributed irregularly through the tissue.
(b)
High-power view shows
benign columnar epithelium lining the glands (arrow), indicating endo
metriosis rather than carcinoma.

AUTOPSY
AUTOPSY
In the past, autopsies (postmortems) allowed physicians and scientists to improve their knowledge of the human body and various diseases. The main reason for an autopsy is to confirm the cause of  death, but autopsies remain very useful for medical education and audit. In the UK there are two main types. The first is the coroner’s autopsy , when the coroner decides that there is a legal requirement to establish the cause of  death, e.g. unexpected death or death during surgery or soon afterwards. Consent from relatives is not necessary . The second type is the hospital autopsy , which requires relatives’ consent. For various reasons hospital autopsies are considerably less common than in the past. in situ
Methodology for
assessment
IHC
See
Summary box 11.17
PCR
NGS
Ampli
/f_i
cation predicts response to anti-HER2
IHC
therapy, e.g. trastuzumab, pertuzumab
FISH/CISH
NGS
Predicts response to immune checkpoint
IHC
inhibitors
(not required for
melanoma)
Predicts response to tyrosine kinase inhibitors PCR
NGS
Predicts response to anti-BRAF therapy
PCR
Prognosis
NGS
Predicts resistance to EGFR inhibitors PCR
NGS
Predicts response to tyrosine kinase inhibitors
NGS
Predicts response to tyrosine kinase inhibitors
IHC screen
FISH
NGS
Predicts response to tyrosine kinase inhibitors
NGS
Predicts response to immune checkpoint
NGS
inhibitors
hybridisation; IHC, immunohistochemistry;
AUTOPSY
In the past, autopsies (postmortems) allowed physicians and scientists to improve their knowledge of the human body and various diseases. The main reason for an autopsy is to confirm the cause of  death, but autopsies remain very useful for medical education and audit. In the UK there are two main types. The first is the coroner’s autopsy , when the coroner decides that there is a legal requirement to establish the cause of  death, e.g. unexpected death or death during surgery or soon afterwards. Consent from relatives is not necessary . The second type is the hospital autopsy , which requires relatives’ consent. For various reasons hospital autopsies are considerably less common than in the past. in situ
Methodology for
assessment
IHC
See
Summary box 11.17
PCR
NGS
Ampli
/f_i
cation predicts response to anti-HER2
IHC
therapy, e.g. trastuzumab, pertuzumab
FISH/CISH
NGS
Predicts response to immune checkpoint
IHC
inhibitors
(not required for
melanoma)
Predicts response to tyrosine kinase inhibitors PCR
NGS
Predicts response to anti-BRAF therapy
PCR
Prognosis
NGS
Predicts resistance to EGFR inhibitors PCR
NGS
Predicts response to tyrosine kinase inhibitors
NGS
Predicts response to tyrosine kinase inhibitors
IHC screen
FISH
NGS
Predicts response to tyrosine kinase inhibitors
NGS
Predicts response to immune checkpoint
NGS
inhibitors
hybridisation; IHC, immunohistochemistry;
AUTOPSY
In the past, autopsies (postmortems) allowed physicians and scientists to improve their knowledge of the human body and various diseases. The main reason for an autopsy is to confirm the cause of  death, but autopsies remain very useful for medical education and audit. In the UK there are two main types. The first is the coroner’s autopsy , when the coroner decides that there is a legal requirement to establish the cause of  death, e.g. unexpected death or death during surgery or soon afterwards. Consent from relatives is not necessary . The second type is the hospital autopsy , which requires relatives’ consent. For various reasons hospital autopsies are considerably less common than in the past. in situ
Methodology for
assessment
IHC
See
Summary box 11.17
PCR
NGS
Ampli
/f_i
cation predicts response to anti-HER2
IHC
therapy, e.g. trastuzumab, pertuzumab
FISH/CISH
NGS
Predicts response to immune checkpoint
IHC
inhibitors
(not required for
melanoma)
Predicts response to tyrosine kinase inhibitors PCR
NGS
Predicts response to anti-BRAF therapy
PCR
Prognosis
NGS
Predicts resistance to EGFR inhibitors PCR
NGS
Predicts response to tyrosine kinase inhibitors
NGS
Predicts response to tyrosine kinase inhibitors
IHC screen
FISH
NGS
Predicts response to tyrosine kinase inhibitors
NGS
Predicts response to immune checkpoint
NGS
inhibitors
hybridisation; IHC, immunohistochemistry;

BRAF V600E mutation
BRAF V600E mutation
for detecting antigens in haematological neoplasms, usually in - blood samples, and for determining ploidy , i.e. the number of sets of  chromosomes in the nucleus of  a cell. Although tradi - tional flow cytometry is of  limited value for tissue analysis, new applications of  image cytometric DNA analysis allow detection of  aneuploidy in tissue sections of  gastrointestinal cancers.
Figure 11.29
Sanger sequencing showing wild-type
BRAF
(a)
and
a
BRAF
V600E mutation
(b)
(courtesy of Dr M Rodriguez-Justo,
UCL-AD, Cancer Institute, London, UK).
BRAF V600E mutation
for detecting antigens in haematological neoplasms, usually in - blood samples, and for determining ploidy , i.e. the number of sets of  chromosomes in the nucleus of  a cell. Although tradi - tional flow cytometry is of  limited value for tissue analysis, new applications of  image cytometric DNA analysis allow detection of  aneuploidy in tissue sections of  gastrointestinal cancers.
Figure 11.29
Sanger sequencing showing wild-type
BRAF
(a)
and
a
BRAF
V600E mutation
(b)
(courtesy of Dr M Rodriguez-Justo,
UCL-AD, Cancer Institute, London, UK).
BRAF V600E mutation
for detecting antigens in haematological neoplasms, usually in - blood samples, and for determining ploidy , i.e. the number of sets of  chromosomes in the nucleus of  a cell. Although tradi - tional flow cytometry is of  limited value for tissue analysis, new applications of  image cytometric DNA analysis allow detection of  aneuploidy in tissue sections of  gastrointestinal cancers.
Figure 11.29
Sanger sequencing showing wild-type
BRAF
(a)
and
a
BRAF
V600E mutation
(b)
(courtesy of Dr M Rodriguez-Justo,
UCL-AD, Cancer Institute, London, UK).

Basic methods in diagnostic molecular pathology
Basic methods in diagnostic molecular pathology
In situ hybridisation H. In situ hybridisation (ISH) uses a labelled oligonucleotide probe - that targets a specific sequence of  RNA or DNA. It allows visu - alisation of  the presence or absence and location of  a particular RNA or DNA sequence in situ in tissue sections. Visualisation may depend on autoradiography , fluorescence microscopy - or bright-field microscopy . Chromogenic in situ hybridisation (CISH) combines ISH and immunohistochemistry for the detection of  specific nucleic acid sequences and is a common alternative to fluorescence in situ hybridisation (FISH) for the detection of HER2 amplification. Viral genomes, e.g. EBV - ( Figure 11.28 ), CMV and high-risk HPV types are detectable using this approach. ISH plays an important role in tissue diagnostics and the management of  tumours.
Figure 11.28
In situ
hybridisation for Epstein–Barr virus (EBV) showing
extensive nuclear positivity (black nuclei) in an EBV-positive gastric
adenocarcinoma.
Basic methods in diagnostic molecular pathology
In situ hybridisation H. In situ hybridisation (ISH) uses a labelled oligonucleotide probe - that targets a specific sequence of  RNA or DNA. It allows visu - alisation of  the presence or absence and location of  a particular RNA or DNA sequence in situ in tissue sections. Visualisation may depend on autoradiography , fluorescence microscopy - or bright-field microscopy . Chromogenic in situ hybridisation (CISH) combines ISH and immunohistochemistry for the detection of  specific nucleic acid sequences and is a common alternative to fluorescence in situ hybridisation (FISH) for the detection of HER2 amplification. Viral genomes, e.g. EBV - ( Figure 11.28 ), CMV and high-risk HPV types are detectable using this approach. ISH plays an important role in tissue diagnostics and the management of  tumours.
Figure 11.28
In situ
hybridisation for Epstein–Barr virus (EBV) showing
extensive nuclear positivity (black nuclei) in an EBV-positive gastric
adenocarcinoma.
Basic methods in diagnostic molecular pathology
In situ hybridisation H. In situ hybridisation (ISH) uses a labelled oligonucleotide probe - that targets a specific sequence of  RNA or DNA. It allows visu - alisation of  the presence or absence and location of  a particular RNA or DNA sequence in situ in tissue sections. Visualisation may depend on autoradiography , fluorescence microscopy - or bright-field microscopy . Chromogenic in situ hybridisation (CISH) combines ISH and immunohistochemistry for the detection of  specific nucleic acid sequences and is a common alternative to fluorescence in situ hybridisation (FISH) for the detection of HER2 amplification. Viral genomes, e.g. EBV - ( Figure 11.28 ), CMV and high-risk HPV types are detectable using this approach. ISH plays an important role in tissue diagnostics and the management of  tumours.
Figure 11.28
In situ
hybridisation for Epstein–Barr virus (EBV) showing
extensive nuclear positivity (black nuclei) in an EBV-positive gastric
adenocarcinoma.

Cancer ‘precision medicine’
Cancer ‘precision medicine’
This refers to the development of  individualised cancer care plans, partly on the basis of molecular abnormalities in a tumour. Germline and somatic mutations may be taken into consideration, with the aim of tailoring treatments and targeting cancer cells precisely . With NGS, analysis of a single sample of  tumour tissue for multiple known mutations that may predict treatment response is possible. In addition, many assays can detect mutations a ff ecting as few as 5% of  neoplas - tic cells. Other techniques may be used at the same time to detect abnormalities in the proteome (protein), transcriptome (mRNA), metabolome (metabolites) or epigenome, sometimes referred to as ‘omics’ assays. Such plans may , inevitably , be ver y complex (see Chapter 12 ). Cancer ‘precision medicine’
This refers to the development of  individualised cancer care plans, partly on the basis of molecular abnormalities in a tumour. Germline and somatic mutations may be taken into consideration, with the aim of tailoring treatments and targeting cancer cells precisely . With NGS, analysis of a single sample of  tumour tissue for multiple known mutations that may predict treatment response is possible. In addition, many assays can detect mutations a ff ecting as few as 5% of  neoplas - tic cells. Other techniques may be used at the same time to detect abnormalities in the proteome (protein), transcriptome (mRNA), metabolome (metabolites) or epigenome, sometimes referred to as ‘omics’ assays. Such plans may , inevitably , be ver y complex (see Chapter 12 ). Cancer ‘precision medicine’
This refers to the development of  individualised cancer care plans, partly on the basis of molecular abnormalities in a tumour. Germline and somatic mutations may be taken into consideration, with the aim of tailoring treatments and targeting cancer cells precisely . With NGS, analysis of a single sample of  tumour tissue for multiple known mutations that may predict treatment response is possible. In addition, many assays can detect mutations a ff ecting as few as 5% of  neoplas - tic cells. Other techniques may be used at the same time to detect abnormalities in the proteome (protein), transcriptome (mRNA), metabolome (metabolites) or epigenome, sometimes referred to as ‘omics’ assays. Such plans may , inevitably , be ver y complex (see Chapter 12 ).

Cytogenetics and fluorescence in situ hybridisation
Cytogenetics and fluorescence in situ hybridisation
Conventional cytogenetics is the microscopic study of  chromo somal changes in individual cells. Newer techniques, including FISH, array comparative genomic hybridisation, RT-PCR and next-generation sequencing (NGS) are increasingly replacing conv entional cytogenetics. Cytogenetic tests seek alterations such as gene amplification, loss of  segments of  chromosomal material, loss of  whole chromosomes (e.g. in renal cell carci noma) and translocations with associated fusion genes (e.g. EWSR1-FLI1 in Ewing’s sarcoma). Cytogenetics and fluorescence in situ hybridisation
Conventional cytogenetics is the microscopic study of  chromo somal changes in individual cells. Newer techniques, including FISH, array comparative genomic hybridisation, RT-PCR and next-generation sequencing (NGS) are increasingly replacing conv entional cytogenetics. Cytogenetic tests seek alterations such as gene amplification, loss of  segments of  chromosomal material, loss of  whole chromosomes (e.g. in renal cell carci noma) and translocations with associated fusion genes (e.g. EWSR1-FLI1 in Ewing’s sarcoma). Cytogenetics and fluorescence in situ hybridisation
Conventional cytogenetics is the microscopic study of  chromo somal changes in individual cells. Newer techniques, including FISH, array comparative genomic hybridisation, RT-PCR and next-generation sequencing (NGS) are increasingly replacing conv entional cytogenetics. Cytogenetic tests seek alterations such as gene amplification, loss of  segments of  chromosomal material, loss of  whole chromosomes (e.g. in renal cell carci noma) and translocations with associated fusion genes (e.g. EWSR1-FLI1 in Ewing’s sarcoma).

Cytological assessment
Cytological assessment
A cytological preparation consists of  a sample of  cells only . Assessment of  architecture is not usually possible because intact tissue is absent or sparse ( Figures 11.9 and 11.22 ) . Therefore, Sodium diphenylbisazobisnaphthylamine sulphonate is a red dye marketed in 1884 by the AGFA company of  Berlin, Germany , using the name ‘ no longer used as a cloth dye owing to the carcinogenic risks of  the benzidine moiety . assessment relies on the characteristics of  the individual cells. Accordingly , diagnosis of  malignancy is often di ffi cult because the pathologist cannot assess certain features that support a diagnosis of  malignancy such as invasiveness. However, cytol - ogy has several potential advantages over a biopsy . Obtaining a specimen may be easier and less traumatic. The area of sampling may be wider. Processing times are usually shorter eport and costs lower. Also, the ability of  non-medical sta ff to r a proportion of  cases reduces costs. Summary box 11.7 Cytology compared with histology /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Congo red ’. It is
(b)
Figure 11.22
(a)
A cytology preparation of a pleural effusion. Numer
ous cells with atypical features are present, forming closely packed
groups of overlapping cells.
(b)
Immunohistochemistry shows positive
staining for carcinoembryonic antigen, favouring carcinoma over
mesothelioma.
Advantages
Wider area of sampling in some cases
Often less invasive
Fast
Cheap
Disadvantages
Cannot assess tissue architecture
Less amenable to further tissue studies
Screening programmes aim to detect and treat premalig nant tissue changes (dysplasia/intraepithelial neoplasia) or early-stage malignancy for which treatment is likely to be curative. The programmes may rely on clinical assessment, imaging and/or pathological assessment. The cervical cancer pr ogramme traditionally relied on cytology , with biopsy and histology follow-up if  appropriate, but the alternative of  HPV testing is increasingly available. The breast cancer screening programme relies on imaging and may use cytology and/or histology to assess possible lesions. The bowel cancer screening programme relies initially on a non-tissue-based test followed, if  appropriate, by lower gastrointestinal endoscopy with or without biopsy of  abnormal areas. Screening for neoplasia in ulcerative colitis and in Barrett’s oesophagus relies on endo scopic assessment and biopsy . Cytological assessment
A cytological preparation consists of  a sample of  cells only . Assessment of  architecture is not usually possible because intact tissue is absent or sparse ( Figures 11.9 and 11.22 ) . Therefore, Sodium diphenylbisazobisnaphthylamine sulphonate is a red dye marketed in 1884 by the AGFA company of  Berlin, Germany , using the name ‘ no longer used as a cloth dye owing to the carcinogenic risks of  the benzidine moiety . assessment relies on the characteristics of  the individual cells. Accordingly , diagnosis of  malignancy is often di ffi cult because the pathologist cannot assess certain features that support a diagnosis of  malignancy such as invasiveness. However, cytol - ogy has several potential advantages over a biopsy . Obtaining a specimen may be easier and less traumatic. The area of sampling may be wider. Processing times are usually shorter eport and costs lower. Also, the ability of  non-medical sta ff to r a proportion of  cases reduces costs. Summary box 11.7 Cytology compared with histology /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Congo red ’. It is
(b)
Figure 11.22
(a)
A cytology preparation of a pleural effusion. Numer
ous cells with atypical features are present, forming closely packed
groups of overlapping cells.
(b)
Immunohistochemistry shows positive
staining for carcinoembryonic antigen, favouring carcinoma over
mesothelioma.
Advantages
Wider area of sampling in some cases
Often less invasive
Fast
Cheap
Disadvantages
Cannot assess tissue architecture
Less amenable to further tissue studies
Screening programmes aim to detect and treat premalig nant tissue changes (dysplasia/intraepithelial neoplasia) or early-stage malignancy for which treatment is likely to be curative. The programmes may rely on clinical assessment, imaging and/or pathological assessment. The cervical cancer pr ogramme traditionally relied on cytology , with biopsy and histology follow-up if  appropriate, but the alternative of  HPV testing is increasingly available. The breast cancer screening programme relies on imaging and may use cytology and/or histology to assess possible lesions. The bowel cancer screening programme relies initially on a non-tissue-based test followed, if  appropriate, by lower gastrointestinal endoscopy with or without biopsy of  abnormal areas. Screening for neoplasia in ulcerative colitis and in Barrett’s oesophagus relies on endo scopic assessment and biopsy . Cytological assessment
A cytological preparation consists of  a sample of  cells only . Assessment of  architecture is not usually possible because intact tissue is absent or sparse ( Figures 11.9 and 11.22 ) . Therefore, Sodium diphenylbisazobisnaphthylamine sulphonate is a red dye marketed in 1884 by the AGFA company of  Berlin, Germany , using the name ‘ no longer used as a cloth dye owing to the carcinogenic risks of  the benzidine moiety . assessment relies on the characteristics of  the individual cells. Accordingly , diagnosis of  malignancy is often di ffi cult because the pathologist cannot assess certain features that support a diagnosis of  malignancy such as invasiveness. However, cytol - ogy has several potential advantages over a biopsy . Obtaining a specimen may be easier and less traumatic. The area of sampling may be wider. Processing times are usually shorter eport and costs lower. Also, the ability of  non-medical sta ff to r a proportion of  cases reduces costs. Summary box 11.7 Cytology compared with histology /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Congo red ’. It is
(b)
Figure 11.22
(a)
A cytology preparation of a pleural effusion. Numer
ous cells with atypical features are present, forming closely packed
groups of overlapping cells.
(b)
Immunohistochemistry shows positive
staining for carcinoembryonic antigen, favouring carcinoma over
mesothelioma.
Advantages
Wider area of sampling in some cases
Often less invasive
Fast
Cheap
Disadvantages
Cannot assess tissue architecture
Less amenable to further tissue studies
Screening programmes aim to detect and treat premalig nant tissue changes (dysplasia/intraepithelial neoplasia) or early-stage malignancy for which treatment is likely to be curative. The programmes may rely on clinical assessment, imaging and/or pathological assessment. The cervical cancer pr ogramme traditionally relied on cytology , with biopsy and histology follow-up if  appropriate, but the alternative of  HPV testing is increasingly available. The breast cancer screening programme relies on imaging and may use cytology and/or histology to assess possible lesions. The bowel cancer screening programme relies initially on a non-tissue-based test followed, if  appropriate, by lower gastrointestinal endoscopy with or without biopsy of  abnormal areas. Screening for neoplasia in ulcerative colitis and in Barrett’s oesophagus relies on endo scopic assessment and biopsy .

Cytology specimen
Cytology specimen
Samples for cytology can be smeared immediately onto glass slides, fixed (usually in alcohol) or air dried and stained imme diately or later. The process usually produces several slides, some of  which are stained with a Papanicolaou (Pap) stain and some with another method such as May–Grünwald–Giemsa (MGG), H&E or Romanowsky ( Figure 11.9 ). Liquid-based thin-layer technology is now r eplacing older methods. For liquid-based cytology , the sampling device is usually washed in a liquid medium and the material obtained is then processed in the laboratory using purpose-built equipment. Cytology specimen
Samples for cytology can be smeared immediately onto glass slides, fixed (usually in alcohol) or air dried and stained imme diately or later. The process usually produces several slides, some of  which are stained with a Papanicolaou (Pap) stain and some with another method such as May–Grünwald–Giemsa (MGG), H&E or Romanowsky ( Figure 11.9 ). Liquid-based thin-layer technology is now r eplacing older methods. For liquid-based cytology , the sampling device is usually washed in a liquid medium and the material obtained is then processed in the laboratory using purpose-built equipment. Cytology specimen
Samples for cytology can be smeared immediately onto glass slides, fixed (usually in alcohol) or air dried and stained imme diately or later. The process usually produces several slides, some of  which are stained with a Papanicolaou (Pap) stain and some with another method such as May–Grünwald–Giemsa (MGG), H&E or Romanowsky ( Figure 11.9 ). Liquid-based thin-layer technology is now r eplacing older methods. For liquid-based cytology , the sampling device is usually washed in a liquid medium and the material obtained is then processed in the laboratory using purpose-built equipment.

Cytology
Cytology
There are various approaches to the procurement of  a cytol ogy sample. Some samples are easy to obtain, e.g. urine and sputum, whereas others require more intervention. A conven tional cervical smear is obtained by sampling the cervical trans formation zone with a brush/br oom. Bronchial aspirates and washings and bronchial, gastrointestinal and biliary brushings sample a relatively wide area and may therefore be useful for the diagnosis of  malignancy . Fine-needle aspiration (FN A) cytology may sample accessi ble sites such as the breast, thyroid and superficial lymph nodes, while ultrasound or CT guidance assists FNA from deeper and less accessible structures, e.g. liver, pancreas , kidney and lung. Ultrasound-guided transbronchial FNA may allow sampling of  mediastinal masses and transmucosal FNA may be appro priate for submucosal gastrointestinal lesions or perivisceral lesions. A biopsy taken at the same procedure may accompany laboratory for cytological assessment. Cytology
There are various approaches to the procurement of  a cytol ogy sample. Some samples are easy to obtain, e.g. urine and sputum, whereas others require more intervention. A conven tional cervical smear is obtained by sampling the cervical trans formation zone with a brush/br oom. Bronchial aspirates and washings and bronchial, gastrointestinal and biliary brushings sample a relatively wide area and may therefore be useful for the diagnosis of  malignancy . Fine-needle aspiration (FN A) cytology may sample accessi ble sites such as the breast, thyroid and superficial lymph nodes, while ultrasound or CT guidance assists FNA from deeper and less accessible structures, e.g. liver, pancreas , kidney and lung. Ultrasound-guided transbronchial FNA may allow sampling of  mediastinal masses and transmucosal FNA may be appro priate for submucosal gastrointestinal lesions or perivisceral lesions. A biopsy taken at the same procedure may accompany laboratory for cytological assessment. Cytology
There are various approaches to the procurement of  a cytol ogy sample. Some samples are easy to obtain, e.g. urine and sputum, whereas others require more intervention. A conven tional cervical smear is obtained by sampling the cervical trans formation zone with a brush/br oom. Bronchial aspirates and washings and bronchial, gastrointestinal and biliary brushings sample a relatively wide area and may therefore be useful for the diagnosis of  malignancy . Fine-needle aspiration (FN A) cytology may sample accessi ble sites such as the breast, thyroid and superficial lymph nodes, while ultrasound or CT guidance assists FNA from deeper and less accessible structures, e.g. liver, pancreas , kidney and lung. Ultrasound-guided transbronchial FNA may allow sampling of  mediastinal masses and transmucosal FNA may be appro priate for submucosal gastrointestinal lesions or perivisceral lesions. A biopsy taken at the same procedure may accompany laboratory for cytological assessment.

DIAGNOSTIC MOLECULAR PATHOLOGY
DIAGNOSTIC MOLECULAR PATHOLOGY
The broad heading of  diagnostic molecular pathology refers to multiple tests that assess molecules (proteins, ribonucleic ), acid [RNA] and deoxyribonucleic acid [DNA]) in tissue. The information that they provide may be useful for diagnosis, classification of  tumours, prognostic predictions, identifying patients with a hereditary cancer risk, determining treatment and identifying residual disease after treatment. Immunohisto - chemistry is conventionally separate from this category . DIAGNOSTIC MOLECULAR PATHOLOGY
The broad heading of  diagnostic molecular pathology refers to multiple tests that assess molecules (proteins, ribonucleic ), acid [RNA] and deoxyribonucleic acid [DNA]) in tissue. The information that they provide may be useful for diagnosis, classification of  tumours, prognostic predictions, identifying patients with a hereditary cancer risk, determining treatment and identifying residual disease after treatment. Immunohisto - chemistry is conventionally separate from this category . DIAGNOSTIC MOLECULAR PATHOLOGY
The broad heading of  diagnostic molecular pathology refers to multiple tests that assess molecules (proteins, ribonucleic ), acid [RNA] and deoxyribonucleic acid [DNA]) in tissue. The information that they provide may be useful for diagnosis, classification of  tumours, prognostic predictions, identifying patients with a hereditary cancer risk, determining treatment and identifying residual disease after treatment. Immunohisto - chemistry is conventionally separate from this category .

DIGITAL PATHOLOGY AND ARTIFICAL INTELLIGENCE
DIGITAL PATHOLOGY AND ARTIFICAL INTELLIGENCE
The term ‘digital pathology’ usually refers to the examination of digitised slides on a workstation (computer) or another device. Uses include education, quality assurance, surveys, research and expert consults. With the development of  high-quality scanners, histopathology departments can scan all slides and store them so that pathologists can access them anywhere. Advantages include more flexible on-site and remote report - ing, easy sharing, a reduction in costs and better recruitment. Disadvantages include the expense of set-up, maintenance and IT and repetitive strain injury . Additionally , diagnostic accuracy may be slightly lower than with glass slides. Some pathologists, particularly cytopathologists, dislike the loss of  a three-dimensional image, and detection of  very small items such as microorganisms can be di ffi cult.
Biomarker
Examples of tumours where relevant Application
Mismatch repair genes
CRC
Gynaecological carcinomas
Other digestive system carcinomas
HER2
Breast carcinoma
Gastric/oesophageal adenocarcinoma
CRC (emerging evidence)
PD-L1
Lung carcinoma
Gastric carcinoma
Bladder/urological carcinoma
Malignant melanoma
Breast carcinoma
Endometrial carcinoma
EGFR
mutation
Lung carcinoma
BRAF
mutation
Malignant melanoma
CRC
KRAS
mutation
CRC
NTRK
fusions
CRC
ALK
fusion
Non-small cell lung carcinoma
Renal cell carcinoma
FGFR
fusions
Bladder carcinoma
Tumour mutation burden
Various
CISH, chromogenic
in situ
hybridisation; CRC, colorectal carcinoma; FISH,
/f_l
uorescence
NGS, next-generation sequencing; PCR, polymerase chain reaction.
DIGITAL PATHOLOGY AND ARTIFICAL INTELLIGENCE
The term ‘digital pathology’ usually refers to the examination of digitised slides on a workstation (computer) or another device. Uses include education, quality assurance, surveys, research and expert consults. With the development of  high-quality scanners, histopathology departments can scan all slides and store them so that pathologists can access them anywhere. Advantages include more flexible on-site and remote report - ing, easy sharing, a reduction in costs and better recruitment. Disadvantages include the expense of set-up, maintenance and IT and repetitive strain injury . Additionally , diagnostic accuracy may be slightly lower than with glass slides. Some pathologists, particularly cytopathologists, dislike the loss of  a three-dimensional image, and detection of  very small items such as microorganisms can be di ffi cult.
Biomarker
Examples of tumours where relevant Application
Mismatch repair genes
CRC
Gynaecological carcinomas
Other digestive system carcinomas
HER2
Breast carcinoma
Gastric/oesophageal adenocarcinoma
CRC (emerging evidence)
PD-L1
Lung carcinoma
Gastric carcinoma
Bladder/urological carcinoma
Malignant melanoma
Breast carcinoma
Endometrial carcinoma
EGFR
mutation
Lung carcinoma
BRAF
mutation
Malignant melanoma
CRC
KRAS
mutation
CRC
NTRK
fusions
CRC
ALK
fusion
Non-small cell lung carcinoma
Renal cell carcinoma
FGFR
fusions
Bladder carcinoma
Tumour mutation burden
Various
CISH, chromogenic
in situ
hybridisation; CRC, colorectal carcinoma; FISH,
/f_l
uorescence
NGS, next-generation sequencing; PCR, polymerase chain reaction.
DIGITAL PATHOLOGY AND ARTIFICAL INTELLIGENCE
The term ‘digital pathology’ usually refers to the examination of digitised slides on a workstation (computer) or another device. Uses include education, quality assurance, surveys, research and expert consults. With the development of  high-quality scanners, histopathology departments can scan all slides and store them so that pathologists can access them anywhere. Advantages include more flexible on-site and remote report - ing, easy sharing, a reduction in costs and better recruitment. Disadvantages include the expense of set-up, maintenance and IT and repetitive strain injury . Additionally , diagnostic accuracy may be slightly lower than with glass slides. Some pathologists, particularly cytopathologists, dislike the loss of  a three-dimensional image, and detection of  very small items such as microorganisms can be di ffi cult.
Biomarker
Examples of tumours where relevant Application
Mismatch repair genes
CRC
Gynaecological carcinomas
Other digestive system carcinomas
HER2
Breast carcinoma
Gastric/oesophageal adenocarcinoma
CRC (emerging evidence)
PD-L1
Lung carcinoma
Gastric carcinoma
Bladder/urological carcinoma
Malignant melanoma
Breast carcinoma
Endometrial carcinoma
EGFR
mutation
Lung carcinoma
BRAF
mutation
Malignant melanoma
CRC
KRAS
mutation
CRC
NTRK
fusions
CRC
ALK
fusion
Non-small cell lung carcinoma
Renal cell carcinoma
FGFR
fusions
Bladder carcinoma
Tumour mutation burden
Various
CISH, chromogenic
in situ
hybridisation; CRC, colorectal carcinoma; FISH,
/f_l
uorescence
NGS, next-generation sequencing; PCR, polymerase chain reaction.

Deeper levels and extra blocks
Deeper levels and extra blocks
The pathologist may request ‘deeper levels’, whereby the BMS cuts further into the para ffi n block to obtain further sections that may provide more information. For example, deeper levels of  an atypical but non-invasive epithelial lesion might show foci of  invasion, allowing a definite diagnosis of  carcinoma. Further sampling of  tissue from a resection specimen (extra Hugo Schi ff , 1834–1915, German biochemist who worked in Florence, Italy . Max Perls , 1843–1881, pathologist, Giessen, Germany . Ira Thompson van Gieson , 1866–1913, American neuropathologist, described this stain in 1889. nodes in a cancer case is insu ffi cient for accurate staging. - Deeper levels and extra blocks
The pathologist may request ‘deeper levels’, whereby the BMS cuts further into the para ffi n block to obtain further sections that may provide more information. For example, deeper levels of  an atypical but non-invasive epithelial lesion might show foci of  invasion, allowing a definite diagnosis of  carcinoma. Further sampling of  tissue from a resection specimen (extra Hugo Schi ff , 1834–1915, German biochemist who worked in Florence, Italy . Max Perls , 1843–1881, pathologist, Giessen, Germany . Ira Thompson van Gieson , 1866–1913, American neuropathologist, described this stain in 1889. nodes in a cancer case is insu ffi cient for accurate staging. - Deeper levels and extra blocks
The pathologist may request ‘deeper levels’, whereby the BMS cuts further into the para ffi n block to obtain further sections that may provide more information. For example, deeper levels of  an atypical but non-invasive epithelial lesion might show foci of  invasion, allowing a definite diagnosis of  carcinoma. Further sampling of  tissue from a resection specimen (extra Hugo Schi ff , 1834–1915, German biochemist who worked in Florence, Italy . Max Perls , 1843–1881, pathologist, Giessen, Germany . Ira Thompson van Gieson , 1866–1913, American neuropathologist, described this stain in 1889. nodes in a cancer case is insu ffi cient for accurate staging. -

Detection of clinically relevant abnormalities in
Detection of clinically relevant abnormalities in genes
There are two broadly related areas of  clinical practice that rely on molecular analysis. First, analysis of tumour DNA may improve diagnostic precision, enhance treatment plans and help predict clinical outcome. Second, it may suggest or detect germline mutations that are characteristic of an inherited disease. This can confirm non-neoplastic conditions, such as cystic fibrosis, or be used to diagnose a hereditary predisposi - tion to cancer, e.g. Lynch syndrome. - Mutational analysis requires extraction of  DNA from tis - sue (or from other sources such as blood) and often includes sequencing-based screening methods (e.g. Sanger sequencing, pyrosequencing) ( Figure 11.29 ) , screening methods compar - ing mutated with normal DNA and targeted m utation detec - tion methods. NGS ( Figure 11.30 ) emerged relatively recently . The term NGS encompasses several platforms, each of  which performs - massively parallel sequencing, allowing simultaneous examina - tion of  millions of  fragments of  DNA for molecular alterations. It is applicable to formalin-fixed tissue, allows evaluation of many DNA regions in a single assay and displays increased analytical sensitivity (i.e . the ability to detect low-frequency alleles) compared with Sanger sequencing or conventional PCR. Widely clinically used targeted NGS panels can identify - multiple known mutations and other variants in 20–500 genes of  interest in a single test. New powerful platforms can detect not only point muta - , RB ), tions but also copy number variants and gene fusions in more than 100 genes involved in human oncogenesis with minimal nuclear acid (DNA and RNA) sample input. Adequate amounts of  good quality tumour DNA are nec - essary for the success of  these techniques. Histolog y samples usually include both non-neoplastic tissue and tumour. The pathologist plays a crucial role in assessing the suitability of - tissue samples for molecular analysis by analysing tumour cell content as a percentage of all cells, cellularity and degree of necrosis. Microdissection of  the area of  interest using conven - tional techniques or laser-assisted approaches improves yields of  tumour-derived DNA. Summary box 11.13 Genes and carcinogenesis /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Indications for molecular analysis of tumour tissue /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Summary box 11.15 Detection methods for main molecular changes /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
(b)
Figure 11.30
Immunohistochemical screening for mismatch repair
gene abnormalities in a carcinoma.
(a)
There is retention of nuclear
MLH1 expression (arrows showing positively staining brown neoplas
tic nuclei).
(b)
In contrast, there is loss of MSH2 expression (no staining
in neoplastic nuclei), suggesting a mismatch r
epair gene abnormality.
Genes
(Proto-) oncogenes
KRAS
BRAF
EGFR
BCL2
Tumour suppressor genes
TP53
BRCA1/2
Pathways
Proliferation and signal transduction
Cell cycle control
DNA repair
Apoptosis
Diagnosis and classi
/f_i
cation
Selection of therapy
Prognosis
Staging
Monitoring disease burden
Screening for germline mutations
Con
/f_i
rmation of neoplasia (e.g. clonality)
Point mutations and small insertions and deletions: NGS, PCR
Fusions: FISH, NGS, PCR
Ampli
/f_i
cations: FISH, NGS
Tumour mutation burden: NGS
Immunohistochemistry may be a very useful initial test, and is
often suf
/f_i
cient

Detection of clinically relevant abnormalities in genes
Detection of clinically relevant abnormalities in genes
There are two broadly related areas of  clinical practice that rely on molecular analysis. First, analysis of tumour DNA may improve diagnostic precision, enhance treatment plans and help predict clinical outcome. Second, it may suggest or detect germline mutations that are characteristic of an inherited disease. This can confirm non-neoplastic conditions, such as cystic fibrosis, or be used to diagnose a hereditary predisposi - tion to cancer, e.g. Lynch syndrome. - Mutational analysis requires extraction of  DNA from tis - sue (or from other sources such as blood) and often includes sequencing-based screening methods (e.g. Sanger sequencing, pyrosequencing) ( Figure 11.29 ) , screening methods compar - ing mutated with normal DNA and targeted m utation detec - tion methods. NGS ( Figure 11.30 ) emerged relatively recently . The term NGS encompasses several platforms, each of  which performs - massively parallel sequencing, allowing simultaneous examina - tion of  millions of  fragments of  DNA for molecular alterations. It is applicable to formalin-fixed tissue, allows evaluation of many DNA regions in a single assay and displays increased analytical sensitivity (i.e . the ability to detect low-frequency alleles) compared with Sanger sequencing or conventional PCR. Widely clinically used targeted NGS panels can identify - multiple known mutations and other variants in 20–500 genes of  interest in a single test. New powerful platforms can detect not only point muta - , RB ), tions but also copy number variants and gene fusions in more than 100 genes involved in human oncogenesis with minimal nuclear acid (DNA and RNA) sample input. Adequate amounts of  good quality tumour DNA are nec - essary for the success of  these techniques. Histolog y samples usually include both non-neoplastic tissue and tumour. The pathologist plays a crucial role in assessing the suitability of - tissue samples for molecular analysis by analysing tumour cell content as a percentage of all cells, cellularity and degree of necrosis. Microdissection of  the area of  interest using conven - tional techniques or laser-assisted approaches improves yields of  tumour-derived DNA. Summary box 11.13 Genes and carcinogenesis /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Indications for molecular analysis of tumour tissue /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Summary box 11.15 Detection methods for main molecular changes /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
(b)
Figure 11.30
Immunohistochemical screening for mismatch repair
gene abnormalities in a carcinoma.
(a)
There is retention of nuclear
MLH1 expression (arrows showing positively staining brown neoplas
tic nuclei).
(b)
In contrast, there is loss of MSH2 expression (no staining
in neoplastic nuclei), suggesting a mismatch r
epair gene abnormality.
Genes
(Proto-) oncogenes
KRAS
BRAF
EGFR
BCL2
Tumour suppressor genes
TP53
BRCA1/2
Pathways
Proliferation and signal transduction
Cell cycle control
DNA repair
Apoptosis
Diagnosis and classi
/f_i
cation
Selection of therapy
Prognosis
Staging
Monitoring disease burden
Screening for germline mutations
Con
/f_i
rmation of neoplasia (e.g. clonality)
Point mutations and small insertions and deletions: NGS, PCR
Fusions: FISH, NGS, PCR
Ampli
/f_i
cations: FISH, NGS
Tumour mutation burden: NGS
Immunohistochemistry may be a very useful initial test, and is
often suf
/f_i
cient

Detection of clinically relevant abnormalities in
Detection of clinically relevant abnormalities in genes
There are two broadly related areas of  clinical practice that rely on molecular analysis. First, analysis of tumour DNA may improve diagnostic precision, enhance treatment plans and help predict clinical outcome. Second, it may suggest or detect germline mutations that are characteristic of an inherited disease. This can confirm non-neoplastic conditions, such as cystic fibrosis, or be used to diagnose a hereditary predisposi - tion to cancer, e.g. Lynch syndrome. - Mutational analysis requires extraction of  DNA from tis - sue (or from other sources such as blood) and often includes sequencing-based screening methods (e.g. Sanger sequencing, pyrosequencing) ( Figure 11.29 ) , screening methods compar - ing mutated with normal DNA and targeted m utation detec - tion methods. NGS ( Figure 11.30 ) emerged relatively recently . The term NGS encompasses several platforms, each of  which performs - massively parallel sequencing, allowing simultaneous examina - tion of  millions of  fragments of  DNA for molecular alterations. It is applicable to formalin-fixed tissue, allows evaluation of many DNA regions in a single assay and displays increased analytical sensitivity (i.e . the ability to detect low-frequency alleles) compared with Sanger sequencing or conventional PCR. Widely clinically used targeted NGS panels can identify - multiple known mutations and other variants in 20–500 genes of  interest in a single test. New powerful platforms can detect not only point muta - , RB ), tions but also copy number variants and gene fusions in more than 100 genes involved in human oncogenesis with minimal nuclear acid (DNA and RNA) sample input. Adequate amounts of  good quality tumour DNA are nec - essary for the success of  these techniques. Histolog y samples usually include both non-neoplastic tissue and tumour. The pathologist plays a crucial role in assessing the suitability of - tissue samples for molecular analysis by analysing tumour cell content as a percentage of all cells, cellularity and degree of necrosis. Microdissection of  the area of  interest using conven - tional techniques or laser-assisted approaches improves yields of  tumour-derived DNA. Summary box 11.13 Genes and carcinogenesis /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Indications for molecular analysis of tumour tissue /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Summary box 11.15 Detection methods for main molecular changes /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
(b)
Figure 11.30
Immunohistochemical screening for mismatch repair
gene abnormalities in a carcinoma.
(a)
There is retention of nuclear
MLH1 expression (arrows showing positively staining brown neoplas
tic nuclei).
(b)
In contrast, there is loss of MSH2 expression (no staining
in neoplastic nuclei), suggesting a mismatch r
epair gene abnormality.
Genes
(Proto-) oncogenes
KRAS
BRAF
EGFR
BCL2
Tumour suppressor genes
TP53
BRCA1/2
Pathways
Proliferation and signal transduction
Cell cycle control
DNA repair
Apoptosis
Diagnosis and classi
/f_i
cation
Selection of therapy
Prognosis
Staging
Monitoring disease burden
Screening for germline mutations
Con
/f_i
rmation of neoplasia (e.g. clonality)
Point mutations and small insertions and deletions: NGS, PCR
Fusions: FISH, NGS, PCR
Ampli
/f_i
cations: FISH, NGS
Tumour mutation burden: NGS
Immunohistochemistry may be a very useful initial test, and is
often suf
/f_i
cient

Electron microscopy
Electron microscopy
Electron microscopy allows visualisation of tissue at very high magnification, e.g. × 1000 to × 500 /uni00A0 000. It may help to decide the lineage of a non-neoplastic or neoplastic cell and may help to determine the nature of  abnormal deposits, e.g. in renal disease. However, it is time-consuming, labour intensive and expensive and consequently has limited applications. Alfred Scott Warthin , 1866–1931, Professor of  Pathology , The University of  Michigan, Ann Arbor, MI, USA. Allen Chronister Starry , 1890–1973, American pathologist.
(b)
Figure 11.24
(a)
A liver biopsy stained with haematoxylin and eosin
in which the severity of
/f_i
brosis cannot be determined.
(b)
A reticulin
stain demonstrates
/f_i
brous bridges (arrows).
Electron microscopy
Electron microscopy allows visualisation of tissue at very high magnification, e.g. × 1000 to × 500 /uni00A0 000. It may help to decide the lineage of a non-neoplastic or neoplastic cell and may help to determine the nature of  abnormal deposits, e.g. in renal disease. However, it is time-consuming, labour intensive and expensive and consequently has limited applications. Alfred Scott Warthin , 1866–1931, Professor of  Pathology , The University of  Michigan, Ann Arbor, MI, USA. Allen Chronister Starry , 1890–1973, American pathologist.
(b)
Figure 11.24
(a)
A liver biopsy stained with haematoxylin and eosin
in which the severity of
/f_i
brosis cannot be determined.
(b)
A reticulin
stain demonstrates
/f_i
brous bridges (arrows).
Electron microscopy
Electron microscopy allows visualisation of tissue at very high magnification, e.g. × 1000 to × 500 /uni00A0 000. It may help to decide the lineage of a non-neoplastic or neoplastic cell and may help to determine the nature of  abnormal deposits, e.g. in renal disease. However, it is time-consuming, labour intensive and expensive and consequently has limited applications. Alfred Scott Warthin , 1866–1931, Professor of  Pathology , The University of  Michigan, Ann Arbor, MI, USA. Allen Chronister Starry , 1890–1973, American pathologist.
(b)
Figure 11.24
(a)
A liver biopsy stained with haematoxylin and eosin
in which the severity of
/f_i
brosis cannot be determined.
(b)
A reticulin
stain demonstrates
/f_i
brous bridges (arrows).

FURTHER READING
FURTHER READING
Brierley JD, Gospodarowicz MK, Wittekind C. TNM classification of malignant tumours , 8th edn. Oxford: Wiley-Blackwell, 2017. Cardesa A, Zidar N, Alos L et al . The Kaiser’s cancer revisited: was Virchow totally wrong? Virchows Arch 2011; 458(6): 649–57. Feakins RM, Allen D, Campbell F et al . Tissue pathways for gastrointes - tinal and pancreatobiliary pathology , 2nd edn. London: Royal College of  Pathologists, 2011. Goldblum JR, Lamps LW , McKenney JK, Myers JL. Rosai and Acker - man’s surgical pathology , 11th edn. Cambridge, MA: Elsevier, 2017. Kumar V , Abbas AK, Aster JC. Robbins and Cotran. Pathologic basis of disease, 10th edn. Philadelphia, PA: Elsevier, 2020. Loughrey MB, Quirke P , Shepherd NA. Dataset for colorectal cancer , 4th edn. London: Royal College of  Pathologists, 2018. World Health Organization Classification of  Tumours Editorial Board. Digestive system tumours , 5th edn. Lyon: International Agency for Research on Cancer, 2019. FURTHER READING
Brierley JD, Gospodarowicz MK, Wittekind C. TNM classification of malignant tumours , 8th edn. Oxford: Wiley-Blackwell, 2017. Cardesa A, Zidar N, Alos L et al . The Kaiser’s cancer revisited: was Virchow totally wrong? Virchows Arch 2011; 458(6): 649–57. Feakins RM, Allen D, Campbell F et al . Tissue pathways for gastrointes - tinal and pancreatobiliary pathology , 2nd edn. London: Royal College of  Pathologists, 2011. Goldblum JR, Lamps LW , McKenney JK, Myers JL. Rosai and Acker - man’s surgical pathology , 11th edn. Cambridge, MA: Elsevier, 2017. Kumar V , Abbas AK, Aster JC. Robbins and Cotran. Pathologic basis of disease, 10th edn. Philadelphia, PA: Elsevier, 2020. Loughrey MB, Quirke P , Shepherd NA. Dataset for colorectal cancer , 4th edn. London: Royal College of  Pathologists, 2018. World Health Organization Classification of  Tumours Editorial Board. Digestive system tumours , 5th edn. Lyon: International Agency for Research on Cancer, 2019. FURTHER READING
Brierley JD, Gospodarowicz MK, Wittekind C. TNM classification of malignant tumours , 8th edn. Oxford: Wiley-Blackwell, 2017. Cardesa A, Zidar N, Alos L et al . The Kaiser’s cancer revisited: was Virchow totally wrong? Virchows Arch 2011; 458(6): 649–57. Feakins RM, Allen D, Campbell F et al . Tissue pathways for gastrointes - tinal and pancreatobiliary pathology , 2nd edn. London: Royal College of  Pathologists, 2011. Goldblum JR, Lamps LW , McKenney JK, Myers JL. Rosai and Acker - man’s surgical pathology , 11th edn. Cambridge, MA: Elsevier, 2017. Kumar V , Abbas AK, Aster JC. Robbins and Cotran. Pathologic basis of disease, 10th edn. Philadelphia, PA: Elsevier, 2020. Loughrey MB, Quirke P , Shepherd NA. Dataset for colorectal cancer , 4th edn. London: Royal College of  Pathologists, 2018. World Health Organization Classification of  Tumours Editorial Board. Digestive system tumours , 5th edn. Lyon: International Agency for Research on Cancer, 2019.

FURTHER WORK
FURTHER WORK
Pathologists request further stains or other tests on a signif - icant minority of  histology specimens. This includes special stains, immunohistochemistry , in situ hybridisation and various molecular pathology techniques. Electron microscopy may assist with renal biopsy assessment. Some of  these additional investigations are also applicable to cytology specimens ( Figure 11.22 ). Su ffi cient tissue is again important for accurate inter - pretation of  some tests, e.g. molecular testing and quantitative immunohistochemical methods. - FURTHER WORK
Pathologists request further stains or other tests on a signif - icant minority of  histology specimens. This includes special stains, immunohistochemistry , in situ hybridisation and various molecular pathology techniques. Electron microscopy may assist with renal biopsy assessment. Some of  these additional investigations are also applicable to cytology specimens ( Figure 11.22 ). Su ffi cient tissue is again important for accurate inter - pretation of  some tests, e.g. molecular testing and quantitative immunohistochemical methods. - FURTHER WORK
Pathologists request further stains or other tests on a signif - icant minority of  histology specimens. This includes special stains, immunohistochemistry , in situ hybridisation and various molecular pathology techniques. Electron microscopy may assist with renal biopsy assessment. Some of  these additional investigations are also applicable to cytology specimens ( Figure 11.22 ). Su ffi cient tissue is again important for accurate inter - pretation of  some tests, e.g. molecular testing and quantitative immunohistochemical methods. -

Flow cytometry
Flow cytometry
Flow cytometry is a laser-based or impedance-based technique used for cell counting, cell sorting, biomarker detection and protein engineering. Cells are suspended in a stream of  fluid and passed by an electronic detection apparatus. It is useful James Ewing , 1866–1943, Professor of  Pathology , Cornell University Medical College, New Y ork, NY , USA, described this type of  sarcoma in 1921. Frederick Sanger , 1918–2013, biochemist, Cambridge University , Cambridge, UK, awarded the Nobel Prize in Chemistry twice: once in 1958 for work on the structure of  proteins and again in 1980 for work on base sequences of  nucleic acids.
(b)
Flow cytometry
Flow cytometry is a laser-based or impedance-based technique used for cell counting, cell sorting, biomarker detection and protein engineering. Cells are suspended in a stream of  fluid and passed by an electronic detection apparatus. It is useful James Ewing , 1866–1943, Professor of  Pathology , Cornell University Medical College, New Y ork, NY , USA, described this type of  sarcoma in 1921. Frederick Sanger , 1918–2013, biochemist, Cambridge University , Cambridge, UK, awarded the Nobel Prize in Chemistry twice: once in 1958 for work on the structure of  proteins and again in 1980 for work on base sequences of  nucleic acids.
(b)
Flow cytometry
Flow cytometry is a laser-based or impedance-based technique used for cell counting, cell sorting, biomarker detection and protein engineering. Cells are suspended in a stream of  fluid and passed by an electronic detection apparatus. It is useful James Ewing , 1866–1943, Professor of  Pathology , Cornell University Medical College, New Y ork, NY , USA, described this type of  sarcoma in 1921. Frederick Sanger , 1918–2013, biochemist, Cambridge University , Cambridge, UK, awarded the Nobel Prize in Chemistry twice: once in 1958 for work on the structure of  proteins and again in 1980 for work on base sequences of  nucleic acids.
(b)

Fresh tissue
Fresh tissue
The most common indication for submission of  a fresh tissue sample (i.e. without the usual formalin or any other fixative) is rapid frozen section diagnosis, usually done intraoperatively . Other indications are microbiological assessment, electron microscopy , chemical analyses (e.g. quantification of  iron in the tissue), research work, tissue banking and some types of molecular pathological analysis. Fresh tissue
The most common indication for submission of  a fresh tissue sample (i.e. without the usual formalin or any other fixative) is rapid frozen section diagnosis, usually done intraoperatively . Other indications are microbiological assessment, electron microscopy , chemical analyses (e.g. quantification of  iron in the tissue), research work, tissue banking and some types of molecular pathological analysis. Fresh tissue
The most common indication for submission of  a fresh tissue sample (i.e. without the usual formalin or any other fixative) is rapid frozen section diagnosis, usually done intraoperatively . Other indications are microbiological assessment, electron microscopy , chemical analyses (e.g. quantification of  iron in the tissue), research work, tissue banking and some types of molecular pathological analysis.

Genomic changes in tumours
Genomic changes in tumours
In normal circumstances, there is precise control of  the divi - sion and proliferation of  human cells. For example, various growth factors influence division by binding to specific cell surface tyrosine kinase receptors, resulting in the initiation of  a complex intracellular cascade of  changes . Damaged - cells may undergo apoptosis, a carefully regulated process of programmed cell death. Tumours require loss of  control of  cell proliferation. Abnormalities of  numerous genes can a ff ect proliferation and fall into facilitate tumour development. The relevant genes two main categories, i.e. proto-oncogenes (which stimulate - cell proliferation) and tumour suppressor genes (which inhibit proliferation) but the picture is not always so straightforward. Activation of  proto-oncogenes by genetic changes may induce or accelerate cell proliferation, while inhibition of  tumour sup - pressor genes may remove the controls that normally prevent or retard proliferation. When a proto-oncogene contributes to cancer development, it is usually known as an oncogene. Other genetic changes can also facilitate tumorigenesis. esis. The classical model for this process is the ‘adenoma– carcinoma sequence’ of  Fearon and V ogelstein, whereby the accumulation of  mutations such as APC , KRAS and TP53 the colorectal mucosa corresponds broadly to the transforma tion of non-neoplastic mucosa into a colorectal adenoma and subsequently a carcinoma. Current models show that the pic ture is often very complex and di ff ers between tumours and that a simple sequence does not operate consistently . Several types of  genetic abnormality can occur during tumorigenesis. The main categories of  abnormalities are point mutations, fusion genes and cop y number changes. Point muta tions ar e single changes in the sequence of  nucleotides in DNA and can be germline, i.e. inherited from a parent and accord ingly present in every cell in the body , or somatic, i.e. acquir at some point during life and a ff ecting only the tumour cells. Deletions and insertions (indels) of  nucleotides result in a frameshift mutation. Examples include TP53 tumour suppres sor gene mutations, causing production of  an abnormal p53 protein that lacks suppressor function; and mutation of  the KIT gene, causing ligand-independent activation of  a growth factor receptor. Fusion genes may be formed by several mechanisms, including translocations and deletions. The translocation t(14:18) in follicular lymphoma results in juxtaposition of  the anti-apoptotic BCL2 to a regulatory region of  an immunoglob ulin heavy chain gene, with subsequent bcl-2 overexpression. Fusion genes can result from various chromosomal changes, e.g. TMPRSS2-ERG gene fusion in prostate adenocarcinoma can occur as a result of  a chromosomal deletion and causes abnormal oncogenic activation of ERG . Gene amplification refers to an increase in copy number, resulting in overexpression of  the gene, and can variably result from abnormalities in DNA replica tion, chromosomal struc ture or telomeres. An example is HER2 amplification, resulting in overexpression of  the growth factor in carcinomas of  breast and stomach. These many types of  abnormality in the genome may ultimately interfere with the function of  proteins involved in regulatory processes: TP53 and KRAS mutations are among the most common. Genetic changes can disrupt various path ways, including signal transduction (e.g. various growth factors and growth factor receptors, intracellular components such as RAS genes, APC gene), cell cycle regulators (e.g. p16 DNA repair pathways (e.g. MMR genes, BRCA1 mutations in breast carcinoma) and apoptosis (e.g. BCL2 , an inhibitor of apoptosis). DNA MMR genes play a vital role in correcting replication errors and other errors. Abnormalities of  MMR genes cause instability of  short tandem repeated sequences of  DNA known as micr osatellites, resulting in MSI. Tumours with this char acteristic are MSI-H (high level of  MSI). The relevant genes are MLH1, MSH2, MSH6 and PMS2 . MSI is a feature of around 15% of  CRCs and can result either from a germline mutation in the MMR gene (Lynch syndrome) or, more often, Eric R Fearon , contemporary , Professor of  Oncology , University of  Michigan, Ann Arbor, MI, USA. Bert Vogelstein , b. 1949, Professor of  Oncology and Pathology , Johns Hopkins Medical School, Baltimore, MD, USA. Henry Thompson Lynch , 1928–2019, Chair of  Preventative Medicine, Creighton University , Omaha, NE, USA. repair gene abnormalities in tumours ). in Epigenetic changes and methylation - Epigenetic factors are external to the gene sequence and can switch it on or o ff . The latter is known as epigenetic silencing - and can result from DNA methylation (addition of  a methyl group to DNA), modifications of  histones and RNA-associated silencing. Loss of  methylation with gene activation can occur in tumours. Conversely , hypermethylation of  tumour suppressor genes or of  the MMR gene MLH1 can reduce or halt their - activity , favouring malignancy . - ed Genomic changes in tumours
In normal circumstances, there is precise control of  the divi - sion and proliferation of  human cells. For example, various growth factors influence division by binding to specific cell surface tyrosine kinase receptors, resulting in the initiation of  a complex intracellular cascade of  changes . Damaged - cells may undergo apoptosis, a carefully regulated process of programmed cell death. Tumours require loss of  control of  cell proliferation. Abnormalities of  numerous genes can a ff ect proliferation and fall into facilitate tumour development. The relevant genes two main categories, i.e. proto-oncogenes (which stimulate - cell proliferation) and tumour suppressor genes (which inhibit proliferation) but the picture is not always so straightforward. Activation of  proto-oncogenes by genetic changes may induce or accelerate cell proliferation, while inhibition of  tumour sup - pressor genes may remove the controls that normally prevent or retard proliferation. When a proto-oncogene contributes to cancer development, it is usually known as an oncogene. Other genetic changes can also facilitate tumorigenesis. esis. The classical model for this process is the ‘adenoma– carcinoma sequence’ of  Fearon and V ogelstein, whereby the accumulation of  mutations such as APC , KRAS and TP53 the colorectal mucosa corresponds broadly to the transforma tion of non-neoplastic mucosa into a colorectal adenoma and subsequently a carcinoma. Current models show that the pic ture is often very complex and di ff ers between tumours and that a simple sequence does not operate consistently . Several types of  genetic abnormality can occur during tumorigenesis. The main categories of  abnormalities are point mutations, fusion genes and cop y number changes. Point muta tions ar e single changes in the sequence of  nucleotides in DNA and can be germline, i.e. inherited from a parent and accord ingly present in every cell in the body , or somatic, i.e. acquir at some point during life and a ff ecting only the tumour cells. Deletions and insertions (indels) of  nucleotides result in a frameshift mutation. Examples include TP53 tumour suppres sor gene mutations, causing production of  an abnormal p53 protein that lacks suppressor function; and mutation of  the KIT gene, causing ligand-independent activation of  a growth factor receptor. Fusion genes may be formed by several mechanisms, including translocations and deletions. The translocation t(14:18) in follicular lymphoma results in juxtaposition of  the anti-apoptotic BCL2 to a regulatory region of  an immunoglob ulin heavy chain gene, with subsequent bcl-2 overexpression. Fusion genes can result from various chromosomal changes, e.g. TMPRSS2-ERG gene fusion in prostate adenocarcinoma can occur as a result of  a chromosomal deletion and causes abnormal oncogenic activation of ERG . Gene amplification refers to an increase in copy number, resulting in overexpression of  the gene, and can variably result from abnormalities in DNA replica tion, chromosomal struc ture or telomeres. An example is HER2 amplification, resulting in overexpression of  the growth factor in carcinomas of  breast and stomach. These many types of  abnormality in the genome may ultimately interfere with the function of  proteins involved in regulatory processes: TP53 and KRAS mutations are among the most common. Genetic changes can disrupt various path ways, including signal transduction (e.g. various growth factors and growth factor receptors, intracellular components such as RAS genes, APC gene), cell cycle regulators (e.g. p16 DNA repair pathways (e.g. MMR genes, BRCA1 mutations in breast carcinoma) and apoptosis (e.g. BCL2 , an inhibitor of apoptosis). DNA MMR genes play a vital role in correcting replication errors and other errors. Abnormalities of  MMR genes cause instability of  short tandem repeated sequences of  DNA known as micr osatellites, resulting in MSI. Tumours with this char acteristic are MSI-H (high level of  MSI). The relevant genes are MLH1, MSH2, MSH6 and PMS2 . MSI is a feature of around 15% of  CRCs and can result either from a germline mutation in the MMR gene (Lynch syndrome) or, more often, Eric R Fearon , contemporary , Professor of  Oncology , University of  Michigan, Ann Arbor, MI, USA. Bert Vogelstein , b. 1949, Professor of  Oncology and Pathology , Johns Hopkins Medical School, Baltimore, MD, USA. Henry Thompson Lynch , 1928–2019, Chair of  Preventative Medicine, Creighton University , Omaha, NE, USA. repair gene abnormalities in tumours ). in Epigenetic changes and methylation - Epigenetic factors are external to the gene sequence and can switch it on or o ff . The latter is known as epigenetic silencing - and can result from DNA methylation (addition of  a methyl group to DNA), modifications of  histones and RNA-associated silencing. Loss of  methylation with gene activation can occur in tumours. Conversely , hypermethylation of  tumour suppressor genes or of  the MMR gene MLH1 can reduce or halt their - activity , favouring malignancy . - ed Genomic changes in tumours
In normal circumstances, there is precise control of  the divi - sion and proliferation of  human cells. For example, various growth factors influence division by binding to specific cell surface tyrosine kinase receptors, resulting in the initiation of  a complex intracellular cascade of  changes . Damaged - cells may undergo apoptosis, a carefully regulated process of programmed cell death. Tumours require loss of  control of  cell proliferation. Abnormalities of  numerous genes can a ff ect proliferation and fall into facilitate tumour development. The relevant genes two main categories, i.e. proto-oncogenes (which stimulate - cell proliferation) and tumour suppressor genes (which inhibit proliferation) but the picture is not always so straightforward. Activation of  proto-oncogenes by genetic changes may induce or accelerate cell proliferation, while inhibition of  tumour sup - pressor genes may remove the controls that normally prevent or retard proliferation. When a proto-oncogene contributes to cancer development, it is usually known as an oncogene. Other genetic changes can also facilitate tumorigenesis. esis. The classical model for this process is the ‘adenoma– carcinoma sequence’ of  Fearon and V ogelstein, whereby the accumulation of  mutations such as APC , KRAS and TP53 the colorectal mucosa corresponds broadly to the transforma tion of non-neoplastic mucosa into a colorectal adenoma and subsequently a carcinoma. Current models show that the pic ture is often very complex and di ff ers between tumours and that a simple sequence does not operate consistently . Several types of  genetic abnormality can occur during tumorigenesis. The main categories of  abnormalities are point mutations, fusion genes and cop y number changes. Point muta tions ar e single changes in the sequence of  nucleotides in DNA and can be germline, i.e. inherited from a parent and accord ingly present in every cell in the body , or somatic, i.e. acquir at some point during life and a ff ecting only the tumour cells. Deletions and insertions (indels) of  nucleotides result in a frameshift mutation. Examples include TP53 tumour suppres sor gene mutations, causing production of  an abnormal p53 protein that lacks suppressor function; and mutation of  the KIT gene, causing ligand-independent activation of  a growth factor receptor. Fusion genes may be formed by several mechanisms, including translocations and deletions. The translocation t(14:18) in follicular lymphoma results in juxtaposition of  the anti-apoptotic BCL2 to a regulatory region of  an immunoglob ulin heavy chain gene, with subsequent bcl-2 overexpression. Fusion genes can result from various chromosomal changes, e.g. TMPRSS2-ERG gene fusion in prostate adenocarcinoma can occur as a result of  a chromosomal deletion and causes abnormal oncogenic activation of ERG . Gene amplification refers to an increase in copy number, resulting in overexpression of  the gene, and can variably result from abnormalities in DNA replica tion, chromosomal struc ture or telomeres. An example is HER2 amplification, resulting in overexpression of  the growth factor in carcinomas of  breast and stomach. These many types of  abnormality in the genome may ultimately interfere with the function of  proteins involved in regulatory processes: TP53 and KRAS mutations are among the most common. Genetic changes can disrupt various path ways, including signal transduction (e.g. various growth factors and growth factor receptors, intracellular components such as RAS genes, APC gene), cell cycle regulators (e.g. p16 DNA repair pathways (e.g. MMR genes, BRCA1 mutations in breast carcinoma) and apoptosis (e.g. BCL2 , an inhibitor of apoptosis). DNA MMR genes play a vital role in correcting replication errors and other errors. Abnormalities of  MMR genes cause instability of  short tandem repeated sequences of  DNA known as micr osatellites, resulting in MSI. Tumours with this char acteristic are MSI-H (high level of  MSI). The relevant genes are MLH1, MSH2, MSH6 and PMS2 . MSI is a feature of around 15% of  CRCs and can result either from a germline mutation in the MMR gene (Lynch syndrome) or, more often, Eric R Fearon , contemporary , Professor of  Oncology , University of  Michigan, Ann Arbor, MI, USA. Bert Vogelstein , b. 1949, Professor of  Oncology and Pathology , Johns Hopkins Medical School, Baltimore, MD, USA. Henry Thompson Lynch , 1928–2019, Chair of  Preventative Medicine, Creighton University , Omaha, NE, USA. repair gene abnormalities in tumours ). in Epigenetic changes and methylation - Epigenetic factors are external to the gene sequence and can switch it on or o ff . The latter is known as epigenetic silencing - and can result from DNA methylation (addition of  a methyl group to DNA), modifications of  histones and RNA-associated silencing. Loss of  methylation with gene activation can occur in tumours. Conversely , hypermethylation of  tumour suppressor genes or of  the MMR gene MLH1 can reduce or halt their - activity , favouring malignancy . - ed

HER2 gene amplification
HER2 gene amplification
HER2 status influences the selection of  therapy for breast cancer and metastatic gastric adenocarcinoma. Tumours with HER2 amplification may be treated with the monoclonal antibodies trastuzumab or pertuzumab, often in combination with other drugs. Recent data suggest that HER2 amplification may be a relevant therapeutic target in metastatic CRCs that are microsatellite stable. Summary box 11.16 Tumour types that may respond to immune checkpoint inhibitor drugs /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
Breast carcinoma
Urothelial carcinoma
Non-small cell lung cancer
Small cell lung cancer
Hepatocellular carcinoma
Malignant melanoma
Translocations can produce novel fusion genes that either produce a chimeric protein, e.g. BCR-ABL t(9:22) in chronic myeloid leukaemia, or may place an active promoter next to a proto-oncogene, causing its activation, e.g. t(14:18) IGH-BCL2 in follicular lymphoma. Translocations that activate tyrosine kinases can result in drug responsiveness, e.g. ALK, RET , NTRK, ROS and FGFR2 . Tumour mutation burden Tumour mutation burden (TMB) is a recently recognised biomarker. A higher number of  mutations within the tumour corresponds to a higher TMB. High levels of  TMB can predict response to ICIs. PD-L1 immunohistochemistry and detection of  MSI-H are also used for this purpose. HER2 gene amplification
HER2 status influences the selection of  therapy for breast cancer and metastatic gastric adenocarcinoma. Tumours with HER2 amplification may be treated with the monoclonal antibodies trastuzumab or pertuzumab, often in combination with other drugs. Recent data suggest that HER2 amplification may be a relevant therapeutic target in metastatic CRCs that are microsatellite stable. Summary box 11.16 Tumour types that may respond to immune checkpoint inhibitor drugs /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
Breast carcinoma
Urothelial carcinoma
Non-small cell lung cancer
Small cell lung cancer
Hepatocellular carcinoma
Malignant melanoma
Translocations can produce novel fusion genes that either produce a chimeric protein, e.g. BCR-ABL t(9:22) in chronic myeloid leukaemia, or may place an active promoter next to a proto-oncogene, causing its activation, e.g. t(14:18) IGH-BCL2 in follicular lymphoma. Translocations that activate tyrosine kinases can result in drug responsiveness, e.g. ALK, RET , NTRK, ROS and FGFR2 . Tumour mutation burden Tumour mutation burden (TMB) is a recently recognised biomarker. A higher number of  mutations within the tumour corresponds to a higher TMB. High levels of  TMB can predict response to ICIs. PD-L1 immunohistochemistry and detection of  MSI-H are also used for this purpose. HER2 gene amplification
HER2 status influences the selection of  therapy for breast cancer and metastatic gastric adenocarcinoma. Tumours with HER2 amplification may be treated with the monoclonal antibodies trastuzumab or pertuzumab, often in combination with other drugs. Recent data suggest that HER2 amplification may be a relevant therapeutic target in metastatic CRCs that are microsatellite stable. Summary box 11.16 Tumour types that may respond to immune checkpoint inhibitor drugs /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
Breast carcinoma
Urothelial carcinoma
Non-small cell lung cancer
Small cell lung cancer
Hepatocellular carcinoma
Malignant melanoma
Translocations can produce novel fusion genes that either produce a chimeric protein, e.g. BCR-ABL t(9:22) in chronic myeloid leukaemia, or may place an active promoter next to a proto-oncogene, causing its activation, e.g. t(14:18) IGH-BCL2 in follicular lymphoma. Translocations that activate tyrosine kinases can result in drug responsiveness, e.g. ALK, RET , NTRK, ROS and FGFR2 . Tumour mutation burden Tumour mutation burden (TMB) is a recently recognised biomarker. A higher number of  mutations within the tumour corresponds to a higher TMB. High levels of  TMB can predict response to ICIs. PD-L1 immunohistochemistry and detection of  MSI-H are also used for this purpose.

Histological assessment
Histological assessment
In a histological preparation, the microscopic structure of  the tissue remains intact, allowing direct visualisation of tissue architecture. Accordingly , the pathologist can see not only the characteristics of  the cells that form the tissue, but also the way in which these cells relate to one another and the structure and arrangement of  the various tissue compartments. Histological assessment
In a histological preparation, the microscopic structure of  the tissue remains intact, allowing direct visualisation of tissue architecture. Accordingly , the pathologist can see not only the characteristics of  the cells that form the tissue, but also the way in which these cells relate to one another and the structure and arrangement of  the various tissue compartments. Histological assessment
In a histological preparation, the microscopic structure of  the tissue remains intact, allowing direct visualisation of tissue architecture. Accordingly , the pathologist can see not only the characteristics of  the cells that form the tissue, but also the way in which these cells relate to one another and the structure and arrangement of  the various tissue compartments.

Histological types of malignancy
Histological types of malignancy
A malignant tumour showing features of  epithelial di ff erentia - tion, and typically arising in an epithelial layer, is a carcinoma. Other important types of  malignancy include malignant mela - noma (melanocytes) ( Figure 11.13b ), lymphoma (lymphoid cells) and sarcoma (mesenchymal cells). Further subclassification is often appropriate and necessary . F or example , categories of carcinoma include squamous cell carcinoma (with evidence of keratinisation) ( Figure 11.15 ), adenocarcinoma (with evidence of glandular di ff erentiation and/or mucin production) ( Figure 11.16 ) or neuroendocrine carcinoma ( Figure 11.13a ) (usually requiring immunohistochemical confirmation of  neuroendo - crine di ff erentiation). Some carcinomas have a pattern that raises a certain di ff erential diagnosis, e.g. clear cell carcinoma ( Figure 11.17 ). There are many other morphological types of carcinoma.
(b)
,
Histological types of malignancy
A malignant tumour showing features of  epithelial di ff erentia - tion, and typically arising in an epithelial layer, is a carcinoma. Other important types of  malignancy include malignant mela - noma (melanocytes) ( Figure 11.13b ), lymphoma (lymphoid cells) and sarcoma (mesenchymal cells). Further subclassification is often appropriate and necessary . F or example , categories of carcinoma include squamous cell carcinoma (with evidence of keratinisation) ( Figure 11.15 ), adenocarcinoma (with evidence of glandular di ff erentiation and/or mucin production) ( Figure 11.16 ) or neuroendocrine carcinoma ( Figure 11.13a ) (usually requiring immunohistochemical confirmation of  neuroendo - crine di ff erentiation). Some carcinomas have a pattern that raises a certain di ff erential diagnosis, e.g. clear cell carcinoma ( Figure 11.17 ). There are many other morphological types of carcinoma.
(b)
,
Histological types of malignancy
A malignant tumour showing features of  epithelial di ff erentia - tion, and typically arising in an epithelial layer, is a carcinoma. Other important types of  malignancy include malignant mela - noma (melanocytes) ( Figure 11.13b ), lymphoma (lymphoid cells) and sarcoma (mesenchymal cells). Further subclassification is often appropriate and necessary . F or example , categories of carcinoma include squamous cell carcinoma (with evidence of keratinisation) ( Figure 11.15 ), adenocarcinoma (with evidence of glandular di ff erentiation and/or mucin production) ( Figure 11.16 ) or neuroendocrine carcinoma ( Figure 11.13a ) (usually requiring immunohistochemical confirmation of  neuroendo - crine di ff erentiation). Some carcinomas have a pattern that raises a certain di ff erential diagnosis, e.g. clear cell carcinoma ( Figure 11.17 ). There are many other morphological types of carcinoma.
(b)
,

Histology specimen
Histology specimen
On arrival in the pathology laboratory , specimens receive a unique identification number, usually with a barcode. They proceed to macroscopic assessment and sampling (colloquially - known as ‘cut up’). The largest specimens require initial open - ing (e.g. gastrointestinal tract) or slicing (e.g. uterus, pancreas, breast) to allow further and adequate fixation in formalin, usually over 24–48 hours ( Figure 11.2 ). When fixation is complete and the specimen is in a suitable condition for cutting - and sampling, a pathologist or BMS describes the appearances and lists the method of  sampling. Specimens a few millimetres in size such as endoscopic biopsies are suitable for submission in their entirety . Small resections, e.g. skin excision biopsies, may be suitable for slicing into two or more pieces and, again, submission in their entirety . For any specimen that is too large for these approaches, the prosector takes representative samples of  areas of  interest or relevance ( Figure 11.3 ). This is traditionally the remit of  the histopathologist, but BMSs or other non-medical sta ff with specific training increasingly contribute. In the UK and many other countries, there is often adher ence to a regional, national or international guideline that includes a protocol for sampling. For e xample, samples from most types of  cancer should include tumour, resection mar gins, lymph nodes, non-neoplastic tissue and any other abnor mal areas. Inks of  various colours help to identify resection margins and surfaces during microscope assessment as they remain in place after processing ( Figure 11.4 ). The prosector places specimens, or samples from speci mens, in plastic cassettes ( Figure 11.5 ). BMSs/technical sta ff then embed the tissue in para ffi n wax while in the cassette to produce a tissue block ( Figure 11.6 ). BMSs then cut sections with a thickness of  approximately 5 /uni00A0 µm from the block using a microtome ( Figure 11.7 ), place the sections on a glass slide and stain them with haemato xylin and eosin (H&E) ( Figure 11.1 ) . These steps require training and skill. A poor quality sec tion may have various artefacts, such as lines, folds and shatter e ff ect, which impede accurate assessment. H&E remains by far the most common initial stain for his topatholog y assessment, probably because it is inexpensive, safe, fast, reliable, familiar and informative. There is a wider variety of  stains for cytolog y preparations including H&E and Giemsa. Traditionally , a pathologist examines stained sections with a microscope ( Figure 11.8 ) and correlates the appearances with the clinical details and the macroscopic description. After special stains, completion of any additional studies such as immunohistochemistry and molecular analysis, the pa thologist enters a report onto a computer system and allocates specific topography and morphology codes that will facilitate future searches. Recent improvements in technology and informa - tion technology (IT) mean that some laboratories use scan - - ning machines to create digital images of  the glass slides that pathologists and others can then access locally or remotely at any time (see Digital pathology and artificial intelligence ). - - Summary box 11.3 Histological processing: sequence of events - /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF - /uni25CF /uni25CF /uni25CF - /uni25CF /uni25CF /uni25CF
Figure 11.2
(a)
A colon from a patient with familial adenomatous polyposis has been opened longitudinally, and the brown appearance re
/f_l
ects
adequate
/f_i
xation. Numerous polyps and a carcinoma are apparent.
after opening. In this example, there is less
/f_i
xation, as a result of which the mucosa in the lower part of the picture remains red rather
brown.
(c)
A uterus and an adjacent cystic lesion after slicing to allow
/f_i
xation (all
/f_i
gures courtesy of Dr J Chin Aleong, Barts Health NHS Trust,
London, UK).
(b)
An oesophagogastrectomy containing a distal oesophageal tumour
than
Receipt of specimen
Macroscopic (gross) description
Sampling of specimen (unless small enough to submit in its
entirety)
Specimen or samples placed in cassette(s)
Production of paraf
/f_i
n wax block(s)
Cutting of 5-µm sections with microtome
Sections placed on glass slides
Sections stained with H&E
Histopathologist examines slides, taking clinical and
macroscopic
/f_i
ndings into account
Further studies on tissue, if necessary
Entry of report onto computer system
Authorisation of report by pathologist
Figure 11.3
A pathologist takes a sample from a resection specimen
with a scalpel and forceps.
(a)
(b)
Figure 11.4
(a)
An unopened pancreatoduodenectomy specimen
(posterior view). Four inks of different colours have been painted onto
separate margins and surfaces.
(b)
Yellow ink on the edge of a histol
ogy section (thick arrow). Tumour (thin arrow) lies close to the surface.
The pathologist can measure the distance between the tumour and a
surface or a resection margin (double-headed arrow).
Figure 11.5
A pathologist places a tissue sample from a resection
specimen in a cassette.
Figure 11.6
Paraf
/f_i
n wax blocks. Cassettes of different colours allow
the organisation of samples and specimens into groups, e.g. accord
ing to specialty or degree of urgency.
Figure 11.7
A section (thick arrow) being cut from a paraf
/f_i
n wax block
(thin arrow) with a microtome.
Figure 11.8
A double-headed microscope allows a consultant histo
pathologist and a trainee to view a slide simultaneously.
Frozen section diagnosis is useful when a very rapid answer is necessary . Surgeons are the main users. The surgeon supplies a small representative fresh tissue sample of  the area of  interest. A BMS freezes the tissue quickly in the pathology laboratory and can produce sections for microscopic examination within several minutes. There are a few disadvantages in comparison with routine processing: fresh tissue carries a higher risk of infection; the quality is inferior to that of  routine material, resulting in a potential reduction in diagnostic accuracy and precision; small but representative samples are necessary; certain types of  tissue (e.g. fat) are di ffi cult to process; and the process is time-consuming and disruptive ( Summary box 11.4 ). Summary box 11.4 Frozen section: advantages and disadvantages /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
Advantages
Quick diagnosis
Disadvantages
Poorer quality sections
Potential reduction in accuracy and precision of histological
diagnosis
Labour intensive
Disruptive
Risk of infection
Small sample required
Some tissue types dif
/f_i
cult to process
Histology specimen
On arrival in the pathology laboratory , specimens receive a unique identification number, usually with a barcode. They proceed to macroscopic assessment and sampling (colloquially - known as ‘cut up’). The largest specimens require initial open - ing (e.g. gastrointestinal tract) or slicing (e.g. uterus, pancreas, breast) to allow further and adequate fixation in formalin, usually over 24–48 hours ( Figure 11.2 ). When fixation is complete and the specimen is in a suitable condition for cutting - and sampling, a pathologist or BMS describes the appearances and lists the method of  sampling. Specimens a few millimetres in size such as endoscopic biopsies are suitable for submission in their entirety . Small resections, e.g. skin excision biopsies, may be suitable for slicing into two or more pieces and, again, submission in their entirety . For any specimen that is too large for these approaches, the prosector takes representative samples of  areas of  interest or relevance ( Figure 11.3 ). This is traditionally the remit of  the histopathologist, but BMSs or other non-medical sta ff with specific training increasingly contribute. In the UK and many other countries, there is often adher ence to a regional, national or international guideline that includes a protocol for sampling. For e xample, samples from most types of  cancer should include tumour, resection mar gins, lymph nodes, non-neoplastic tissue and any other abnor mal areas. Inks of  various colours help to identify resection margins and surfaces during microscope assessment as they remain in place after processing ( Figure 11.4 ). The prosector places specimens, or samples from speci mens, in plastic cassettes ( Figure 11.5 ). BMSs/technical sta ff then embed the tissue in para ffi n wax while in the cassette to produce a tissue block ( Figure 11.6 ). BMSs then cut sections with a thickness of  approximately 5 /uni00A0 µm from the block using a microtome ( Figure 11.7 ), place the sections on a glass slide and stain them with haemato xylin and eosin (H&E) ( Figure 11.1 ) . These steps require training and skill. A poor quality sec tion may have various artefacts, such as lines, folds and shatter e ff ect, which impede accurate assessment. H&E remains by far the most common initial stain for his topatholog y assessment, probably because it is inexpensive, safe, fast, reliable, familiar and informative. There is a wider variety of  stains for cytolog y preparations including H&E and Giemsa. Traditionally , a pathologist examines stained sections with a microscope ( Figure 11.8 ) and correlates the appearances with the clinical details and the macroscopic description. After special stains, completion of any additional studies such as immunohistochemistry and molecular analysis, the pa thologist enters a report onto a computer system and allocates specific topography and morphology codes that will facilitate future searches. Recent improvements in technology and informa - tion technology (IT) mean that some laboratories use scan - - ning machines to create digital images of  the glass slides that pathologists and others can then access locally or remotely at any time (see Digital pathology and artificial intelligence ). - - Summary box 11.3 Histological processing: sequence of events - /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF - /uni25CF /uni25CF /uni25CF - /uni25CF /uni25CF /uni25CF
Figure 11.2
(a)
A colon from a patient with familial adenomatous polyposis has been opened longitudinally, and the brown appearance re
/f_l
ects
adequate
/f_i
xation. Numerous polyps and a carcinoma are apparent.
after opening. In this example, there is less
/f_i
xation, as a result of which the mucosa in the lower part of the picture remains red rather
brown.
(c)
A uterus and an adjacent cystic lesion after slicing to allow
/f_i
xation (all
/f_i
gures courtesy of Dr J Chin Aleong, Barts Health NHS Trust,
London, UK).
(b)
An oesophagogastrectomy containing a distal oesophageal tumour
than
Receipt of specimen
Macroscopic (gross) description
Sampling of specimen (unless small enough to submit in its
entirety)
Specimen or samples placed in cassette(s)
Production of paraf
/f_i
n wax block(s)
Cutting of 5-µm sections with microtome
Sections placed on glass slides
Sections stained with H&E
Histopathologist examines slides, taking clinical and
macroscopic
/f_i
ndings into account
Further studies on tissue, if necessary
Entry of report onto computer system
Authorisation of report by pathologist
Figure 11.3
A pathologist takes a sample from a resection specimen
with a scalpel and forceps.
(a)
(b)
Figure 11.4
(a)
An unopened pancreatoduodenectomy specimen
(posterior view). Four inks of different colours have been painted onto
separate margins and surfaces.
(b)
Yellow ink on the edge of a histol
ogy section (thick arrow). Tumour (thin arrow) lies close to the surface.
The pathologist can measure the distance between the tumour and a
surface or a resection margin (double-headed arrow).
Figure 11.5
A pathologist places a tissue sample from a resection
specimen in a cassette.
Figure 11.6
Paraf
/f_i
n wax blocks. Cassettes of different colours allow
the organisation of samples and specimens into groups, e.g. accord
ing to specialty or degree of urgency.
Figure 11.7
A section (thick arrow) being cut from a paraf
/f_i
n wax block
(thin arrow) with a microtome.
Figure 11.8
A double-headed microscope allows a consultant histo
pathologist and a trainee to view a slide simultaneously.
Frozen section diagnosis is useful when a very rapid answer is necessary . Surgeons are the main users. The surgeon supplies a small representative fresh tissue sample of  the area of  interest. A BMS freezes the tissue quickly in the pathology laboratory and can produce sections for microscopic examination within several minutes. There are a few disadvantages in comparison with routine processing: fresh tissue carries a higher risk of infection; the quality is inferior to that of  routine material, resulting in a potential reduction in diagnostic accuracy and precision; small but representative samples are necessary; certain types of  tissue (e.g. fat) are di ffi cult to process; and the process is time-consuming and disruptive ( Summary box 11.4 ). Summary box 11.4 Frozen section: advantages and disadvantages /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
Advantages
Quick diagnosis
Disadvantages
Poorer quality sections
Potential reduction in accuracy and precision of histological
diagnosis
Labour intensive
Disruptive
Risk of infection
Small sample required
Some tissue types dif
/f_i
cult to process
Histology specimen
On arrival in the pathology laboratory , specimens receive a unique identification number, usually with a barcode. They proceed to macroscopic assessment and sampling (colloquially - known as ‘cut up’). The largest specimens require initial open - ing (e.g. gastrointestinal tract) or slicing (e.g. uterus, pancreas, breast) to allow further and adequate fixation in formalin, usually over 24–48 hours ( Figure 11.2 ). When fixation is complete and the specimen is in a suitable condition for cutting - and sampling, a pathologist or BMS describes the appearances and lists the method of  sampling. Specimens a few millimetres in size such as endoscopic biopsies are suitable for submission in their entirety . Small resections, e.g. skin excision biopsies, may be suitable for slicing into two or more pieces and, again, submission in their entirety . For any specimen that is too large for these approaches, the prosector takes representative samples of  areas of  interest or relevance ( Figure 11.3 ). This is traditionally the remit of  the histopathologist, but BMSs or other non-medical sta ff with specific training increasingly contribute. In the UK and many other countries, there is often adher ence to a regional, national or international guideline that includes a protocol for sampling. For e xample, samples from most types of  cancer should include tumour, resection mar gins, lymph nodes, non-neoplastic tissue and any other abnor mal areas. Inks of  various colours help to identify resection margins and surfaces during microscope assessment as they remain in place after processing ( Figure 11.4 ). The prosector places specimens, or samples from speci mens, in plastic cassettes ( Figure 11.5 ). BMSs/technical sta ff then embed the tissue in para ffi n wax while in the cassette to produce a tissue block ( Figure 11.6 ). BMSs then cut sections with a thickness of  approximately 5 /uni00A0 µm from the block using a microtome ( Figure 11.7 ), place the sections on a glass slide and stain them with haemato xylin and eosin (H&E) ( Figure 11.1 ) . These steps require training and skill. A poor quality sec tion may have various artefacts, such as lines, folds and shatter e ff ect, which impede accurate assessment. H&E remains by far the most common initial stain for his topatholog y assessment, probably because it is inexpensive, safe, fast, reliable, familiar and informative. There is a wider variety of  stains for cytolog y preparations including H&E and Giemsa. Traditionally , a pathologist examines stained sections with a microscope ( Figure 11.8 ) and correlates the appearances with the clinical details and the macroscopic description. After special stains, completion of any additional studies such as immunohistochemistry and molecular analysis, the pa thologist enters a report onto a computer system and allocates specific topography and morphology codes that will facilitate future searches. Recent improvements in technology and informa - tion technology (IT) mean that some laboratories use scan - - ning machines to create digital images of  the glass slides that pathologists and others can then access locally or remotely at any time (see Digital pathology and artificial intelligence ). - - Summary box 11.3 Histological processing: sequence of events - /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF - /uni25CF /uni25CF /uni25CF - /uni25CF /uni25CF /uni25CF
Figure 11.2
(a)
A colon from a patient with familial adenomatous polyposis has been opened longitudinally, and the brown appearance re
/f_l
ects
adequate
/f_i
xation. Numerous polyps and a carcinoma are apparent.
after opening. In this example, there is less
/f_i
xation, as a result of which the mucosa in the lower part of the picture remains red rather
brown.
(c)
A uterus and an adjacent cystic lesion after slicing to allow
/f_i
xation (all
/f_i
gures courtesy of Dr J Chin Aleong, Barts Health NHS Trust,
London, UK).
(b)
An oesophagogastrectomy containing a distal oesophageal tumour
than
Receipt of specimen
Macroscopic (gross) description
Sampling of specimen (unless small enough to submit in its
entirety)
Specimen or samples placed in cassette(s)
Production of paraf
/f_i
n wax block(s)
Cutting of 5-µm sections with microtome
Sections placed on glass slides
Sections stained with H&E
Histopathologist examines slides, taking clinical and
macroscopic
/f_i
ndings into account
Further studies on tissue, if necessary
Entry of report onto computer system
Authorisation of report by pathologist
Figure 11.3
A pathologist takes a sample from a resection specimen
with a scalpel and forceps.
(a)
(b)
Figure 11.4
(a)
An unopened pancreatoduodenectomy specimen
(posterior view). Four inks of different colours have been painted onto
separate margins and surfaces.
(b)
Yellow ink on the edge of a histol
ogy section (thick arrow). Tumour (thin arrow) lies close to the surface.
The pathologist can measure the distance between the tumour and a
surface or a resection margin (double-headed arrow).
Figure 11.5
A pathologist places a tissue sample from a resection
specimen in a cassette.
Figure 11.6
Paraf
/f_i
n wax blocks. Cassettes of different colours allow
the organisation of samples and specimens into groups, e.g. accord
ing to specialty or degree of urgency.
Figure 11.7
A section (thick arrow) being cut from a paraf
/f_i
n wax block
(thin arrow) with a microtome.
Figure 11.8
A double-headed microscope allows a consultant histo
pathologist and a trainee to view a slide simultaneously.
Frozen section diagnosis is useful when a very rapid answer is necessary . Surgeons are the main users. The surgeon supplies a small representative fresh tissue sample of  the area of  interest. A BMS freezes the tissue quickly in the pathology laboratory and can produce sections for microscopic examination within several minutes. There are a few disadvantages in comparison with routine processing: fresh tissue carries a higher risk of infection; the quality is inferior to that of  routine material, resulting in a potential reduction in diagnostic accuracy and precision; small but representative samples are necessary; certain types of  tissue (e.g. fat) are di ffi cult to process; and the process is time-consuming and disruptive ( Summary box 11.4 ). Summary box 11.4 Frozen section: advantages and disadvantages /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
Advantages
Quick diagnosis
Disadvantages
Poorer quality sections
Potential reduction in accuracy and precision of histological
diagnosis
Labour intensive
Disruptive
Risk of infection
Small sample required
Some tissue types dif
/f_i
cult to process

Histology
Histology
Specimens for histology are classified as biopsies and resec - tions, although strictly speaking all samples are biopsies. The reasons for taking small biopsies include diagnosis, further assessment and prognostic prediction. Types of  small biopsy include punch biopsy , needle core biopsy and m ucosal biopsy ( Summary box 11.2 ). - The purpose of  a resection is usually treatment of  a lesion (e.g. a tumour) by removing it. Other reasons for a resection exist, e.g. sleeve gastrectomy for obesity or creation of  an ile - - ostomy or colostomy . The pa thologist’s approach depends on the reason for surgery . For example, assessment of  a cancer resection has multiple purposes, including confirmation of  the diagnosis, classifica tion, grading, staging, determination of  fur - ther management and prediction of  outcome. - An excision biopsy is larger than the common types of small biopsy and serves as both a diagnostic biopsy and as a resection. For example, excision of a small skin lesion achiev es its removal and also allows histological diagnosis and classifi - - cation. Ultrasound-guided and computed tomography (CT) - guided biopsies of  focal and less accessible lesions have become more common and may pose challenges to the pathologist - because of  limited sample size. Material from biopsies or r esections is usually suitable for molecular analysis. Increasingly , the role of  pathologists - includes the identification of  appropriate material for various Common types of tissue sample /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF molecular tests and assessment of  tissue suitability for molec ular testing. For example, some tumour biopsies may contain insu ffi cient tumour for molecular testing. Of  course, correct diagnosis and grading are essential before molecular testing occurs, and attempts to bypass this step and take tissue for molecular testing without including the ste p of  histological assessment run many unnecessary risks such as absence of tumour in the sample or the presence of  a tumour that is dif ferent from that expected clinically . All samples for routine histology are immediately placed in a fixative, usually formalin (10% formaldehyde), by the surgical team or by other clinical sta ff to preserve morphology . T usually happens before delivery to the pathology laboratory .
Histology
Formalin-
/f_i
xed tissue
Biopsy
Mucosal, e.g. gastrointestinal, bronchial, oral
Punch, e.g. skin
Needle (core), e.g. liver
Curettings, e.g. endometrium, prostate
Excision biopsy
Resection
Fresh tissue
Frozen section diagnosis
Research
Tissue banking
Occasional special stains that require fresh tissue
Cytology
Cervical
Washings, brushings, scrapes
Fine-needle aspirate
Fluids, e.g. ascites, pleural
/f_l
uid
Sputum
Histology
Specimens for histology are classified as biopsies and resec - tions, although strictly speaking all samples are biopsies. The reasons for taking small biopsies include diagnosis, further assessment and prognostic prediction. Types of  small biopsy include punch biopsy , needle core biopsy and m ucosal biopsy ( Summary box 11.2 ). - The purpose of  a resection is usually treatment of  a lesion (e.g. a tumour) by removing it. Other reasons for a resection exist, e.g. sleeve gastrectomy for obesity or creation of  an ile - - ostomy or colostomy . The pa thologist’s approach depends on the reason for surgery . For example, assessment of  a cancer resection has multiple purposes, including confirmation of  the diagnosis, classifica tion, grading, staging, determination of  fur - ther management and prediction of  outcome. - An excision biopsy is larger than the common types of small biopsy and serves as both a diagnostic biopsy and as a resection. For example, excision of a small skin lesion achiev es its removal and also allows histological diagnosis and classifi - - cation. Ultrasound-guided and computed tomography (CT) - guided biopsies of  focal and less accessible lesions have become more common and may pose challenges to the pathologist - because of  limited sample size. Material from biopsies or r esections is usually suitable for molecular analysis. Increasingly , the role of  pathologists - includes the identification of  appropriate material for various Common types of tissue sample /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF molecular tests and assessment of  tissue suitability for molec ular testing. For example, some tumour biopsies may contain insu ffi cient tumour for molecular testing. Of  course, correct diagnosis and grading are essential before molecular testing occurs, and attempts to bypass this step and take tissue for molecular testing without including the ste p of  histological assessment run many unnecessary risks such as absence of tumour in the sample or the presence of  a tumour that is dif ferent from that expected clinically . All samples for routine histology are immediately placed in a fixative, usually formalin (10% formaldehyde), by the surgical team or by other clinical sta ff to preserve morphology . T usually happens before delivery to the pathology laboratory .
Histology
Formalin-
/f_i
xed tissue
Biopsy
Mucosal, e.g. gastrointestinal, bronchial, oral
Punch, e.g. skin
Needle (core), e.g. liver
Curettings, e.g. endometrium, prostate
Excision biopsy
Resection
Fresh tissue
Frozen section diagnosis
Research
Tissue banking
Occasional special stains that require fresh tissue
Cytology
Cervical
Washings, brushings, scrapes
Fine-needle aspirate
Fluids, e.g. ascites, pleural
/f_l
uid
Sputum
Histology
Specimens for histology are classified as biopsies and resec - tions, although strictly speaking all samples are biopsies. The reasons for taking small biopsies include diagnosis, further assessment and prognostic prediction. Types of  small biopsy include punch biopsy , needle core biopsy and m ucosal biopsy ( Summary box 11.2 ). - The purpose of  a resection is usually treatment of  a lesion (e.g. a tumour) by removing it. Other reasons for a resection exist, e.g. sleeve gastrectomy for obesity or creation of  an ile - - ostomy or colostomy . The pa thologist’s approach depends on the reason for surgery . For example, assessment of  a cancer resection has multiple purposes, including confirmation of  the diagnosis, classifica tion, grading, staging, determination of  fur - ther management and prediction of  outcome. - An excision biopsy is larger than the common types of small biopsy and serves as both a diagnostic biopsy and as a resection. For example, excision of a small skin lesion achiev es its removal and also allows histological diagnosis and classifi - - cation. Ultrasound-guided and computed tomography (CT) - guided biopsies of  focal and less accessible lesions have become more common and may pose challenges to the pathologist - because of  limited sample size. Material from biopsies or r esections is usually suitable for molecular analysis. Increasingly , the role of  pathologists - includes the identification of  appropriate material for various Common types of tissue sample /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF molecular tests and assessment of  tissue suitability for molec ular testing. For example, some tumour biopsies may contain insu ffi cient tumour for molecular testing. Of  course, correct diagnosis and grading are essential before molecular testing occurs, and attempts to bypass this step and take tissue for molecular testing without including the ste p of  histological assessment run many unnecessary risks such as absence of tumour in the sample or the presence of  a tumour that is dif ferent from that expected clinically . All samples for routine histology are immediately placed in a fixative, usually formalin (10% formaldehyde), by the surgical team or by other clinical sta ff to preserve morphology . T usually happens before delivery to the pathology laboratory .
Histology
Formalin-
/f_i
xed tissue
Biopsy
Mucosal, e.g. gastrointestinal, bronchial, oral
Punch, e.g. skin
Needle (core), e.g. liver
Curettings, e.g. endometrium, prostate
Excision biopsy
Resection
Fresh tissue
Frozen section diagnosis
Research
Tissue banking
Occasional special stains that require fresh tissue
Cytology
Cervical
Washings, brushings, scrapes
Fine-needle aspirate
Fluids, e.g. ascites, pleural
/f_l
uid
Sputum

Immunohistochemistry  tumour pathology
Immunohistochemistry: tumour pathology
Immunohistochemistry has multiple applications in tumour pathology , including elucidation of  site of  origin and determi nation of  cell type/direction of  di ff erentiation. Immunohisto chemistry may also help to confirm neoplasia, determine the selection of  treatment, refine prognostic predictions and screen for known underlying genetic changes. Numerous immunohistochemical stains help to deter mine cell type in tumours . Epithelial cells express cytokera tins. Therefore, cytokeratin positivity , though not diagnostic, favours carcinoma ( Figure 11.25 ) over other types of malig nancy . Lymphoid markers include the panlymphoid marker CD45, the T-lymphocyte marker CD3 and the B-lymphocyte marker CD20. Markers of melanocytic di ff erentiation include S100, MelanA and HMB45. Chromogranin, synaptophysin Summary box 11.11 Some immunohistochemical stains used for tumours /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF typically expresses CD117 ( Figure 11.26 ) and DOG-1. Endo - thelial cell markers include CD31, which may confirm a diag - nosis of  vascular neoplasia or highlight vascular invasion by tumours. H&E appearances may indicate or suggest the anatomical site of  origin of  a metastatic tumour. For example, an adeno - carcinoma has sev eral possible sources such as gastrointestinal tract, pancr eatobiliary system, bronchus, breast and gynaeco - ) is often of logical tract. A clear cell carcinoma ( Figure 11.17 renal origin but could be from the liver, pancreas, parathyroid or endometrium, among other sites. Immunohistochemical stains often provide valuable further information about ana - tomical origin. Some are highly specific for a particular site , e.g. prostate-specific antigen (PSA) and thyroglobulin. Others are somewhat less specific, e.g. thyroid transcription factor-1 (TTF-1), a marker of  bronchogenic or thyroid origin; hepato - cyte-specific antigen, suggesting hepatocellular origin; and cytokeratin 20, typically expressed by colorectal epithelium. veral types of Carcinoembryonic antigen (CEA) is present in se carcinoma ( Figure 11.22b ). In practice, pathologists encoun - - tering a neoplasm of  uncertain origin or uncertain phenotype - usually request a panel of  markers relevant to the clinical set - , espe - ting and to the H&E appearances. Some malignancies cially poorly di ff erentiated examples, do not conform to the - - -
Cell type/site of origin
Epithelial (carcinoma): cytokeratins
Lymphoid (lymphoma): CD45, CD3 (T cells), CD20 (B cells)
Melanocytic (melanoma): S100, HMB45, Melan A
Neuroendocrine: synaptophysin, chromogranin
Vascular: CD31
Myoid: desmin, actin
Site of origin/cell type
Prostate: prostate-speci
/f_i
c antigen (PSA)
Lung: thyroid transcription factor-1 (TTF-1)
Thyroid: thyroglobulin
Colorectum: cytokeratin 20 (CK20), CDX2
Liver: hepatocyte-speci
/f_i
c antigen (HSA)
Gastrointestinal stromal tumour (GIST): CD117, DOG-1
Prognosis and treatment
Breast carcinoma and gastric carcinoma: HER-2
Neuroendocrine tumours: Ki67 proliferation index
Screening for mutations
Colorectal carcinoma: mismatch repair proteins (MLH1,
MSH2, MSH6, PMS2)
(a)
(b)
Figure 11.26
(a)
A metastatic tumour composed of spindle cells. The
clinical team suspected a diagnosis of gastrointestinal stromal tumour
(GIST).
(b)
Positive immunohistochemistry for CD117, supporting a
diagnosis of GIST.
typical immunohistochemical profiles. In all circumstances, interpretation takes place in the light of the clinical picture and imaging findings. Less often, immunohistochemistry helps to confirm malig nancy . For example, kappa or lambda light chain restriction (expression of  only one immunoglobulin light chain) in lym phoid proliferations suggests clonality and, in turn, neoplasia rather than a reactive process . In general, immunohistochem istry does not distinguish well between benign and malignant. Immunohistochemistry also plays a role in the selection of tr eatment and in predicting prognosis. For example, assessment of  oestrogen receptor (ER) and human epidermal gro wth fac tor receptor-2 (HER2) status is routine for carcinomas of  the breast (see Immunohistochemistry: tumour pathology while lymphomas are typically subjected to a comprehensive panel of  mar kers that help determine treatment and prognosis. Ki67 proliferative index is an important prognostic factor for neuroendocrine neoplasms ( Figure 11.27 ) . Immunohistochemistry: infections and other applications There are antibodies to many infective agents, including cytomegalovirus (CMV), Epstein–Barr virus (EBV), herpes simplex virus, human herpes virus 8 (HHV8), hepatitis B virus and Helicobacter pylori. Some of  these organisms, e.g. pylori and CMV , may be obvious or suspected on H&E exam ination, while others, e.g. EBV and HHV8, always require immunohistochemistry or other techniques for their detection. Immunohistochemistry can also detect immunoglobulin and complement expression (e.g. in lymphomas or renal biop sies); confirm the abnormal accumulation of  various proteins such as alpha-1-antitrypsin (A1AT); and help to c haracterise amyloid. Newer immunohistochemical markers that detect specific gene mutations are appearing and may become useful in clin ical practice in the futur e. An important example is screening for MMR gene mutations in most gastrointestinal carcinomas Sir Michael Anthony Epstein , b.1921, Professor of  Pathology , University of  Bristol, Bristol, UK. Yvonne Barr , 1931–2016, Irish born virologist who emigrated to Australia. Epstein and Barr discovered this virus in 1964. BRAF V600E can replace mutational analysis in some settings. The major advantages of immunohistochemistry over other molecular tests for detecting genetic alterations are lower cost and faster turnaround. Summary box 11.12 Uses of immunohistochemistry /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF - /uni25CF /uni25CF - -
Figure 11.27
Immunohistochemistry for Ki67. The proliferative index
is approximately 35% in this
/f_i
eld.
Cell type
Neoplasia
Direction of differentiation/phenotype
Determination of anatomical site of origin
Con
/f_i
rmation of neoplasia
Grading
Selection of treatment
Detection of/screening for mutations
Prognosis
Microorganisms – detection
Other
Amyloid
Immunoglobulins
Complement
Immunohistochemistry: tumour pathology
Immunohistochemistry has multiple applications in tumour pathology , including elucidation of  site of  origin and determi nation of  cell type/direction of  di ff erentiation. Immunohisto chemistry may also help to confirm neoplasia, determine the selection of  treatment, refine prognostic predictions and screen for known underlying genetic changes. Numerous immunohistochemical stains help to deter mine cell type in tumours . Epithelial cells express cytokera tins. Therefore, cytokeratin positivity , though not diagnostic, favours carcinoma ( Figure 11.25 ) over other types of malig nancy . Lymphoid markers include the panlymphoid marker CD45, the T-lymphocyte marker CD3 and the B-lymphocyte marker CD20. Markers of melanocytic di ff erentiation include S100, MelanA and HMB45. Chromogranin, synaptophysin Summary box 11.11 Some immunohistochemical stains used for tumours /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF typically expresses CD117 ( Figure 11.26 ) and DOG-1. Endo - thelial cell markers include CD31, which may confirm a diag - nosis of  vascular neoplasia or highlight vascular invasion by tumours. H&E appearances may indicate or suggest the anatomical site of  origin of  a metastatic tumour. For example, an adeno - carcinoma has sev eral possible sources such as gastrointestinal tract, pancr eatobiliary system, bronchus, breast and gynaeco - ) is often of logical tract. A clear cell carcinoma ( Figure 11.17 renal origin but could be from the liver, pancreas, parathyroid or endometrium, among other sites. Immunohistochemical stains often provide valuable further information about ana - tomical origin. Some are highly specific for a particular site , e.g. prostate-specific antigen (PSA) and thyroglobulin. Others are somewhat less specific, e.g. thyroid transcription factor-1 (TTF-1), a marker of  bronchogenic or thyroid origin; hepato - cyte-specific antigen, suggesting hepatocellular origin; and cytokeratin 20, typically expressed by colorectal epithelium. veral types of Carcinoembryonic antigen (CEA) is present in se carcinoma ( Figure 11.22b ). In practice, pathologists encoun - - tering a neoplasm of  uncertain origin or uncertain phenotype - usually request a panel of  markers relevant to the clinical set - , espe - ting and to the H&E appearances. Some malignancies cially poorly di ff erentiated examples, do not conform to the - - -
Cell type/site of origin
Epithelial (carcinoma): cytokeratins
Lymphoid (lymphoma): CD45, CD3 (T cells), CD20 (B cells)
Melanocytic (melanoma): S100, HMB45, Melan A
Neuroendocrine: synaptophysin, chromogranin
Vascular: CD31
Myoid: desmin, actin
Site of origin/cell type
Prostate: prostate-speci
/f_i
c antigen (PSA)
Lung: thyroid transcription factor-1 (TTF-1)
Thyroid: thyroglobulin
Colorectum: cytokeratin 20 (CK20), CDX2
Liver: hepatocyte-speci
/f_i
c antigen (HSA)
Gastrointestinal stromal tumour (GIST): CD117, DOG-1
Prognosis and treatment
Breast carcinoma and gastric carcinoma: HER-2
Neuroendocrine tumours: Ki67 proliferation index
Screening for mutations
Colorectal carcinoma: mismatch repair proteins (MLH1,
MSH2, MSH6, PMS2)
(a)
(b)
Figure 11.26
(a)
A metastatic tumour composed of spindle cells. The
clinical team suspected a diagnosis of gastrointestinal stromal tumour
(GIST).
(b)
Positive immunohistochemistry for CD117, supporting a
diagnosis of GIST.
typical immunohistochemical profiles. In all circumstances, interpretation takes place in the light of the clinical picture and imaging findings. Less often, immunohistochemistry helps to confirm malig nancy . For example, kappa or lambda light chain restriction (expression of  only one immunoglobulin light chain) in lym phoid proliferations suggests clonality and, in turn, neoplasia rather than a reactive process . In general, immunohistochem istry does not distinguish well between benign and malignant. Immunohistochemistry also plays a role in the selection of tr eatment and in predicting prognosis. For example, assessment of  oestrogen receptor (ER) and human epidermal gro wth fac tor receptor-2 (HER2) status is routine for carcinomas of  the breast (see Immunohistochemistry: tumour pathology while lymphomas are typically subjected to a comprehensive panel of  mar kers that help determine treatment and prognosis. Ki67 proliferative index is an important prognostic factor for neuroendocrine neoplasms ( Figure 11.27 ) . Immunohistochemistry: infections and other applications There are antibodies to many infective agents, including cytomegalovirus (CMV), Epstein–Barr virus (EBV), herpes simplex virus, human herpes virus 8 (HHV8), hepatitis B virus and Helicobacter pylori. Some of  these organisms, e.g. pylori and CMV , may be obvious or suspected on H&E exam ination, while others, e.g. EBV and HHV8, always require immunohistochemistry or other techniques for their detection. Immunohistochemistry can also detect immunoglobulin and complement expression (e.g. in lymphomas or renal biop sies); confirm the abnormal accumulation of  various proteins such as alpha-1-antitrypsin (A1AT); and help to c haracterise amyloid. Newer immunohistochemical markers that detect specific gene mutations are appearing and may become useful in clin ical practice in the futur e. An important example is screening for MMR gene mutations in most gastrointestinal carcinomas Sir Michael Anthony Epstein , b.1921, Professor of  Pathology , University of  Bristol, Bristol, UK. Yvonne Barr , 1931–2016, Irish born virologist who emigrated to Australia. Epstein and Barr discovered this virus in 1964. BRAF V600E can replace mutational analysis in some settings. The major advantages of immunohistochemistry over other molecular tests for detecting genetic alterations are lower cost and faster turnaround. Summary box 11.12 Uses of immunohistochemistry /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF - /uni25CF /uni25CF - -
Figure 11.27
Immunohistochemistry for Ki67. The proliferative index
is approximately 35% in this
/f_i
eld.
Cell type
Neoplasia
Direction of differentiation/phenotype
Determination of anatomical site of origin
Con
/f_i
rmation of neoplasia
Grading
Selection of treatment
Detection of/screening for mutations
Prognosis
Microorganisms – detection
Other
Amyloid
Immunoglobulins
Complement
Immunohistochemistry: tumour pathology
Immunohistochemistry has multiple applications in tumour pathology , including elucidation of  site of  origin and determi nation of  cell type/direction of  di ff erentiation. Immunohisto chemistry may also help to confirm neoplasia, determine the selection of  treatment, refine prognostic predictions and screen for known underlying genetic changes. Numerous immunohistochemical stains help to deter mine cell type in tumours . Epithelial cells express cytokera tins. Therefore, cytokeratin positivity , though not diagnostic, favours carcinoma ( Figure 11.25 ) over other types of malig nancy . Lymphoid markers include the panlymphoid marker CD45, the T-lymphocyte marker CD3 and the B-lymphocyte marker CD20. Markers of melanocytic di ff erentiation include S100, MelanA and HMB45. Chromogranin, synaptophysin Summary box 11.11 Some immunohistochemical stains used for tumours /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF typically expresses CD117 ( Figure 11.26 ) and DOG-1. Endo - thelial cell markers include CD31, which may confirm a diag - nosis of  vascular neoplasia or highlight vascular invasion by tumours. H&E appearances may indicate or suggest the anatomical site of  origin of  a metastatic tumour. For example, an adeno - carcinoma has sev eral possible sources such as gastrointestinal tract, pancr eatobiliary system, bronchus, breast and gynaeco - ) is often of logical tract. A clear cell carcinoma ( Figure 11.17 renal origin but could be from the liver, pancreas, parathyroid or endometrium, among other sites. Immunohistochemical stains often provide valuable further information about ana - tomical origin. Some are highly specific for a particular site , e.g. prostate-specific antigen (PSA) and thyroglobulin. Others are somewhat less specific, e.g. thyroid transcription factor-1 (TTF-1), a marker of  bronchogenic or thyroid origin; hepato - cyte-specific antigen, suggesting hepatocellular origin; and cytokeratin 20, typically expressed by colorectal epithelium. veral types of Carcinoembryonic antigen (CEA) is present in se carcinoma ( Figure 11.22b ). In practice, pathologists encoun - - tering a neoplasm of  uncertain origin or uncertain phenotype - usually request a panel of  markers relevant to the clinical set - , espe - ting and to the H&E appearances. Some malignancies cially poorly di ff erentiated examples, do not conform to the - - -
Cell type/site of origin
Epithelial (carcinoma): cytokeratins
Lymphoid (lymphoma): CD45, CD3 (T cells), CD20 (B cells)
Melanocytic (melanoma): S100, HMB45, Melan A
Neuroendocrine: synaptophysin, chromogranin
Vascular: CD31
Myoid: desmin, actin
Site of origin/cell type
Prostate: prostate-speci
/f_i
c antigen (PSA)
Lung: thyroid transcription factor-1 (TTF-1)
Thyroid: thyroglobulin
Colorectum: cytokeratin 20 (CK20), CDX2
Liver: hepatocyte-speci
/f_i
c antigen (HSA)
Gastrointestinal stromal tumour (GIST): CD117, DOG-1
Prognosis and treatment
Breast carcinoma and gastric carcinoma: HER-2
Neuroendocrine tumours: Ki67 proliferation index
Screening for mutations
Colorectal carcinoma: mismatch repair proteins (MLH1,
MSH2, MSH6, PMS2)
(a)
(b)
Figure 11.26
(a)
A metastatic tumour composed of spindle cells. The
clinical team suspected a diagnosis of gastrointestinal stromal tumour
(GIST).
(b)
Positive immunohistochemistry for CD117, supporting a
diagnosis of GIST.
typical immunohistochemical profiles. In all circumstances, interpretation takes place in the light of the clinical picture and imaging findings. Less often, immunohistochemistry helps to confirm malig nancy . For example, kappa or lambda light chain restriction (expression of  only one immunoglobulin light chain) in lym phoid proliferations suggests clonality and, in turn, neoplasia rather than a reactive process . In general, immunohistochem istry does not distinguish well between benign and malignant. Immunohistochemistry also plays a role in the selection of tr eatment and in predicting prognosis. For example, assessment of  oestrogen receptor (ER) and human epidermal gro wth fac tor receptor-2 (HER2) status is routine for carcinomas of  the breast (see Immunohistochemistry: tumour pathology while lymphomas are typically subjected to a comprehensive panel of  mar kers that help determine treatment and prognosis. Ki67 proliferative index is an important prognostic factor for neuroendocrine neoplasms ( Figure 11.27 ) . Immunohistochemistry: infections and other applications There are antibodies to many infective agents, including cytomegalovirus (CMV), Epstein–Barr virus (EBV), herpes simplex virus, human herpes virus 8 (HHV8), hepatitis B virus and Helicobacter pylori. Some of  these organisms, e.g. pylori and CMV , may be obvious or suspected on H&E exam ination, while others, e.g. EBV and HHV8, always require immunohistochemistry or other techniques for their detection. Immunohistochemistry can also detect immunoglobulin and complement expression (e.g. in lymphomas or renal biop sies); confirm the abnormal accumulation of  various proteins such as alpha-1-antitrypsin (A1AT); and help to c haracterise amyloid. Newer immunohistochemical markers that detect specific gene mutations are appearing and may become useful in clin ical practice in the futur e. An important example is screening for MMR gene mutations in most gastrointestinal carcinomas Sir Michael Anthony Epstein , b.1921, Professor of  Pathology , University of  Bristol, Bristol, UK. Yvonne Barr , 1931–2016, Irish born virologist who emigrated to Australia. Epstein and Barr discovered this virus in 1964. BRAF V600E can replace mutational analysis in some settings. The major advantages of immunohistochemistry over other molecular tests for detecting genetic alterations are lower cost and faster turnaround. Summary box 11.12 Uses of immunohistochemistry /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF - /uni25CF /uni25CF - -
Figure 11.27
Immunohistochemistry for Ki67. The proliferative index
is approximately 35% in this
/f_i
eld.
Cell type
Neoplasia
Direction of differentiation/phenotype
Determination of anatomical site of origin
Con
/f_i
rmation of neoplasia
Grading
Selection of treatment
Detection of/screening for mutations
Prognosis
Microorganisms – detection
Other
Amyloid
Immunoglobulins
Complement

Immunohistochemistry
Immunohistochemistry
Immunohistochemistry emerged in the 1970s and has had a major impact on histopathological diagnosis. The technique detects a specific antigen using an antibody . The antibody is ). labelled with a dye and after binding to its target antigen is visible in the tissue section as a coloured stain, often brown ( Figure 11.25 ) . This allows the pathologist to confirm or exclude the presence of  an antigen as well as determine its tissue distribution and cellular localisation. Quantification - xample, Ki67 is a cell cycle marker may also be possible. For e In situ that allows the pathologist to calculate a proliferative index, which in turn has prognostic value for neuroendocrine neoplasms and other lesions. Immunohistochemistry is appli - cable to fixed and frozen tissue and to cytological preparations ( Figure 11.22b ). It is safe, quick and relatively inexpensive and is often specific. However, false-positive results can result from non-specific staining or from cross-reaction with similar antigens. Excessive reliance on immunohistochemistry can lead to errors.
Figure 11.25
Diffuse immunohistochemical staining (brown) for a
pancytokeratin marker in a malignancy, favouring carcinoma over
other tumours.
Immunohistochemistry
Immunohistochemistry emerged in the 1970s and has had a major impact on histopathological diagnosis. The technique detects a specific antigen using an antibody . The antibody is ). labelled with a dye and after binding to its target antigen is visible in the tissue section as a coloured stain, often brown ( Figure 11.25 ) . This allows the pathologist to confirm or exclude the presence of  an antigen as well as determine its tissue distribution and cellular localisation. Quantification - xample, Ki67 is a cell cycle marker may also be possible. For e In situ that allows the pathologist to calculate a proliferative index, which in turn has prognostic value for neuroendocrine neoplasms and other lesions. Immunohistochemistry is appli - cable to fixed and frozen tissue and to cytological preparations ( Figure 11.22b ). It is safe, quick and relatively inexpensive and is often specific. However, false-positive results can result from non-specific staining or from cross-reaction with similar antigens. Excessive reliance on immunohistochemistry can lead to errors.
Figure 11.25
Diffuse immunohistochemical staining (brown) for a
pancytokeratin marker in a malignancy, favouring carcinoma over
other tumours.
Immunohistochemistry
Immunohistochemistry emerged in the 1970s and has had a major impact on histopathological diagnosis. The technique detects a specific antigen using an antibody . The antibody is ). labelled with a dye and after binding to its target antigen is visible in the tissue section as a coloured stain, often brown ( Figure 11.25 ) . This allows the pathologist to confirm or exclude the presence of  an antigen as well as determine its tissue distribution and cellular localisation. Quantification - xample, Ki67 is a cell cycle marker may also be possible. For e In situ that allows the pathologist to calculate a proliferative index, which in turn has prognostic value for neuroendocrine neoplasms and other lesions. Immunohistochemistry is appli - cable to fixed and frozen tissue and to cytological preparations ( Figure 11.22b ). It is safe, quick and relatively inexpensive and is often specific. However, false-positive results can result from non-specific staining or from cross-reaction with similar antigens. Excessive reliance on immunohistochemistry can lead to errors.
Figure 11.25
Diffuse immunohistochemical staining (brown) for a
pancytokeratin marker in a malignancy, favouring carcinoma over
other tumours.

Introduction
INTRODUCTION
Pre-nineteenth century tissue diagnosis depended on naked eye examination of  autopsy material and of  a small selection of surgical specimens. The development of the light micro scope allowed closer examination of  tissue from autopsies and surgical procedures, with visualisation of  cells, nuclei and tissue structure. Microscopic diagnosis was initially controversial, partly as a result of the ‘Kaiser’s cancer’ (a histological diag nosis by Virchow of  a non-malignant laryngeal lesion, after which Kaiser Friedrich III died of  laryngeal malignancy), but the medical and surgical community eventually accepted its value. Tissue analysis is now an integ ral and routine element of clinical practice. It is heavily dependent on microscopic assess ment, although newer methods of tissue analysis will increas ing ly provide additional information. Assessment of  tissue is usually the responsibility of  a histopathologist/cellular pathol ogist (a medically qualified practitioner), who depends on support from technical sta ff . In the UK, the sta ff responsible for tissue processing and the production of sections on glass slides are known as biomedical scientists (BMSs). The specialty variably known as Histopatholog y , Anatomic Pathology or Cellular Pathology encompasses histopathology , cytopathol ogy , autopsy work and molecular tissue diagnosis. Developments and changes in cellular pathology are con tinuous. The volume of  biopsies continues to increase as a result of  increasing clinical demands, expectations of  greater diagnostic precision, widespread flexible endoscopy and an ageing population with a higher pr evalence of  cancer and other illnesses. Cancer screening programmes also have an impact as they often depend heavily on cellular pathology . New techniques to refine histological assessment require addi tional resources. There is an increasing obligation to comply Rudolf  Ludwig Carl Virchow , 1821–1902, pathologist, Charité Hospital, Berlin, Germany , known as the ‘father of  modern pathology’. with national or international standards of  reporting, e.g. for cancer, and participation by pathologists in multidisciplinary team meetings is now routine rather than occasional. Other developments may reduce activity . Newer, less invasive meth - - ods may replace tissue analysis, e.g. human papilloma virus (HPV) testing for cervical pre-neoplastic lesions is replacing cytological assessment. New methods in imaging may reduce the need for tissue analysis. - The location of  a modern cellular pathology department is usually within or near a medium-sized or lar ge hospital or in a purpose-built o ff -site centre. Typically , more than 80% of specimens are from the gastrointestinal tract, gynaecological tract, skin or urological system. In line with clinical services, highly specialised work such as neuropathology takes place in - major regional centres. Consolidation of  clinical services may - result in reconfiguration of  relevant pathology services and molecular testing facilities. -
To be aware of:
The principles of microscopic diagnosis
•
The features of neoplasia
•
The importance of clinicopathological correlation
•
The role of additional techniques, including special stains,
•
immunohistochemistry and molecular pathology

Learning objectives
Learning objectives
To understand: The value and limitations of tissue diagnosis • Approaches to tissue processing • Learning objectives
To understand: The value and limitations of tissue diagnosis • Approaches to tissue processing • Learning objectives
To understand: The value and limitations of tissue diagnosis • Approaches to tissue processing •

Microscopic features of inflammation
Microscopic features of inflammation
Acute inflammation is characterised histologically by neutro - phils (polymorphonuclear leukocytes), erosion or ulceration ( Figure 11.18 ) and chronic inflammation by lymphocytes and plasma cells. Other inflammatory cells include eosino - phils ( Figure 11.19 ) , mast cells and histiocytes. Granulomas (collections of  epithelioid histiocytes) ( Figure 11.20a ) raise the possibility of  mycobacterial infection ( Figure 11.20b ), fungal ’s disease or a reaction infection, parasites, sarcoidosis, Crohn to foreign material, among numerous other possible causes. s may reflect parasitic infection or Eosinophils in large number allergy . Interpretation depends heavily on the site and clinical setting.
Figure 11.18
An acute in
/f_l
ammatory process characterised by numer
ous neutrophils. Note the typical multilobated nuclei (arrows).
Figure 11.19
Oesophageal mucosa in
/f_i
ltrated by numerous eosino
phils with bright red cytoplasm, many of which are forming clusters.
Eosinophils may re
/f_l
ect allergy, parasitic infection or a wide variety of
other causes. In this example, the clinicopathological diagnosis was
eosinophilic oesophagitis.
Microscopic features of inflammation
Acute inflammation is characterised histologically by neutro - phils (polymorphonuclear leukocytes), erosion or ulceration ( Figure 11.18 ) and chronic inflammation by lymphocytes and plasma cells. Other inflammatory cells include eosino - phils ( Figure 11.19 ) , mast cells and histiocytes. Granulomas (collections of  epithelioid histiocytes) ( Figure 11.20a ) raise the possibility of  mycobacterial infection ( Figure 11.20b ), fungal ’s disease or a reaction infection, parasites, sarcoidosis, Crohn to foreign material, among numerous other possible causes. s may reflect parasitic infection or Eosinophils in large number allergy . Interpretation depends heavily on the site and clinical setting.
Figure 11.18
An acute in
/f_l
ammatory process characterised by numer
ous neutrophils. Note the typical multilobated nuclei (arrows).
Figure 11.19
Oesophageal mucosa in
/f_i
ltrated by numerous eosino
phils with bright red cytoplasm, many of which are forming clusters.
Eosinophils may re
/f_l
ect allergy, parasitic infection or a wide variety of
other causes. In this example, the clinicopathological diagnosis was
eosinophilic oesophagitis.
Microscopic features of inflammation
Acute inflammation is characterised histologically by neutro - phils (polymorphonuclear leukocytes), erosion or ulceration ( Figure 11.18 ) and chronic inflammation by lymphocytes and plasma cells. Other inflammatory cells include eosino - phils ( Figure 11.19 ) , mast cells and histiocytes. Granulomas (collections of  epithelioid histiocytes) ( Figure 11.20a ) raise the possibility of  mycobacterial infection ( Figure 11.20b ), fungal ’s disease or a reaction infection, parasites, sarcoidosis, Crohn to foreign material, among numerous other possible causes. s may reflect parasitic infection or Eosinophils in large number allergy . Interpretation depends heavily on the site and clinical setting.
Figure 11.18
An acute in
/f_l
ammatory process characterised by numer
ous neutrophils. Note the typical multilobated nuclei (arrows).
Figure 11.19
Oesophageal mucosa in
/f_i
ltrated by numerous eosino
phils with bright red cytoplasm, many of which are forming clusters.
Eosinophils may re
/f_l
ect allergy, parasitic infection or a wide variety of
other causes. In this example, the clinicopathological diagnosis was
eosinophilic oesophagitis.

Mismatch repair gene abnormalities in tumours
Mismatch repair gene abnormalities in tumours
High levels of  microsatellite instability (MSI-H), also known as deficient mismatch repair (D-MMR), occur as a result of  germ line mutations or acquired somatic events in the MMR genes ( MLH1, MSH2, MSH6 and PMS2 ). The former is referred to as Lynch syndrome (previously known as hereditary non polyposis colorectal carcinoma) and is an autosomal dominant condition with predisposition to colorectal, gynaecological and other tumours (often at an early age). Summary box 11.17 Microsatellite instability and mismatch repair genes /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF for MMR mutations, although some centres use PCR-based microsatellite testing for screening. Loss of  immunohistochem - ical staining by neoplastic cells is a marker for a gene defect, an indication for further testing and may lead to genetic testing for Lynch syndrome ( Figure 11.30 ). In most patients, a detectable MMR abnormality is sporadic and does not represent Lynch syndrome . MMR gene defects in CRC also identify sporadic tumours with di ff erent phenotypic and genetic characteristics. For example, BRAF V600E mutations are frequent in these cases and such cancers develop via the serrated polyp pathway rather than from adenomas (see Chapter 77 ). MMR abnormalities generally predict lower recurrence rates, better survival rates and a lack of  need for 5-fluorouracil.
Microsatellite instability (MSI)
Regulated by four main genes:
MLH1, PMS2, MSH2, MSH6
Genetic changes responsible for MSI
Sporadic hypermethylation of
MLH1
(more common; 85%)
Germline mutation, i.e. Lynch syndrome (less common)
Microsatellite unstable (MSI-H) tumours
15% of colorectal carcinoma (CRC)
30% of endometrial carcinoma
Tests
Immunohistochemistry
Recommended for all newly diagnosed CRCs
The preferred initial test in most centres
PCR-based microsatellite testing
NGS
Clinicopathological correlation: MSI-H CRC
Typically right sided
More likely to have mucinous element histologically
Likely to have
BRAF
V600E mutation
Clinical value
Phenotypic classi
/f_i
cation, e.g. medullary CRC is typically
MSI-H
Prognosis, e.g. MSI-H better prognosis overall
Selection of drug therapy, e.g. MSI-H CRC responds better
to ICIs and has no response to 5-
/f_l
uorouracil
Screening for germline mutation, i.e. Lynch syndrome
Mismatch repair gene abnormalities in tumours
High levels of  microsatellite instability (MSI-H), also known as deficient mismatch repair (D-MMR), occur as a result of  germ line mutations or acquired somatic events in the MMR genes ( MLH1, MSH2, MSH6 and PMS2 ). The former is referred to as Lynch syndrome (previously known as hereditary non polyposis colorectal carcinoma) and is an autosomal dominant condition with predisposition to colorectal, gynaecological and other tumours (often at an early age). Summary box 11.17 Microsatellite instability and mismatch repair genes /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF for MMR mutations, although some centres use PCR-based microsatellite testing for screening. Loss of  immunohistochem - ical staining by neoplastic cells is a marker for a gene defect, an indication for further testing and may lead to genetic testing for Lynch syndrome ( Figure 11.30 ). In most patients, a detectable MMR abnormality is sporadic and does not represent Lynch syndrome . MMR gene defects in CRC also identify sporadic tumours with di ff erent phenotypic and genetic characteristics. For example, BRAF V600E mutations are frequent in these cases and such cancers develop via the serrated polyp pathway rather than from adenomas (see Chapter 77 ). MMR abnormalities generally predict lower recurrence rates, better survival rates and a lack of  need for 5-fluorouracil.
Microsatellite instability (MSI)
Regulated by four main genes:
MLH1, PMS2, MSH2, MSH6
Genetic changes responsible for MSI
Sporadic hypermethylation of
MLH1
(more common; 85%)
Germline mutation, i.e. Lynch syndrome (less common)
Microsatellite unstable (MSI-H) tumours
15% of colorectal carcinoma (CRC)
30% of endometrial carcinoma
Tests
Immunohistochemistry
Recommended for all newly diagnosed CRCs
The preferred initial test in most centres
PCR-based microsatellite testing
NGS
Clinicopathological correlation: MSI-H CRC
Typically right sided
More likely to have mucinous element histologically
Likely to have
BRAF
V600E mutation
Clinical value
Phenotypic classi
/f_i
cation, e.g. medullary CRC is typically
MSI-H
Prognosis, e.g. MSI-H better prognosis overall
Selection of drug therapy, e.g. MSI-H CRC responds better
to ICIs and has no response to 5-
/f_l
uorouracil
Screening for germline mutation, i.e. Lynch syndrome
Mismatch repair gene abnormalities in tumours
High levels of  microsatellite instability (MSI-H), also known as deficient mismatch repair (D-MMR), occur as a result of  germ line mutations or acquired somatic events in the MMR genes ( MLH1, MSH2, MSH6 and PMS2 ). The former is referred to as Lynch syndrome (previously known as hereditary non polyposis colorectal carcinoma) and is an autosomal dominant condition with predisposition to colorectal, gynaecological and other tumours (often at an early age). Summary box 11.17 Microsatellite instability and mismatch repair genes /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF for MMR mutations, although some centres use PCR-based microsatellite testing for screening. Loss of  immunohistochem - ical staining by neoplastic cells is a marker for a gene defect, an indication for further testing and may lead to genetic testing for Lynch syndrome ( Figure 11.30 ). In most patients, a detectable MMR abnormality is sporadic and does not represent Lynch syndrome . MMR gene defects in CRC also identify sporadic tumours with di ff erent phenotypic and genetic characteristics. For example, BRAF V600E mutations are frequent in these cases and such cancers develop via the serrated polyp pathway rather than from adenomas (see Chapter 77 ). MMR abnormalities generally predict lower recurrence rates, better survival rates and a lack of  need for 5-fluorouracil.
Microsatellite instability (MSI)
Regulated by four main genes:
MLH1, PMS2, MSH2, MSH6
Genetic changes responsible for MSI
Sporadic hypermethylation of
MLH1
(more common; 85%)
Germline mutation, i.e. Lynch syndrome (less common)
Microsatellite unstable (MSI-H) tumours
15% of colorectal carcinoma (CRC)
30% of endometrial carcinoma
Tests
Immunohistochemistry
Recommended for all newly diagnosed CRCs
The preferred initial test in most centres
PCR-based microsatellite testing
NGS
Clinicopathological correlation: MSI-H CRC
Typically right sided
More likely to have mucinous element histologically
Likely to have
BRAF
V600E mutation
Clinical value
Phenotypic classi
/f_i
cation, e.g. medullary CRC is typically
MSI-H
Prognosis, e.g. MSI-H better prognosis overall
Selection of drug therapy, e.g. MSI-H CRC responds better
to ICIs and has no response to 5-
/f_l
uorouracil
Screening for germline mutation, i.e. Lynch syndrome

Molecular changes and drug therapy
Molecular changes and drug therapy
An increasingly common reason for molecular testing and related immunohistochemistry is the prediction of  the response of  advanced malignant tumours to specific drugs whose target is usually known (‘theranostics’). For example, tumours with tyrosine kinase gene fusions that result in activation of  the kinase are more likely than their counterparts to respond to tyrosine kinase inhibitors. A newer class of  drugs known collectively as immune checkpoint inhibitors (ICIs) is highly successful for the treatment of  a variety of  advanced malignancies. Detection of any of  several biomarkers may predict responsiveness to ICIs.
Molecular changes and drug therapy
An increasingly common reason for molecular testing and related immunohistochemistry is the prediction of  the response of  advanced malignant tumours to specific drugs whose target is usually known (‘theranostics’). For example, tumours with tyrosine kinase gene fusions that result in activation of  the kinase are more likely than their counterparts to respond to tyrosine kinase inhibitors. A newer class of  drugs known collectively as immune checkpoint inhibitors (ICIs) is highly successful for the treatment of  a variety of  advanced malignancies. Detection of any of  several biomarkers may predict responsiveness to ICIs.
Molecular changes and drug therapy
An increasingly common reason for molecular testing and related immunohistochemistry is the prediction of  the response of  advanced malignant tumours to specific drugs whose target is usually known (‘theranostics’). For example, tumours with tyrosine kinase gene fusions that result in activation of  the kinase are more likely than their counterparts to respond to tyrosine kinase inhibitors. A newer class of  drugs known collectively as immune checkpoint inhibitors (ICIs) is highly successful for the treatment of  a variety of  advanced malignancies. Detection of any of  several biomarkers may predict responsiveness to ICIs.

Molecular profile  examples of specific tumours
Molecular profile: examples of specific tumours
Colorectal carcinoma In CRC, the anti-EGFR monoclonal antibodies cetuximab and panitumumab are used in combination with chemotherapy for metastatic disease. These drugs are less likely to be e ff ective if KRAS or NRAS mutations are present than if  a tumour is ‘wild type’ (i.e. has no RAS mutation). Various other genetic changes assist with selection of therapy and making prognostic predictions ( Summary boxes 11.17 and 11.18 ). Molecular analysis in colorectal carcinoma /uni25CF /uni25CF Summary box 11.17 /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Bronchial (lung) carcinoma In non-small cell lung cancer, specific EGFR mutations occur in a minority of  lesions and identification predicts a response to the anti-EGFR tyrosine kinase inhibitor gefitinib, while ALK gene rearrangement predicts a response to the anaplastic lymphoma kinase (ALK) inhibitor crizotinib (see Chapter 60 ). Summary box 11.19 shows other relevant molecular changes. Summary box 11.19 Molecular and related changes in non-small cell lung carcinoma /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Gynaecological carcinoma MMR status is increasingly important for the classification and management of ovarian and endometrial carcinomas. Other molecular changes that are important for prognosis and selection of  therapy in endometrial cancer include polymerase ε (POL ε ) and TP53 abnormalities. HER2 amplification and PD-1 expression may be relevant in some settings. Classification influences prognostic predictions and now depends not only on traditional histology but also on a number of  molecular changes (many of  which are detectable using immunohisto - chemistr y) (see Chapter 87 ). Breast carcinoma The most important ancillary tests for breast carcinoma remain ER immunohistochemistry and HER2 testing. As for many advanced malignancies, ICIs may be useful and accordingly PD-L1 immunostaining (with the appropriate antibody clone) may help predict outcome (see Chapter 58 ). Lymphoma The distinction between benign and malignant lymphoid proliferations is sometimes di ffi cult. Clonal immunoglobulin heavy chain (IgH) gene rearrangements in B-cell proliferations and clonal T-cell receptor gene rearrangements in T-cell proliferations favour lymphoma over reactive proliferations. Identification of  characteristic cytogenetic abnormalities plays an important role in diagnosis, classification and management of  several haematological neoplasms. PCR-based tests help detect minimal residual disease after therapy . Gastrointestinal stromal tumour, soft-tissue tumours and malignant melanoma Most GISTs have either a KIT gene mutation or a PDGFRA gene mutation, more often the former. A few have defects in succinate dehydrogenase ( SDH ), BRAF or NF1 genes. Iden - tification of  known mutations helps confirm the diagnosis. Mutational profile also helps predict clinical outcome and response to chemotherapy . For example, imatinib, a tyrosine kinase inhibitor, is a useful drug for advanced GIST but is ine ff ective in those with SDH mutations. Molecular testing assists the diagnosis and classification of many types of  soft-tissue tumour. Examples include Ewing’s sarcoma and alveolar rhabdomyosarcoma, in which specific fusion genes are diagnostic. FISH testing detects c haracteristic cytogenetic changes. In metastatic malignant melanoma, specific BRAF muta - tions predict response to the BRAF kinase inhibitor vemu - rafenib. Table 11.1 outlines the clinical applications of  some bio - markers in tumours.
Mismatch repair gene abnormalities
Multiple considerations (see
)
KRAS or NRAS
mutation
Predicts resistance to EGFR inhibitors
Tumour mutation burden
Predicts response to ICI therapy
BRAF
V600E mutation
Poor prognosis in metastatic CRC
Predictive of response to therapy
NTRK
fusion
Uncommon (<1% CRC)
Usually MSI-H
Poor prognosis
Speci
/f_i
c therapy available: tyrosine kinase inhibitors
Prediction of response to tyrosine kinase therapy
Mutations
EGFR
KRAS
BRAF
V600E
Fusions
ALK
RET
NTRK
Prediction of response to immune checkpoint inhibitors
PD-L1 expression (in a subgroup)
Molecular profile: examples of specific tumours
Colorectal carcinoma In CRC, the anti-EGFR monoclonal antibodies cetuximab and panitumumab are used in combination with chemotherapy for metastatic disease. These drugs are less likely to be e ff ective if KRAS or NRAS mutations are present than if  a tumour is ‘wild type’ (i.e. has no RAS mutation). Various other genetic changes assist with selection of therapy and making prognostic predictions ( Summary boxes 11.17 and 11.18 ). Molecular analysis in colorectal carcinoma /uni25CF /uni25CF Summary box 11.17 /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Bronchial (lung) carcinoma In non-small cell lung cancer, specific EGFR mutations occur in a minority of  lesions and identification predicts a response to the anti-EGFR tyrosine kinase inhibitor gefitinib, while ALK gene rearrangement predicts a response to the anaplastic lymphoma kinase (ALK) inhibitor crizotinib (see Chapter 60 ). Summary box 11.19 shows other relevant molecular changes. Summary box 11.19 Molecular and related changes in non-small cell lung carcinoma /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Gynaecological carcinoma MMR status is increasingly important for the classification and management of ovarian and endometrial carcinomas. Other molecular changes that are important for prognosis and selection of  therapy in endometrial cancer include polymerase ε (POL ε ) and TP53 abnormalities. HER2 amplification and PD-1 expression may be relevant in some settings. Classification influences prognostic predictions and now depends not only on traditional histology but also on a number of  molecular changes (many of  which are detectable using immunohisto - chemistr y) (see Chapter 87 ). Breast carcinoma The most important ancillary tests for breast carcinoma remain ER immunohistochemistry and HER2 testing. As for many advanced malignancies, ICIs may be useful and accordingly PD-L1 immunostaining (with the appropriate antibody clone) may help predict outcome (see Chapter 58 ). Lymphoma The distinction between benign and malignant lymphoid proliferations is sometimes di ffi cult. Clonal immunoglobulin heavy chain (IgH) gene rearrangements in B-cell proliferations and clonal T-cell receptor gene rearrangements in T-cell proliferations favour lymphoma over reactive proliferations. Identification of  characteristic cytogenetic abnormalities plays an important role in diagnosis, classification and management of  several haematological neoplasms. PCR-based tests help detect minimal residual disease after therapy . Gastrointestinal stromal tumour, soft-tissue tumours and malignant melanoma Most GISTs have either a KIT gene mutation or a PDGFRA gene mutation, more often the former. A few have defects in succinate dehydrogenase ( SDH ), BRAF or NF1 genes. Iden - tification of  known mutations helps confirm the diagnosis. Mutational profile also helps predict clinical outcome and response to chemotherapy . For example, imatinib, a tyrosine kinase inhibitor, is a useful drug for advanced GIST but is ine ff ective in those with SDH mutations. Molecular testing assists the diagnosis and classification of many types of  soft-tissue tumour. Examples include Ewing’s sarcoma and alveolar rhabdomyosarcoma, in which specific fusion genes are diagnostic. FISH testing detects c haracteristic cytogenetic changes. In metastatic malignant melanoma, specific BRAF muta - tions predict response to the BRAF kinase inhibitor vemu - rafenib. Table 11.1 outlines the clinical applications of  some bio - markers in tumours.
Mismatch repair gene abnormalities
Multiple considerations (see
)
KRAS or NRAS
mutation
Predicts resistance to EGFR inhibitors
Tumour mutation burden
Predicts response to ICI therapy
BRAF
V600E mutation
Poor prognosis in metastatic CRC
Predictive of response to therapy
NTRK
fusion
Uncommon (<1% CRC)
Usually MSI-H
Poor prognosis
Speci
/f_i
c therapy available: tyrosine kinase inhibitors
Prediction of response to tyrosine kinase therapy
Mutations
EGFR
KRAS
BRAF
V600E
Fusions
ALK
RET
NTRK
Prediction of response to immune checkpoint inhibitors
PD-L1 expression (in a subgroup)
Molecular profile: examples of specific tumours
Colorectal carcinoma In CRC, the anti-EGFR monoclonal antibodies cetuximab and panitumumab are used in combination with chemotherapy for metastatic disease. These drugs are less likely to be e ff ective if KRAS or NRAS mutations are present than if  a tumour is ‘wild type’ (i.e. has no RAS mutation). Various other genetic changes assist with selection of therapy and making prognostic predictions ( Summary boxes 11.17 and 11.18 ). Molecular analysis in colorectal carcinoma /uni25CF /uni25CF Summary box 11.17 /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Bronchial (lung) carcinoma In non-small cell lung cancer, specific EGFR mutations occur in a minority of  lesions and identification predicts a response to the anti-EGFR tyrosine kinase inhibitor gefitinib, while ALK gene rearrangement predicts a response to the anaplastic lymphoma kinase (ALK) inhibitor crizotinib (see Chapter 60 ). Summary box 11.19 shows other relevant molecular changes. Summary box 11.19 Molecular and related changes in non-small cell lung carcinoma /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Gynaecological carcinoma MMR status is increasingly important for the classification and management of ovarian and endometrial carcinomas. Other molecular changes that are important for prognosis and selection of  therapy in endometrial cancer include polymerase ε (POL ε ) and TP53 abnormalities. HER2 amplification and PD-1 expression may be relevant in some settings. Classification influences prognostic predictions and now depends not only on traditional histology but also on a number of  molecular changes (many of  which are detectable using immunohisto - chemistr y) (see Chapter 87 ). Breast carcinoma The most important ancillary tests for breast carcinoma remain ER immunohistochemistry and HER2 testing. As for many advanced malignancies, ICIs may be useful and accordingly PD-L1 immunostaining (with the appropriate antibody clone) may help predict outcome (see Chapter 58 ). Lymphoma The distinction between benign and malignant lymphoid proliferations is sometimes di ffi cult. Clonal immunoglobulin heavy chain (IgH) gene rearrangements in B-cell proliferations and clonal T-cell receptor gene rearrangements in T-cell proliferations favour lymphoma over reactive proliferations. Identification of  characteristic cytogenetic abnormalities plays an important role in diagnosis, classification and management of  several haematological neoplasms. PCR-based tests help detect minimal residual disease after therapy . Gastrointestinal stromal tumour, soft-tissue tumours and malignant melanoma Most GISTs have either a KIT gene mutation or a PDGFRA gene mutation, more often the former. A few have defects in succinate dehydrogenase ( SDH ), BRAF or NF1 genes. Iden - tification of  known mutations helps confirm the diagnosis. Mutational profile also helps predict clinical outcome and response to chemotherapy . For example, imatinib, a tyrosine kinase inhibitor, is a useful drug for advanced GIST but is ine ff ective in those with SDH mutations. Molecular testing assists the diagnosis and classification of many types of  soft-tissue tumour. Examples include Ewing’s sarcoma and alveolar rhabdomyosarcoma, in which specific fusion genes are diagnostic. FISH testing detects c haracteristic cytogenetic changes. In metastatic malignant melanoma, specific BRAF muta - tions predict response to the BRAF kinase inhibitor vemu - rafenib. Table 11.1 outlines the clinical applications of  some bio - markers in tumours.
Mismatch repair gene abnormalities
Multiple considerations (see
)
KRAS or NRAS
mutation
Predicts resistance to EGFR inhibitors
Tumour mutation burden
Predicts response to ICI therapy
BRAF
V600E mutation
Poor prognosis in metastatic CRC
Predictive of response to therapy
NTRK
fusion
Uncommon (<1% CRC)
Usually MSI-H
Poor prognosis
Speci
/f_i
c therapy available: tyrosine kinase inhibitors
Prediction of response to tyrosine kinase therapy
Mutations
EGFR
KRAS
BRAF
V600E
Fusions
ALK
RET
NTRK
Prediction of response to immune checkpoint inhibitors
PD-L1 expression (in a subgroup)

Non-neoplastic and inflammatory conditions
Non-neoplastic and inflammatory conditions
The diagnosis, assessment and management of  non-neoplastic disease generates numerous pathology specimens. Examples include appendectomy for appendicitis, cholecystectomy for gallstone disease, hysterectomy for fibroids, skin excision for various lesions such as sebaceous cysts and prostatic chippings from glands with hyperplasia. Thorough histological examina - tion helps to confirm or refute the provisional clinical diagnosis and also to exclude other conditions, some of  which may be allbladder, malignant entirely incidental, e.g. neoplasia of the g change in uterine fibroids or carcinoma in prostatic chippings. Surgical and medical teams also generate a very large number of biopsies with the purpose of diagnosing and assessing non-neoplastic disease. In this setting, correlation with the clinical picture may be very important. For example, clinical details are essential for meaningful interpretation of  inflamma - tory bowel disease biopsies, inflammatory skin biopsies, renal biopsies and medical liver biopsies. Non-neoplastic and inflammatory conditions
The diagnosis, assessment and management of  non-neoplastic disease generates numerous pathology specimens. Examples include appendectomy for appendicitis, cholecystectomy for gallstone disease, hysterectomy for fibroids, skin excision for various lesions such as sebaceous cysts and prostatic chippings from glands with hyperplasia. Thorough histological examina - tion helps to confirm or refute the provisional clinical diagnosis and also to exclude other conditions, some of  which may be allbladder, malignant entirely incidental, e.g. neoplasia of the g change in uterine fibroids or carcinoma in prostatic chippings. Surgical and medical teams also generate a very large number of biopsies with the purpose of diagnosing and assessing non-neoplastic disease. In this setting, correlation with the clinical picture may be very important. For example, clinical details are essential for meaningful interpretation of  inflamma - tory bowel disease biopsies, inflammatory skin biopsies, renal biopsies and medical liver biopsies. Non-neoplastic and inflammatory conditions
The diagnosis, assessment and management of  non-neoplastic disease generates numerous pathology specimens. Examples include appendectomy for appendicitis, cholecystectomy for gallstone disease, hysterectomy for fibroids, skin excision for various lesions such as sebaceous cysts and prostatic chippings from glands with hyperplasia. Thorough histological examina - tion helps to confirm or refute the provisional clinical diagnosis and also to exclude other conditions, some of  which may be allbladder, malignant entirely incidental, e.g. neoplasia of the g change in uterine fibroids or carcinoma in prostatic chippings. Surgical and medical teams also generate a very large number of biopsies with the purpose of diagnosing and assessing non-neoplastic disease. In this setting, correlation with the clinical picture may be very important. For example, clinical details are essential for meaningful interpretation of  inflamma - tory bowel disease biopsies, inflammatory skin biopsies, renal biopsies and medical liver biopsies.

Other terms
Other terms
Other specific tissue abnormalities are also detectable by microscopy . Histopathologists may use specific terms. Some examples are as follows. /uni25CF Hyperplasia: an increase in cell number. /uni25CF Hypertrophy: an increase in cell size. /uni25CF Atrophy: may refer to a reduction in cell number or cell size or a diminution in size of  a structure (e.g. a duodenal villus undergoing atrophy in coeliac disease). /uni25CF Metaplasia: a change from one mature cell type to another, e.g. columnar metaplasia in the oesophagus (Barrett’s oesophagus), whereby metaplastic gastric or intestinal-type epithelium replaces normal squamous epithelium. /uni25CF Necrosis: cell or tissue death, typically because of factors external to the cell, and associated with cell swelling, Theodor Langhans , 1839–1915, Professor of  Pathological Anatomy , University of  Bern, Bern, Switzerland. Franz Heinrich Paul Ziehl , 1859–1926, neurologist, Lübeck, Germany . Friedrich Carl Adolf  Neelsen , 1854–1894, pathologist, prosector, the Stadt-Krankenhaus, Dresden, Germany . inflammation and eventual disappearance of  cells ( Figure 11.20 ). /uni25CF Apoptosis: a process of  programmed cell death that occurs because of  internal signals, and on histological examina - tion typically manifests as cell shrinkage and nuclear chro - matin condensation.
(b)
Figure 11.20
(a)
A granuloma with necrosis, suggesting tuberculosis.
Multinucleate giant cells of Langhans type are also present (arrow).
(b)
A Ziehl–Neelsen stain (from a different case) shows numerous pink
acid-fast rod-shaped bacilli, con
/f_i
rming mycobacterial infection.
Other terms
Other specific tissue abnormalities are also detectable by microscopy . Histopathologists may use specific terms. Some examples are as follows. /uni25CF Hyperplasia: an increase in cell number. /uni25CF Hypertrophy: an increase in cell size. /uni25CF Atrophy: may refer to a reduction in cell number or cell size or a diminution in size of  a structure (e.g. a duodenal villus undergoing atrophy in coeliac disease). /uni25CF Metaplasia: a change from one mature cell type to another, e.g. columnar metaplasia in the oesophagus (Barrett’s oesophagus), whereby metaplastic gastric or intestinal-type epithelium replaces normal squamous epithelium. /uni25CF Necrosis: cell or tissue death, typically because of factors external to the cell, and associated with cell swelling, Theodor Langhans , 1839–1915, Professor of  Pathological Anatomy , University of  Bern, Bern, Switzerland. Franz Heinrich Paul Ziehl , 1859–1926, neurologist, Lübeck, Germany . Friedrich Carl Adolf  Neelsen , 1854–1894, pathologist, prosector, the Stadt-Krankenhaus, Dresden, Germany . inflammation and eventual disappearance of  cells ( Figure 11.20 ). /uni25CF Apoptosis: a process of  programmed cell death that occurs because of  internal signals, and on histological examina - tion typically manifests as cell shrinkage and nuclear chro - matin condensation.
(b)
Figure 11.20
(a)
A granuloma with necrosis, suggesting tuberculosis.
Multinucleate giant cells of Langhans type are also present (arrow).
(b)
A Ziehl–Neelsen stain (from a different case) shows numerous pink
acid-fast rod-shaped bacilli, con
/f_i
rming mycobacterial infection.
Other terms
Other specific tissue abnormalities are also detectable by microscopy . Histopathologists may use specific terms. Some examples are as follows. /uni25CF Hyperplasia: an increase in cell number. /uni25CF Hypertrophy: an increase in cell size. /uni25CF Atrophy: may refer to a reduction in cell number or cell size or a diminution in size of  a structure (e.g. a duodenal villus undergoing atrophy in coeliac disease). /uni25CF Metaplasia: a change from one mature cell type to another, e.g. columnar metaplasia in the oesophagus (Barrett’s oesophagus), whereby metaplastic gastric or intestinal-type epithelium replaces normal squamous epithelium. /uni25CF Necrosis: cell or tissue death, typically because of factors external to the cell, and associated with cell swelling, Theodor Langhans , 1839–1915, Professor of  Pathological Anatomy , University of  Bern, Bern, Switzerland. Franz Heinrich Paul Ziehl , 1859–1926, neurologist, Lübeck, Germany . Friedrich Carl Adolf  Neelsen , 1854–1894, pathologist, prosector, the Stadt-Krankenhaus, Dresden, Germany . inflammation and eventual disappearance of  cells ( Figure 11.20 ). /uni25CF Apoptosis: a process of  programmed cell death that occurs because of  internal signals, and on histological examina - tion typically manifests as cell shrinkage and nuclear chro - matin condensation.
(b)
Figure 11.20
(a)
A granuloma with necrosis, suggesting tuberculosis.
Multinucleate giant cells of Langhans type are also present (arrow).
(b)
A Ziehl–Neelsen stain (from a different case) shows numerous pink
acid-fast rod-shaped bacilli, con
/f_i
rming mycobacterial infection.

PRINCIPLES OF MICROSCOPIC DIAGNOSIS Diagnosis of m
PRINCIPLES OF MICROSCOPIC DIAGNOSIS Diagnosis of malignancy
Neoplasia is a broad term that includes benign and malignant tumours and precursors of  malignancy . The word ‘cancer’ is not precise, derives from observations of the similarities - between crabs and tumours by ancient Greek physicians such as Hippocrates and usually refers to all malignancies (rather than carcinoma alone). Classification of a tumour as malignant implies that it can behave aggressively . The main features of malignancy are metastasis and invasion and there are charac - teristic architectural and cytological abnormalities. However, the criteria for a diagnosis of malignancy di ff er between anatomical sites and between tumour types. Sometimes, the traditional concept of benign and malignant is not applicable and instead there is a classification that identifies a spectrum of  tumours from well di ff erentiated to poorly di ff erentiated or from low grade to high grade depending on known clinical behaviour. Microscopic features of malignancy /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Microscopic evidence of  aggressive behaviour by the tumour is usually su ffi cient for a malignant label. For example, metastasis to another organ such as lymph nodes or liver is diagnostic of malignancy . Invasion of surrounding structures, perineural invasion ( Figure 11.10 ) and vascular spread or invasion ( Figure 11.11 ) strongly suggest malignancy . Other microscopic features that are typical of  malig nancy include derangement of  the usual tissue architecture, an increase in the number of  mitotic figures, atypical mitotic figures and necrosis (tissue death) ( Figure 11.12 ) . Changes in . cytological changes, the appearances of  individual cells, i.e include nuclear enlargement, an increase in the nuclear: cytoplasmic ratio, nuclear pleomorphism (variation in nuclear appearance) and n uclear hyperchromasia (dark colour) 11.13a ) . Multiplicity , irregularity and enlargement of  nucleoli may also be apparent ( Figure 11.13b ). However, none of  these features is diagnostic of  malignancy in isolation. or a histological diagnosis of  malignancy vary The criteria f according to the site and type of  tissue. Carcinoma is by far the most common type of  malignancy , and in many settings Nerve Tumour Necrosis Viable tumour - ( Figure is diagnosable when epithelial cells invade beyond their nor - mal boundaries. However, the categorisation of some types of non-epithelial proliferations (e.g. lymphoid or mesenchy - chitec - mal) as malignant may rely on cytological and/or ar tural features rather than on invasiveness. In some cases, e.g. phaeochromocytoma, reliable histological distinction between e.g. gas - benign and malignant is not possible. In other cases, trointestinal stromal tumours (GISTs), there are risk catego - ries based on combinations of histological features that help to predict the likelihood of aggressive behaviour rather than . Additional techniques such benign or malignant designations as immunohistochemistry and clonality studies occasionally help to confirm or support a diagnosis of  neoplasia or malig - nancy (see Immunohistochemistry: tumour pathology and Diagnostic molecular pathology ). The term ‘dysplasia’ usually indicates that microscopic fea - e is tures similar to those of  carcinoma are present but that ther no invasion. The term ‘intraepithelial neoplasia’ is analogous to dysplasia. Examples include cervical intraepithelial neopla - sia (CIN) and gastrointestinal dysplasia ( Figure 11.14 ). Grad - ysplasia may be as low grade/high grade or as mild/ ing of  d moderate/severe while grading of  intraepithelial neoplasia may be numerical (e.g. CIN 1, CIN 2 and CIN 3).
Metastasis
Invasion
Of surrounding tissue
Vascular (intraluminal tumour and/or tumour in blood vessel
wall)
Perineural
Architectural abnormalities
Necrosis
Numerous mitotic
/f_i
gures
Atypical mitotic
/f_i
gures
Nuclear abnormalities
Pleomorphism
Enlargement
Hyperchromaticity
Chromatin clumping
Nucleolar enlargement and multiplicity
Figure 11.10
Perineural invasion. A nerve is almost surrounded by
adenocarcinoma.
Figure 11.11
Vascular invasion. Aggregates of carcinoma cells are
present within blood vessels. The tumour is poorly differentiated.
Figure 11.12
An area of necrosis in a poorly differentiated carcinoma.
malignancy . These include contamination of  a specimen with tumour from elsewhere, interchanging of  specimens, observer error and histological mimicry . A false-negative diagnosis, i.e. a failure to diagnose malignancy when present, may reflect absence of tumour in the specimen or failure of the pathologist to recognise the changes as neoplastic. Several conditions can resemble malignancy histologically . For example, radiation e ff ect can produce cytological atypia that mimics malignancy , and the epithelial changes in regen - erating tissue adjacent to a mucosal ulcer may show features reminiscent of  neoplasia. The risk of  interpretative error by the histopathologist is likely to be lower if  there is thor ough training of  pathologists, regular updating of  knowledge, dis - cussion of  di ffi cult cases with colleagues and avoidance of excessive workloads. The surgeon also helps to minimise errors by supplying good clinical details. Summary box 11.6 Causes of false-positive diagnoses of malignancy /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
(b)
Figure 11.13
Cellular features of malignancy.
(a)
A neuroendocrine
carcinoma showing nuclear pleomorphism (variation in shape) and
variation in nuclear size. There are several mitotic
/f_i
gures (arrows).
A malignant melanoma showing nuclear pleomorphism and prominent
nucleoli (arrow) (courtesy of Dr E Husain, Aberdeen Royal In
/f_i
rmary
Aberdeen, UK).
Figure 11.14
A colonic biopsy from a tubular adenoma with low-
grade dysplasia. A non-dysplastic crypt is apparent at lower right.
The remaining crypts mostly show features of dysplasia, including
nuclear strati
/f_i
cation (multilayering), nuclear enlargement and nuclear
hyperchromaticity (dark colour).
Interchanged samples
Contamination
Interpretative error
Treatment-induced change, e.g. radiotherapy
Ulceration
PRINCIPLES OF MICROSCOPIC DIAGNOSIS Diagnosis of malignancy
Neoplasia is a broad term that includes benign and malignant tumours and precursors of  malignancy . The word ‘cancer’ is not precise, derives from observations of the similarities - between crabs and tumours by ancient Greek physicians such as Hippocrates and usually refers to all malignancies (rather than carcinoma alone). Classification of a tumour as malignant implies that it can behave aggressively . The main features of malignancy are metastasis and invasion and there are charac - teristic architectural and cytological abnormalities. However, the criteria for a diagnosis of malignancy di ff er between anatomical sites and between tumour types. Sometimes, the traditional concept of benign and malignant is not applicable and instead there is a classification that identifies a spectrum of  tumours from well di ff erentiated to poorly di ff erentiated or from low grade to high grade depending on known clinical behaviour. Microscopic features of malignancy /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Microscopic evidence of  aggressive behaviour by the tumour is usually su ffi cient for a malignant label. For example, metastasis to another organ such as lymph nodes or liver is diagnostic of malignancy . Invasion of surrounding structures, perineural invasion ( Figure 11.10 ) and vascular spread or invasion ( Figure 11.11 ) strongly suggest malignancy . Other microscopic features that are typical of  malig nancy include derangement of  the usual tissue architecture, an increase in the number of  mitotic figures, atypical mitotic figures and necrosis (tissue death) ( Figure 11.12 ) . Changes in . cytological changes, the appearances of  individual cells, i.e include nuclear enlargement, an increase in the nuclear: cytoplasmic ratio, nuclear pleomorphism (variation in nuclear appearance) and n uclear hyperchromasia (dark colour) 11.13a ) . Multiplicity , irregularity and enlargement of  nucleoli may also be apparent ( Figure 11.13b ). However, none of  these features is diagnostic of  malignancy in isolation. or a histological diagnosis of  malignancy vary The criteria f according to the site and type of  tissue. Carcinoma is by far the most common type of  malignancy , and in many settings Nerve Tumour Necrosis Viable tumour - ( Figure is diagnosable when epithelial cells invade beyond their nor - mal boundaries. However, the categorisation of some types of non-epithelial proliferations (e.g. lymphoid or mesenchy - chitec - mal) as malignant may rely on cytological and/or ar tural features rather than on invasiveness. In some cases, e.g. phaeochromocytoma, reliable histological distinction between e.g. gas - benign and malignant is not possible. In other cases, trointestinal stromal tumours (GISTs), there are risk catego - ries based on combinations of histological features that help to predict the likelihood of aggressive behaviour rather than . Additional techniques such benign or malignant designations as immunohistochemistry and clonality studies occasionally help to confirm or support a diagnosis of  neoplasia or malig - nancy (see Immunohistochemistry: tumour pathology and Diagnostic molecular pathology ). The term ‘dysplasia’ usually indicates that microscopic fea - e is tures similar to those of  carcinoma are present but that ther no invasion. The term ‘intraepithelial neoplasia’ is analogous to dysplasia. Examples include cervical intraepithelial neopla - sia (CIN) and gastrointestinal dysplasia ( Figure 11.14 ). Grad - ysplasia may be as low grade/high grade or as mild/ ing of  d moderate/severe while grading of  intraepithelial neoplasia may be numerical (e.g. CIN 1, CIN 2 and CIN 3).
Metastasis
Invasion
Of surrounding tissue
Vascular (intraluminal tumour and/or tumour in blood vessel
wall)
Perineural
Architectural abnormalities
Necrosis
Numerous mitotic
/f_i
gures
Atypical mitotic
/f_i
gures
Nuclear abnormalities
Pleomorphism
Enlargement
Hyperchromaticity
Chromatin clumping
Nucleolar enlargement and multiplicity
Figure 11.10
Perineural invasion. A nerve is almost surrounded by
adenocarcinoma.
Figure 11.11
Vascular invasion. Aggregates of carcinoma cells are
present within blood vessels. The tumour is poorly differentiated.
Figure 11.12
An area of necrosis in a poorly differentiated carcinoma.
malignancy . These include contamination of  a specimen with tumour from elsewhere, interchanging of  specimens, observer error and histological mimicry . A false-negative diagnosis, i.e. a failure to diagnose malignancy when present, may reflect absence of tumour in the specimen or failure of the pathologist to recognise the changes as neoplastic. Several conditions can resemble malignancy histologically . For example, radiation e ff ect can produce cytological atypia that mimics malignancy , and the epithelial changes in regen - erating tissue adjacent to a mucosal ulcer may show features reminiscent of  neoplasia. The risk of  interpretative error by the histopathologist is likely to be lower if  there is thor ough training of  pathologists, regular updating of  knowledge, dis - cussion of  di ffi cult cases with colleagues and avoidance of excessive workloads. The surgeon also helps to minimise errors by supplying good clinical details. Summary box 11.6 Causes of false-positive diagnoses of malignancy /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
(b)
Figure 11.13
Cellular features of malignancy.
(a)
A neuroendocrine
carcinoma showing nuclear pleomorphism (variation in shape) and
variation in nuclear size. There are several mitotic
/f_i
gures (arrows).
A malignant melanoma showing nuclear pleomorphism and prominent
nucleoli (arrow) (courtesy of Dr E Husain, Aberdeen Royal In
/f_i
rmary
Aberdeen, UK).
Figure 11.14
A colonic biopsy from a tubular adenoma with low-
grade dysplasia. A non-dysplastic crypt is apparent at lower right.
The remaining crypts mostly show features of dysplasia, including
nuclear strati
/f_i
cation (multilayering), nuclear enlargement and nuclear
hyperchromaticity (dark colour).
Interchanged samples
Contamination
Interpretative error
Treatment-induced change, e.g. radiotherapy
Ulceration

PRINCIPLES OF MICROSCOPIC DIAGNOSIS Diagnosis of malignancy
PRINCIPLES OF MICROSCOPIC DIAGNOSIS Diagnosis of malignancy
Neoplasia is a broad term that includes benign and malignant tumours and precursors of  malignancy . The word ‘cancer’ is not precise, derives from observations of the similarities - between crabs and tumours by ancient Greek physicians such as Hippocrates and usually refers to all malignancies (rather than carcinoma alone). Classification of a tumour as malignant implies that it can behave aggressively . The main features of malignancy are metastasis and invasion and there are charac - teristic architectural and cytological abnormalities. However, the criteria for a diagnosis of malignancy di ff er between anatomical sites and between tumour types. Sometimes, the traditional concept of benign and malignant is not applicable and instead there is a classification that identifies a spectrum of  tumours from well di ff erentiated to poorly di ff erentiated or from low grade to high grade depending on known clinical behaviour. Microscopic features of malignancy /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Microscopic evidence of  aggressive behaviour by the tumour is usually su ffi cient for a malignant label. For example, metastasis to another organ such as lymph nodes or liver is diagnostic of malignancy . Invasion of surrounding structures, perineural invasion ( Figure 11.10 ) and vascular spread or invasion ( Figure 11.11 ) strongly suggest malignancy . Other microscopic features that are typical of  malig nancy include derangement of  the usual tissue architecture, an increase in the number of  mitotic figures, atypical mitotic figures and necrosis (tissue death) ( Figure 11.12 ) . Changes in . cytological changes, the appearances of  individual cells, i.e include nuclear enlargement, an increase in the nuclear: cytoplasmic ratio, nuclear pleomorphism (variation in nuclear appearance) and n uclear hyperchromasia (dark colour) 11.13a ) . Multiplicity , irregularity and enlargement of  nucleoli may also be apparent ( Figure 11.13b ). However, none of  these features is diagnostic of  malignancy in isolation. or a histological diagnosis of  malignancy vary The criteria f according to the site and type of  tissue. Carcinoma is by far the most common type of  malignancy , and in many settings Nerve Tumour Necrosis Viable tumour - ( Figure is diagnosable when epithelial cells invade beyond their nor - mal boundaries. However, the categorisation of some types of non-epithelial proliferations (e.g. lymphoid or mesenchy - chitec - mal) as malignant may rely on cytological and/or ar tural features rather than on invasiveness. In some cases, e.g. phaeochromocytoma, reliable histological distinction between e.g. gas - benign and malignant is not possible. In other cases, trointestinal stromal tumours (GISTs), there are risk catego - ries based on combinations of histological features that help to predict the likelihood of aggressive behaviour rather than . Additional techniques such benign or malignant designations as immunohistochemistry and clonality studies occasionally help to confirm or support a diagnosis of  neoplasia or malig - nancy (see Immunohistochemistry: tumour pathology and Diagnostic molecular pathology ). The term ‘dysplasia’ usually indicates that microscopic fea - e is tures similar to those of  carcinoma are present but that ther no invasion. The term ‘intraepithelial neoplasia’ is analogous to dysplasia. Examples include cervical intraepithelial neopla - sia (CIN) and gastrointestinal dysplasia ( Figure 11.14 ). Grad - ysplasia may be as low grade/high grade or as mild/ ing of  d moderate/severe while grading of  intraepithelial neoplasia may be numerical (e.g. CIN 1, CIN 2 and CIN 3).
Metastasis
Invasion
Of surrounding tissue
Vascular (intraluminal tumour and/or tumour in blood vessel
wall)
Perineural
Architectural abnormalities
Necrosis
Numerous mitotic
/f_i
gures
Atypical mitotic
/f_i
gures
Nuclear abnormalities
Pleomorphism
Enlargement
Hyperchromaticity
Chromatin clumping
Nucleolar enlargement and multiplicity
Figure 11.10
Perineural invasion. A nerve is almost surrounded by
adenocarcinoma.
Figure 11.11
Vascular invasion. Aggregates of carcinoma cells are
present within blood vessels. The tumour is poorly differentiated.
Figure 11.12
An area of necrosis in a poorly differentiated carcinoma.
malignancy . These include contamination of  a specimen with tumour from elsewhere, interchanging of  specimens, observer error and histological mimicry . A false-negative diagnosis, i.e. a failure to diagnose malignancy when present, may reflect absence of tumour in the specimen or failure of the pathologist to recognise the changes as neoplastic. Several conditions can resemble malignancy histologically . For example, radiation e ff ect can produce cytological atypia that mimics malignancy , and the epithelial changes in regen - erating tissue adjacent to a mucosal ulcer may show features reminiscent of  neoplasia. The risk of  interpretative error by the histopathologist is likely to be lower if  there is thor ough training of  pathologists, regular updating of  knowledge, dis - cussion of  di ffi cult cases with colleagues and avoidance of excessive workloads. The surgeon also helps to minimise errors by supplying good clinical details. Summary box 11.6 Causes of false-positive diagnoses of malignancy /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
(b)
Figure 11.13
Cellular features of malignancy.
(a)
A neuroendocrine
carcinoma showing nuclear pleomorphism (variation in shape) and
variation in nuclear size. There are several mitotic
/f_i
gures (arrows).
A malignant melanoma showing nuclear pleomorphism and prominent
nucleoli (arrow) (courtesy of Dr E Husain, Aberdeen Royal In
/f_i
rmary
Aberdeen, UK).
Figure 11.14
A colonic biopsy from a tubular adenoma with low-
grade dysplasia. A non-dysplastic crypt is apparent at lower right.
The remaining crypts mostly show features of dysplasia, including
nuclear strati
/f_i
cation (multilayering), nuclear enlargement and nuclear
hyperchromaticity (dark colour).
Interchanged samples
Contamination
Interpretative error
Treatment-induced change, e.g. radiotherapy
Ulceration

Polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction (PCR) amplifies DNA, yield ing millions of  copies from a single copy of  a selected target. Amplification of  RNA is also possible, using the technique of  reverse transcriptase PCR (RT-PCR). It is worth noting that r eal-time PCR (RTPCR) is a di ff erent method, typically used for quantification, with a very similar abbreviation. PCR is fast and safe and can be performed on homogenised fresh or formalin-fixed tissue. PCR-based methods have numer ous applications in oncology (see Detection of clinically relevant abnormalities in genes ), including mutational analysis ( Figure 11.29 ), testing for clonality , detection of fusion transcripts resulting from cytogenetic c hanges, detection of  amplifications, demonstration of  MSI and detection of gene hypermethylation. PCR-based methods can also detect microorganisms in tissue but this is not a common application because of  the risk of  false positives. Polymerase chain reaction
The polymerase chain reaction (PCR) amplifies DNA, yield ing millions of  copies from a single copy of  a selected target. Amplification of  RNA is also possible, using the technique of  reverse transcriptase PCR (RT-PCR). It is worth noting that r eal-time PCR (RTPCR) is a di ff erent method, typically used for quantification, with a very similar abbreviation. PCR is fast and safe and can be performed on homogenised fresh or formalin-fixed tissue. PCR-based methods have numer ous applications in oncology (see Detection of clinically relevant abnormalities in genes ), including mutational analysis ( Figure 11.29 ), testing for clonality , detection of fusion transcripts resulting from cytogenetic c hanges, detection of  amplifications, demonstration of  MSI and detection of gene hypermethylation. PCR-based methods can also detect microorganisms in tissue but this is not a common application because of  the risk of  false positives. Polymerase chain reaction
The polymerase chain reaction (PCR) amplifies DNA, yield ing millions of  copies from a single copy of  a selected target. Amplification of  RNA is also possible, using the technique of  reverse transcriptase PCR (RT-PCR). It is worth noting that r eal-time PCR (RTPCR) is a di ff erent method, typically used for quantification, with a very similar abbreviation. PCR is fast and safe and can be performed on homogenised fresh or formalin-fixed tissue. PCR-based methods have numer ous applications in oncology (see Detection of clinically relevant abnormalities in genes ), including mutational analysis ( Figure 11.29 ), testing for clonality , detection of fusion transcripts resulting from cytogenetic c hanges, detection of  amplifications, demonstration of  MSI and detection of gene hypermethylation. PCR-based methods can also detect microorganisms in tissue but this is not a common application because of  the risk of  false positives.

Prognosis
Prognosis
Tests that help determine the selection of  therapy for tumours may also have additional prognostic value. For example, a BRAF mutation in metastatic CRC is associated with a very - poor prognosis. Commercially available multiple molecular marker tests may provide prognostic information (see Chapter 58 ). - Prognosis
Tests that help determine the selection of  therapy for tumours may also have additional prognostic value. For example, a BRAF mutation in metastatic CRC is associated with a very - poor prognosis. Commercially available multiple molecular marker tests may provide prognostic information (see Chapter 58 ). - Prognosis
Tests that help determine the selection of  therapy for tumours may also have additional prognostic value. For example, a BRAF mutation in metastatic CRC is associated with a very - poor prognosis. Commercially available multiple molecular marker tests may provide prognostic information (see Chapter 58 ). -

Prognostic factors for malignant tumours
Prognostic factors for malignant tumours
Tissue assessment is important for cancer prognosis. Stage is generally the most important prognostic factor for carcinomas. The internationally accepted Union for International Cancer Control (UICC)/American Joint Committee on Cancer (AJCC) staging schemes depend heavily on the histopatho - logical TNM (Tumour Node Metastasis) category (pTNM), although the overall stage and in particular the M category are also ev aluated clinically and on imaging and the final stage is derived from a combination of  clinical, imaging, pathological and other assessments. The degree of  di ff erentiation may also be prognostic and is usually determined microscopically . As a of  their non-neoplastic tissue counterparts ( Figures 11.15 and 11.16 ) , whereas poorly di ff erentiated tumours do not ( Figures 11.11 and 11.12 ) . Other histological features associated with a worse prognosis include vascular invasion ( Figure 11.11 ), perineural in vasion ( Figure 11.10 ) and positive resection margins. The prognostic value of  these factors di ff ers between tumour types and sites. - There is an increasing number of  potential prognostic fac tors for a wide range of  malignancies and preneoplastic lesions. These include immunohistochemical tests and molecular tests that may aim to detect underlying genetic changes such as or amplifications or may help to refine grading. For mutations example, immunohistochemistry for the proliferation marker Ki67 is now essential for grading and prediction of  behaviour of  well-di ff erentiated neuroendocrine neoplasms. Screen - ing for mismatch repair (MMR) gene abnormalities using immunohistochemistry helps to predict response to therapy , outcome and the need for genetic testing for familial disease. Although controversial, immunohistochemical staining help to predict the behaviour of preneoplastic lesions might such as Barrett’s oesophagus (p53 staining) or cervical/anal intraepithelial neoplasia (p16 staining).
Figure 11.15
A well-differentiated squamous cell carcinoma. Irregular
nests of squamous cells are present. They include foci of keratinisa
tion (arrows).
Figure 11.16
A well-differentiated adenocarcinoma. Gland formation
(arrow) is obvious.
Figure 11.17
A metastatic clear cell carcinoma composed of sheets
of cells with clear cytoplasm. A tumour with this appearance is most
likely to be of renal origin but could have other sources such as liver,
parathyroid gland, gynaecological tract and gastrointestinal tract.
Prognostic factors for malignant tumours
Tissue assessment is important for cancer prognosis. Stage is generally the most important prognostic factor for carcinomas. The internationally accepted Union for International Cancer Control (UICC)/American Joint Committee on Cancer (AJCC) staging schemes depend heavily on the histopatho - logical TNM (Tumour Node Metastasis) category (pTNM), although the overall stage and in particular the M category are also ev aluated clinically and on imaging and the final stage is derived from a combination of  clinical, imaging, pathological and other assessments. The degree of  di ff erentiation may also be prognostic and is usually determined microscopically . As a of  their non-neoplastic tissue counterparts ( Figures 11.15 and 11.16 ) , whereas poorly di ff erentiated tumours do not ( Figures 11.11 and 11.12 ) . Other histological features associated with a worse prognosis include vascular invasion ( Figure 11.11 ), perineural in vasion ( Figure 11.10 ) and positive resection margins. The prognostic value of  these factors di ff ers between tumour types and sites. - There is an increasing number of  potential prognostic fac tors for a wide range of  malignancies and preneoplastic lesions. These include immunohistochemical tests and molecular tests that may aim to detect underlying genetic changes such as or amplifications or may help to refine grading. For mutations example, immunohistochemistry for the proliferation marker Ki67 is now essential for grading and prediction of  behaviour of  well-di ff erentiated neuroendocrine neoplasms. Screen - ing for mismatch repair (MMR) gene abnormalities using immunohistochemistry helps to predict response to therapy , outcome and the need for genetic testing for familial disease. Although controversial, immunohistochemical staining help to predict the behaviour of preneoplastic lesions might such as Barrett’s oesophagus (p53 staining) or cervical/anal intraepithelial neoplasia (p16 staining).
Figure 11.15
A well-differentiated squamous cell carcinoma. Irregular
nests of squamous cells are present. They include foci of keratinisa
tion (arrows).
Figure 11.16
A well-differentiated adenocarcinoma. Gland formation
(arrow) is obvious.
Figure 11.17
A metastatic clear cell carcinoma composed of sheets
of cells with clear cytoplasm. A tumour with this appearance is most
likely to be of renal origin but could have other sources such as liver,
parathyroid gland, gynaecological tract and gastrointestinal tract.
Prognostic factors for malignant tumours
Tissue assessment is important for cancer prognosis. Stage is generally the most important prognostic factor for carcinomas. The internationally accepted Union for International Cancer Control (UICC)/American Joint Committee on Cancer (AJCC) staging schemes depend heavily on the histopatho - logical TNM (Tumour Node Metastasis) category (pTNM), although the overall stage and in particular the M category are also ev aluated clinically and on imaging and the final stage is derived from a combination of  clinical, imaging, pathological and other assessments. The degree of  di ff erentiation may also be prognostic and is usually determined microscopically . As a of  their non-neoplastic tissue counterparts ( Figures 11.15 and 11.16 ) , whereas poorly di ff erentiated tumours do not ( Figures 11.11 and 11.12 ) . Other histological features associated with a worse prognosis include vascular invasion ( Figure 11.11 ), perineural in vasion ( Figure 11.10 ) and positive resection margins. The prognostic value of  these factors di ff ers between tumour types and sites. - There is an increasing number of  potential prognostic fac tors for a wide range of  malignancies and preneoplastic lesions. These include immunohistochemical tests and molecular tests that may aim to detect underlying genetic changes such as or amplifications or may help to refine grading. For mutations example, immunohistochemistry for the proliferation marker Ki67 is now essential for grading and prediction of  behaviour of  well-di ff erentiated neuroendocrine neoplasms. Screen - ing for mismatch repair (MMR) gene abnormalities using immunohistochemistry helps to predict response to therapy , outcome and the need for genetic testing for familial disease. Although controversial, immunohistochemical staining help to predict the behaviour of preneoplastic lesions might such as Barrett’s oesophagus (p53 staining) or cervical/anal intraepithelial neoplasia (p16 staining).
Figure 11.15
A well-differentiated squamous cell carcinoma. Irregular
nests of squamous cells are present. They include foci of keratinisa
tion (arrows).
Figure 11.16
A well-differentiated adenocarcinoma. Gland formation
(arrow) is obvious.
Figure 11.17
A metastatic clear cell carcinoma composed of sheets
of cells with clear cytoplasm. A tumour with this appearance is most
likely to be of renal origin but could have other sources such as liver,
parathyroid gland, gynaecological tract and gastrointestinal tract.

REASONS FOR ASSESSMENT OF TISSUE
REASONS FOR ASSESSMENT OF TISSUE
The contributions that tissue analysis makes to clinical - management include diagnosis, staging, prediction of  outcome and assistance with selection of  therapy . These are often - interrelated. The process of  tissue assessment may make a new diagnosis or may confirm or refute a suspected or existing clinical diagnosis. There may be pointers towards a cause. Analysis may also reveal additional diagnoses that may be unsuspected. As an example, pathological assessment of  an appendicec - tomy specimen most often confirms a suspected clinical diag - - nosis of  acute appendicitis. However, the appendix sometimes contains an incidental neuroendocrine neoplasm, mucinous contains granulomas, raising the possibility of  Crohn’s disease or infection. Also, a specific cause of abdominal pain other than appendicitis, e.g. endometriosis, may be apparent in the appendiceal tissue. Absence of  any histological abnormality raises the possibility of  an extra-appendiceal cause. Similarly , biopsies from a patient with inflammatory bowel disease may confirm the diagnosis but may sometimes reveal or suggest an alternative cause of  intestinal inflammation such as tuberculo sis, amoebiasis, ischaemia or mucosal prolapse. Summary box 11.1 Reasons for analysis of tissue /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Tissue analysis also helps, increasingly , to determine or refine treatment and prognosis. For example, the assessment of a breast, lung, colorectal or other major cancer resection spec imen helps to confirms the diagnosis but, more importantly , provides crucial information about features such as tumour stage, vascular invasion, perineural invasion and resection mar gin involv ement, which in turn help to predict clinical outcome and determine postoperative treatment. The degree of  tumour regression in a resection after neoadjuvant therapy may also have prognostic value. Additionally , pathological assessment of resections helps surgeons and radiologists to audit their accu racy and performance. Molecular pathological analysis of  cancer tissue (see Diagnostic molecular pathology ) increasingly contributes to management, including diagnostic categorisation, prognos tic predictions and selection of drug therapy . The molecular test or group of  tests that an oncologist chooses depends on patient status, tumour location, tumour morphology and stage, among other factors. The identification of a particular bio marker may provide an indication for targeted therapy . For example, detection of  high microsatellite instability (MSI) in metastatic colorectal carcinoma (CRC) may predict respon siveness to immune checkpoint inhibition. Burrill Bernard Crohn , 1884–1983, gastroenterologist, Mount Sinai Hospital, New Y ork, NY , USA. Norman Rupert Barrett , 1903–1979, surgeon, St Thomas’s Hospital, London, UK. patient. Correlation with the clinical picture and the macro - scopic findings enhances the interpretation of  pathological changes considerably . Ther efore, absence of  relevant details may cause unnecessary delays and even er rors. For example, radiation therapy can have profound e ff ects on tissue morphol - ogy , including mimicry of  other inflammatory conditions or neoplasia. Accordingly , a request form with adequate informa - - tion should accompany all specimens. Examples of important details include site of  biopsy/resection, clinical setting, reasons for the procedure, patient details, medications, relevant risk factors and past medical and surgical history , including pre - vious chemotherapy and radiotherapy . A request form stating ‘cancer’ or ‘Crohn’s’ is better than a form with no details but is clearly not su ffi cient. For small and large resection specimens, good quality mac - roscopic assessment and sampling is an important precursor to microscopic assessment (see Specimen processing ).
Diagnosis
Con
/f_i
rmation/rejection of a clinical diagnosis
Additional diagnoses
Classi
/f_i
cation of neoplasia
Classi
/f_i
cation of non-neoplastic disease
Staging of malignancy
Prognosis
Management
Selection of therapy
Assessment of response to treatment
Cancer screening programmes and related programmes
Cervical, bowel, breast, in
/f_l
ammatory bowel disease,
Barrett’s oesophagus
Clinical trial support
Audit
REASONS FOR ASSESSMENT OF TISSUE
The contributions that tissue analysis makes to clinical - management include diagnosis, staging, prediction of  outcome and assistance with selection of  therapy . These are often - interrelated. The process of  tissue assessment may make a new diagnosis or may confirm or refute a suspected or existing clinical diagnosis. There may be pointers towards a cause. Analysis may also reveal additional diagnoses that may be unsuspected. As an example, pathological assessment of  an appendicec - tomy specimen most often confirms a suspected clinical diag - - nosis of  acute appendicitis. However, the appendix sometimes contains an incidental neuroendocrine neoplasm, mucinous contains granulomas, raising the possibility of  Crohn’s disease or infection. Also, a specific cause of abdominal pain other than appendicitis, e.g. endometriosis, may be apparent in the appendiceal tissue. Absence of  any histological abnormality raises the possibility of  an extra-appendiceal cause. Similarly , biopsies from a patient with inflammatory bowel disease may confirm the diagnosis but may sometimes reveal or suggest an alternative cause of  intestinal inflammation such as tuberculo sis, amoebiasis, ischaemia or mucosal prolapse. Summary box 11.1 Reasons for analysis of tissue /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Tissue analysis also helps, increasingly , to determine or refine treatment and prognosis. For example, the assessment of a breast, lung, colorectal or other major cancer resection spec imen helps to confirms the diagnosis but, more importantly , provides crucial information about features such as tumour stage, vascular invasion, perineural invasion and resection mar gin involv ement, which in turn help to predict clinical outcome and determine postoperative treatment. The degree of  tumour regression in a resection after neoadjuvant therapy may also have prognostic value. Additionally , pathological assessment of resections helps surgeons and radiologists to audit their accu racy and performance. Molecular pathological analysis of  cancer tissue (see Diagnostic molecular pathology ) increasingly contributes to management, including diagnostic categorisation, prognos tic predictions and selection of drug therapy . The molecular test or group of  tests that an oncologist chooses depends on patient status, tumour location, tumour morphology and stage, among other factors. The identification of a particular bio marker may provide an indication for targeted therapy . For example, detection of  high microsatellite instability (MSI) in metastatic colorectal carcinoma (CRC) may predict respon siveness to immune checkpoint inhibition. Burrill Bernard Crohn , 1884–1983, gastroenterologist, Mount Sinai Hospital, New Y ork, NY , USA. Norman Rupert Barrett , 1903–1979, surgeon, St Thomas’s Hospital, London, UK. patient. Correlation with the clinical picture and the macro - scopic findings enhances the interpretation of  pathological changes considerably . Ther efore, absence of  relevant details may cause unnecessary delays and even er rors. For example, radiation therapy can have profound e ff ects on tissue morphol - ogy , including mimicry of  other inflammatory conditions or neoplasia. Accordingly , a request form with adequate informa - - tion should accompany all specimens. Examples of important details include site of  biopsy/resection, clinical setting, reasons for the procedure, patient details, medications, relevant risk factors and past medical and surgical history , including pre - vious chemotherapy and radiotherapy . A request form stating ‘cancer’ or ‘Crohn’s’ is better than a form with no details but is clearly not su ffi cient. For small and large resection specimens, good quality mac - roscopic assessment and sampling is an important precursor to microscopic assessment (see Specimen processing ).
Diagnosis
Con
/f_i
rmation/rejection of a clinical diagnosis
Additional diagnoses
Classi
/f_i
cation of neoplasia
Classi
/f_i
cation of non-neoplastic disease
Staging of malignancy
Prognosis
Management
Selection of therapy
Assessment of response to treatment
Cancer screening programmes and related programmes
Cervical, bowel, breast, in
/f_l
ammatory bowel disease,
Barrett’s oesophagus
Clinical trial support
Audit
REASONS FOR ASSESSMENT OF TISSUE
The contributions that tissue analysis makes to clinical - management include diagnosis, staging, prediction of  outcome and assistance with selection of  therapy . These are often - interrelated. The process of  tissue assessment may make a new diagnosis or may confirm or refute a suspected or existing clinical diagnosis. There may be pointers towards a cause. Analysis may also reveal additional diagnoses that may be unsuspected. As an example, pathological assessment of  an appendicec - tomy specimen most often confirms a suspected clinical diag - - nosis of  acute appendicitis. However, the appendix sometimes contains an incidental neuroendocrine neoplasm, mucinous contains granulomas, raising the possibility of  Crohn’s disease or infection. Also, a specific cause of abdominal pain other than appendicitis, e.g. endometriosis, may be apparent in the appendiceal tissue. Absence of  any histological abnormality raises the possibility of  an extra-appendiceal cause. Similarly , biopsies from a patient with inflammatory bowel disease may confirm the diagnosis but may sometimes reveal or suggest an alternative cause of  intestinal inflammation such as tuberculo sis, amoebiasis, ischaemia or mucosal prolapse. Summary box 11.1 Reasons for analysis of tissue /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Tissue analysis also helps, increasingly , to determine or refine treatment and prognosis. For example, the assessment of a breast, lung, colorectal or other major cancer resection spec imen helps to confirms the diagnosis but, more importantly , provides crucial information about features such as tumour stage, vascular invasion, perineural invasion and resection mar gin involv ement, which in turn help to predict clinical outcome and determine postoperative treatment. The degree of  tumour regression in a resection after neoadjuvant therapy may also have prognostic value. Additionally , pathological assessment of resections helps surgeons and radiologists to audit their accu racy and performance. Molecular pathological analysis of  cancer tissue (see Diagnostic molecular pathology ) increasingly contributes to management, including diagnostic categorisation, prognos tic predictions and selection of drug therapy . The molecular test or group of  tests that an oncologist chooses depends on patient status, tumour location, tumour morphology and stage, among other factors. The identification of a particular bio marker may provide an indication for targeted therapy . For example, detection of  high microsatellite instability (MSI) in metastatic colorectal carcinoma (CRC) may predict respon siveness to immune checkpoint inhibition. Burrill Bernard Crohn , 1884–1983, gastroenterologist, Mount Sinai Hospital, New Y ork, NY , USA. Norman Rupert Barrett , 1903–1979, surgeon, St Thomas’s Hospital, London, UK. patient. Correlation with the clinical picture and the macro - scopic findings enhances the interpretation of  pathological changes considerably . Ther efore, absence of  relevant details may cause unnecessary delays and even er rors. For example, radiation therapy can have profound e ff ects on tissue morphol - ogy , including mimicry of  other inflammatory conditions or neoplasia. Accordingly , a request form with adequate informa - - tion should accompany all specimens. Examples of important details include site of  biopsy/resection, clinical setting, reasons for the procedure, patient details, medications, relevant risk factors and past medical and surgical history , including pre - vious chemotherapy and radiotherapy . A request form stating ‘cancer’ or ‘Crohn’s’ is better than a form with no details but is clearly not su ffi cient. For small and large resection specimens, good quality mac - roscopic assessment and sampling is an important precursor to microscopic assessment (see Specimen processing ).
Diagnosis
Con
/f_i
rmation/rejection of a clinical diagnosis
Additional diagnoses
Classi
/f_i
cation of neoplasia
Classi
/f_i
cation of non-neoplastic disease
Staging of malignancy
Prognosis
Management
Selection of therapy
Assessment of response to treatment
Cancer screening programmes and related programmes
Cervical, bowel, breast, in
/f_l
ammatory bowel disease,
Barrett’s oesophagus
Clinical trial support
Audit

RISK MANAGEMENT
RISK MANAGEMENT
Safety and risk management are priorities in the labora - tory . The use of  warning labels helps to reduce the risk of contamination by transmissible infection, e.g. hepatitis B virus or human immunodeficiency virus (HIV). This is especially important when submitting and handling fresh (unfixed) tissue . Formalin kills many microorganisms, but a risk of transmissible infection still requires notification. Also, formalin itself  is toxic to the eyes and skin. Accordingly , laboratory sta ff and indeed any sta ff should discard leaking or faulty specimen containers and deal immediately with formalin spillages. Patient details should be present on all specimen containers so as to avoid errors of  identity ( Figure 11.1 ). Rigorous systems are in place to avoid interchange of specimens or confusion between di ff er - - ent patients’ samples. - his - -
Figure 11.1
Sections on glass slides stained with haematoxylin
and eosin. Each slide has a unique specimen identifying number
(06S022081), a letter corresponding to the biopsy site (A–F) and a site
label (e.g. DUOBX for duodenal biopsy).
RISK MANAGEMENT
Safety and risk management are priorities in the labora - tory . The use of  warning labels helps to reduce the risk of contamination by transmissible infection, e.g. hepatitis B virus or human immunodeficiency virus (HIV). This is especially important when submitting and handling fresh (unfixed) tissue . Formalin kills many microorganisms, but a risk of transmissible infection still requires notification. Also, formalin itself  is toxic to the eyes and skin. Accordingly , laboratory sta ff and indeed any sta ff should discard leaking or faulty specimen containers and deal immediately with formalin spillages. Patient details should be present on all specimen containers so as to avoid errors of  identity ( Figure 11.1 ). Rigorous systems are in place to avoid interchange of specimens or confusion between di ff er - - ent patients’ samples. - his - -
Figure 11.1
Sections on glass slides stained with haematoxylin
and eosin. Each slide has a unique specimen identifying number
(06S022081), a letter corresponding to the biopsy site (A–F) and a site
label (e.g. DUOBX for duodenal biopsy).
RISK MANAGEMENT
Safety and risk management are priorities in the labora - tory . The use of  warning labels helps to reduce the risk of contamination by transmissible infection, e.g. hepatitis B virus or human immunodeficiency virus (HIV). This is especially important when submitting and handling fresh (unfixed) tissue . Formalin kills many microorganisms, but a risk of transmissible infection still requires notification. Also, formalin itself  is toxic to the eyes and skin. Accordingly , laboratory sta ff and indeed any sta ff should discard leaking or faulty specimen containers and deal immediately with formalin spillages. Patient details should be present on all specimen containers so as to avoid errors of  identity ( Figure 11.1 ). Rigorous systems are in place to avoid interchange of specimens or confusion between di ff er - - ent patients’ samples. - his - -
Figure 11.1
Sections on glass slides stained with haematoxylin
and eosin. Each slide has a unique specimen identifying number
(06S022081), a letter corresponding to the biopsy site (A–F) and a site
label (e.g. DUOBX for duodenal biopsy).

SPECIMEN PROCESSING
SPECIMEN PROCESSING
SPECIMEN PROCESSING
SPECIMEN PROCESSING

Special stains
Special stains
A ‘special stain’ is a stain that is not routine, i.e. not an H&E stain. Immunohistochemical stains are conventionally separate from this category . Some special stains demonstrate normal substances in increased quantities or in abnormal locations. The periodic acid–Schi ff (PAS) stain demonstrates both - glycogen and mucin, whereas a diastase PAS (D-PAS) stain demonstrates mucin, e.g. in an adenocarcinoma. Perls Prussian blue stain demonstrates iron accumulation ( Figure 11.23 ), e.g. in haemochromatosis. A reticulin stain helps to demonstrate fibrosis ( Figure 11.24 ). Elastic stains also show fibrosis and can highlight blood vessels by outlining their elastic laminae. Special stains can also re veal the accumulation of abnormal substances, e.g . a Congo red stain for amyloid. Summary box 11.9 Additional techniques for assessing tissue /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Summary box 11.10 Common special stains /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
Special stains
Immunohistochemistry
Electron microscopy
In situ
hybridisation, including
/f_l
uorescence
in situ
hybridisation
(FISH)
Molecular pathology techniques (including single biomarker
polymerase chain reaction [PCR] and next-generation
sequencing [NGS])
PAS: glycogen, fungi
D-PAS: mucin
Perls Prussian blue: iron
Reticulin: reticulin
/f_i
bres,
/f_i
brosis
van Gieson: collagen
Congo red: amyloid
Ziehl–Neelsen: mycobacteria
Special stains are also useful for the diagnosis of  infection. Some microorganisms are not visible on routine H&E slides but are demonstrable with a stain. For example, a Ziehl–Neelsen stain demonstrates acid-fast bacilli, particularly mycobacteria, by staining them bright red on a blue background ( Figure 11.20 Other microorganisms may be detectable on H&E but are easier to see with a special stain, e.g. fungi (PAS or Grocott stain), protozoa (Giemsa stain) and spirochaetes (Warthin– in situ hybridisation Starry stain). Immunohistochemistry and also help to detect some microorganisms (see Immunohisto chemistry: infections and other applications and hybridisation ).
(b)
Figure 11.23
(a)
Brown pigment in a biopsy.
(b)
A Perls stain is posi
tive, indicating that the pigment is iron.
Special stains
A ‘special stain’ is a stain that is not routine, i.e. not an H&E stain. Immunohistochemical stains are conventionally separate from this category . Some special stains demonstrate normal substances in increased quantities or in abnormal locations. The periodic acid–Schi ff (PAS) stain demonstrates both - glycogen and mucin, whereas a diastase PAS (D-PAS) stain demonstrates mucin, e.g. in an adenocarcinoma. Perls Prussian blue stain demonstrates iron accumulation ( Figure 11.23 ), e.g. in haemochromatosis. A reticulin stain helps to demonstrate fibrosis ( Figure 11.24 ). Elastic stains also show fibrosis and can highlight blood vessels by outlining their elastic laminae. Special stains can also re veal the accumulation of abnormal substances, e.g . a Congo red stain for amyloid. Summary box 11.9 Additional techniques for assessing tissue /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Summary box 11.10 Common special stains /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
Special stains
Immunohistochemistry
Electron microscopy
In situ
hybridisation, including
/f_l
uorescence
in situ
hybridisation
(FISH)
Molecular pathology techniques (including single biomarker
polymerase chain reaction [PCR] and next-generation
sequencing [NGS])
PAS: glycogen, fungi
D-PAS: mucin
Perls Prussian blue: iron
Reticulin: reticulin
/f_i
bres,
/f_i
brosis
van Gieson: collagen
Congo red: amyloid
Ziehl–Neelsen: mycobacteria
Special stains are also useful for the diagnosis of  infection. Some microorganisms are not visible on routine H&E slides but are demonstrable with a stain. For example, a Ziehl–Neelsen stain demonstrates acid-fast bacilli, particularly mycobacteria, by staining them bright red on a blue background ( Figure 11.20 Other microorganisms may be detectable on H&E but are easier to see with a special stain, e.g. fungi (PAS or Grocott stain), protozoa (Giemsa stain) and spirochaetes (Warthin– in situ hybridisation Starry stain). Immunohistochemistry and also help to detect some microorganisms (see Immunohisto chemistry: infections and other applications and hybridisation ).
(b)
Figure 11.23
(a)
Brown pigment in a biopsy.
(b)
A Perls stain is posi
tive, indicating that the pigment is iron.
Special stains
A ‘special stain’ is a stain that is not routine, i.e. not an H&E stain. Immunohistochemical stains are conventionally separate from this category . Some special stains demonstrate normal substances in increased quantities or in abnormal locations. The periodic acid–Schi ff (PAS) stain demonstrates both - glycogen and mucin, whereas a diastase PAS (D-PAS) stain demonstrates mucin, e.g. in an adenocarcinoma. Perls Prussian blue stain demonstrates iron accumulation ( Figure 11.23 ), e.g. in haemochromatosis. A reticulin stain helps to demonstrate fibrosis ( Figure 11.24 ). Elastic stains also show fibrosis and can highlight blood vessels by outlining their elastic laminae. Special stains can also re veal the accumulation of abnormal substances, e.g . a Congo red stain for amyloid. Summary box 11.9 Additional techniques for assessing tissue /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF Summary box 11.10 Common special stains /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
Special stains
Immunohistochemistry
Electron microscopy
In situ
hybridisation, including
/f_l
uorescence
in situ
hybridisation
(FISH)
Molecular pathology techniques (including single biomarker
polymerase chain reaction [PCR] and next-generation
sequencing [NGS])
PAS: glycogen, fungi
D-PAS: mucin
Perls Prussian blue: iron
Reticulin: reticulin
/f_i
bres,
/f_i
brosis
van Gieson: collagen
Congo red: amyloid
Ziehl–Neelsen: mycobacteria
Special stains are also useful for the diagnosis of  infection. Some microorganisms are not visible on routine H&E slides but are demonstrable with a stain. For example, a Ziehl–Neelsen stain demonstrates acid-fast bacilli, particularly mycobacteria, by staining them bright red on a blue background ( Figure 11.20 Other microorganisms may be detectable on H&E but are easier to see with a special stain, e.g. fungi (PAS or Grocott stain), protozoa (Giemsa stain) and spirochaetes (Warthin– in situ hybridisation Starry stain). Immunohistochemistry and also help to detect some microorganisms (see Immunohisto chemistry: infections and other applications and hybridisation ).
(b)
Figure 11.23
(a)
Brown pigment in a biopsy.
(b)
A Perls stain is posi
tive, indicating that the pigment is iron.

Specimen adequacy
Specimen adequacy
There are many reasons for an inadequate specimen. The operator may fail to sample the target organ or lesion or may take a sample that is too small to include or reveal a hetero geneous abnormality . A sample from the centre of a necrotic or ulcerated lesion might include no viable tissue. Superficial biopsies from a carcinoma may fail to distinguish dysplasia ( Figure 11.14 ) from invasive car cinoma. Cautery and crush artefact are sometimes severe enough to impede assessment. Suboptimal laboratory processing can also cause problems with interpretation. Summary box 11.8 Reasons for an inadequate sample /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
Histology and cytology
Failure or inability to sample the intended area
Sample too small
Sample unrepresentative
Non-viable tissue, e.g. ulcer or necrosis
Histology
Sample too super
/f_i
cial to detect deeper layers
Cautery artefact
Crush artefact
Specimen adequacy
There are many reasons for an inadequate specimen. The operator may fail to sample the target organ or lesion or may take a sample that is too small to include or reveal a hetero geneous abnormality . A sample from the centre of a necrotic or ulcerated lesion might include no viable tissue. Superficial biopsies from a carcinoma may fail to distinguish dysplasia ( Figure 11.14 ) from invasive car cinoma. Cautery and crush artefact are sometimes severe enough to impede assessment. Suboptimal laboratory processing can also cause problems with interpretation. Summary box 11.8 Reasons for an inadequate sample /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
Histology and cytology
Failure or inability to sample the intended area
Sample too small
Sample unrepresentative
Non-viable tissue, e.g. ulcer or necrosis
Histology
Sample too super
/f_i
cial to detect deeper layers
Cautery artefact
Crush artefact
Specimen adequacy
There are many reasons for an inadequate specimen. The operator may fail to sample the target organ or lesion or may take a sample that is too small to include or reveal a hetero geneous abnormality . A sample from the centre of a necrotic or ulcerated lesion might include no viable tissue. Superficial biopsies from a carcinoma may fail to distinguish dysplasia ( Figure 11.14 ) from invasive car cinoma. Cautery and crush artefact are sometimes severe enough to impede assessment. Suboptimal laboratory processing can also cause problems with interpretation. Summary box 11.8 Reasons for an inadequate sample /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF
Histology and cytology
Failure or inability to sample the intended area
Sample too small
Sample unrepresentative
Non-viable tissue, e.g. ulcer or necrosis
Histology
Sample too super
/f_i
cial to detect deeper layers
Cautery artefact
Crush artefact

Storage
Storage
Resection specimens are generally stored for about 4–6 weeks. Tissue blocks and slides are retained for as long as space George Nicholas Papanicolaou , 1883–1962, Professor of  Anatomy , Cornell University , New Y ork, NY , USA. Richard May , 1863–1936, Professor of  Medicine, Munich, Germany . Ludwig Grünwald , 1863–1927, otolaryngologist, Munich, Germany . Gustav Giemsa , 1867–1948, chemist and bacteriologist, Hamburg, Germany . Dmitri Leonidovich Romanowsky , 1861–1921, Professor of  Medicine, St Petersburg, Russia. Hippocrates of  Kos , Greek physician and surgeon, and by common consent ‘the father of medicine’, was born on the island of  Kos, o ff Turkey , about 460 /uni00A0/b.sc/c.sc/e.sc and probably died in 375 /uni00A0 /b.sc/c.sc/e.sc . permits. Fresh tissue can be frozen for future clinical review , teaching, audit or research. Many countries now have formal tissue-banking processes that allow construction of  an archive of  cases. In many countries, the storage, transport and subse - quent use of  tissue is subject to many legal constraints.
Figure 11.9
A cervical smear stained with a Papanicolaou stain.
Numerous cells are present (courtesy of Professor MT Sheaff, Barts
Health NHS Trust).
Storage
Resection specimens are generally stored for about 4–6 weeks. Tissue blocks and slides are retained for as long as space George Nicholas Papanicolaou , 1883–1962, Professor of  Anatomy , Cornell University , New Y ork, NY , USA. Richard May , 1863–1936, Professor of  Medicine, Munich, Germany . Ludwig Grünwald , 1863–1927, otolaryngologist, Munich, Germany . Gustav Giemsa , 1867–1948, chemist and bacteriologist, Hamburg, Germany . Dmitri Leonidovich Romanowsky , 1861–1921, Professor of  Medicine, St Petersburg, Russia. Hippocrates of  Kos , Greek physician and surgeon, and by common consent ‘the father of medicine’, was born on the island of  Kos, o ff Turkey , about 460 /uni00A0/b.sc/c.sc/e.sc and probably died in 375 /uni00A0 /b.sc/c.sc/e.sc . permits. Fresh tissue can be frozen for future clinical review , teaching, audit or research. Many countries now have formal tissue-banking processes that allow construction of  an archive of  cases. In many countries, the storage, transport and subse - quent use of  tissue is subject to many legal constraints.
Figure 11.9
A cervical smear stained with a Papanicolaou stain.
Numerous cells are present (courtesy of Professor MT Sheaff, Barts
Health NHS Trust).
Storage
Resection specimens are generally stored for about 4–6 weeks. Tissue blocks and slides are retained for as long as space George Nicholas Papanicolaou , 1883–1962, Professor of  Anatomy , Cornell University , New Y ork, NY , USA. Richard May , 1863–1936, Professor of  Medicine, Munich, Germany . Ludwig Grünwald , 1863–1927, otolaryngologist, Munich, Germany . Gustav Giemsa , 1867–1948, chemist and bacteriologist, Hamburg, Germany . Dmitri Leonidovich Romanowsky , 1861–1921, Professor of  Medicine, St Petersburg, Russia. Hippocrates of  Kos , Greek physician and surgeon, and by common consent ‘the father of medicine’, was born on the island of  Kos, o ff Turkey , about 460 /uni00A0/b.sc/c.sc/e.sc and probably died in 375 /uni00A0 /b.sc/c.sc/e.sc . permits. Fresh tissue can be frozen for future clinical review , teaching, audit or research. Many countries now have formal tissue-banking processes that allow construction of  an archive of  cases. In many countries, the storage, transport and subse - quent use of  tissue is subject to many legal constraints.
Figure 11.9
A cervical smear stained with a Papanicolaou stain.
Numerous cells are present (courtesy of Professor MT Sheaff, Barts
Health NHS Trust).

TISSUE SPECIMENS
TISSUE SPECIMENS
Routine tissue specimens received by a histopathology depart - ment include those intended for histopathological analysis and those for cytopathological assessment. These may overlap, and ‘cytology’ preparations sometimes undergo reprocessing to become histological specimens. TISSUE SPECIMENS
Routine tissue specimens received by a histopathology depart - ment include those intended for histopathological analysis and those for cytopathological assessment. These may overlap, and ‘cytology’ preparations sometimes undergo reprocessing to become histological specimens. TISSUE SPECIMENS
Routine tissue specimens received by a histopathology depart - ment include those intended for histopathological analysis and those for cytopathological assessment. These may overlap, and ‘cytology’ preparations sometimes undergo reprocessing to become histological specimens.