Tissue and Molecular
diagnosis
Dr.Niharika Gajjela
Dept. of General Surgery
Junior Resident -1st year
Introduction
Tissue diagnosis depended on
Pre-nineteenth Naked eye examination
Century Of autopsy material / small selection of surgical
specimens
After the
It allowed closer examination of tissue from
development of
autopsies and surgical procedures, with visualisation
Light
of cells, nuclei and tissue structure.
microscope
• Tissue analysis is now an integral and routine element of clinical
practice. It is heavily dependent on microscopic assessment.
• Developments and changes in cellular pathology are continuous. 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 prevalence of cancer and other illnesses.
Reasons for analysis of tissue
1. Diagnosis
• Confirmation /rejection of a clinical
diagnosis
• Additional diagnoses
• Classification of neoplasia
• Classification of non-neoplastic disease
2. Staging of malignancy
3. Prognosis
4. Management
• Selection of therapy
• Assessment of response to treatment
5. Cancer screening programmes and related programmes
• Cervical, bowel, breast, infammatory bowel disease, Barrett’s
oesophagus
6.Clinical trial support
7.Audit
Tissue specimens
pecimens received by a histopathology department include those intended for h
ose for cytopathological assessment.
rlap, and ‘cytology’ preparations sometimes undergo reprocessing to become hi
Risk management
• Safety and risk management are priorities
in the laboratory.
• The use of warning labels helps to reduce
the risk of contamination by transmissible
infection, e.g. hepatitis B virus or human
immunodefciency virus (HIV).
• This is especially important when
submitting and handling fresh (unfxed)
tissue.
• Patient details should be present on all
specimen containers so as to avoid errors
of identity
Specimen processing
Histology specimen
• The largest specimens require initial opening (e.g. gastrointestinal
tract) or slicing (e.g. uterus, pancreas, breast) to allow further and
adequate fixation in formalin, usually over 24–48 hours
• For any specimen that is too • Inks of various colours help
large for these approaches, to identify resection margins
the prosector takes and surfaces during
representative samples of microscope assessment as
areas of interest or they remain in place after
relevance processing
• The prosector places specimens, • BMSs/technical staff then embed
or samples from specimens, in the tissue in paraffin wax while
plastic cassettes in the cassette to produce a
tissue block
Histological processing: sequence of events
● 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 paraffin 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 fndings into account
● Further studies on tissue, if necessary
● Entry of report onto computer system
● Authorisation of report by pathologist
Frozen section specimen
Advantages and disadvantages
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 diffcult to process
PRINCIPLES OF MICROSCOPIC DIAGNOSIS
Diagnosis of malignancy
Microscopic features of malignancy
● Metastasis
● Invasion
-of surrounding tissue
-Vascular ( intraluminal tumour and/or tumour in blood vessel wall)
- Perineural
● Architectural abnormalities
● Necrosis
● Numerous mitotic figures
● Atypical mitotic figures
● Nuclear abnormalities
-Pleomorphism
-Enlargement
-Hyperchromaticity
-Chromatin clumping
● Nucleolar enlargement and multiplicity
Causes of false-positive diagnoses of malignancy
● Interchanged samples
● Contamination
● Interpretative error
● Treatment-induced change, e.g. radiotherapy
● Ulceration
Cytology compared with histology
Advantages
● Wider area of sampling in some cases
● Often less invasive
● Fast
● Cheap
Disadvantages
● Cannot assess tissue architecture
● Less amenable to further tissue studies
Reasons for an inadequate sample
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 superficial to detect deeper layers
● Cautery artefact
● Crush artefact
Additional techniques for assessing tissue
● Special stains
● Immunohistochemistry
● Electron microscopy
● In situ hybridisation, including fuorescence in situ hybridisation (FISH)
● Molecular pathology techniques (including single biomarker polymerase
chain reaction [PCR] and next-generation sequencing [NGS])
Common 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.
● PAS: glycogen, fungi
● D-PAS: mucin
● Perls Prussian blue: iron
● Reticulin: reticulin fbres, fbrosis
● van Gieson: collagen
● Congo red: amyloid
● Ziehl–Neelsen: mycobacteria
hemistry
hemistry emerged in the 1970s and has had a major impact on histopathological
cts a specifc antigen using an antibody
is labelled with a dye and after binding to its target antigen is visible in the tissu
n, often brown
Some immunohistochemical stains used for tumours
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-specific antigen (PSA)
● Lung: thyroid transcription factor-1 (TTF-1)
● Thyroid: thyroglobulin
● Colorectum: cytokeratin 20 (CK20), CDX2
● Liver: hepatocyte-specific 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)
Uses of immunohistochemistry
● Cell type
● Neoplasia
Direction of differentiation/phenotype
Determination of anatomical site of origin
Confirmation of neoplasia
Grading
Selection of treatment
Detection of/screening for mutations
Prognosis
● Microorganisms – detection
● Other
Amyloid
Immunoglobulins
Complement
DIAGNOSTIC MOLECULAR
PATHOLOGY
Basic methods in diagnostic molecular pathology
In situ hybridisation
▪️In situ hybridisation (ISH) uses a labelled oligonucleotide probe that
targets a specific sequence of RNA or DNA.
▪️It allows visualisation 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
Polymerase chain reaction
• The polymerase chain reaction (PCR)
amplifies DNA, yielding 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).
• PCR-based methods have numerous
applications in oncology
• PCR-based methods can also detect
microorganisms in tissue but this is not a
common application because of the risk
of false positives.
Cytogenetics and fluorescence in situ
hybridisation
• Conventional cytogenetics is the microscopic study of chromosomal
changes in individual cells.
• Newer techniques, including FISH, array comparative genomic
hybridisation, RT-PCR and next-generation sequencing (NGS) are
increasingly replacing conventional cytogenetics.
• Cytogenetic tests seek alterations such as gene amplifcation, loss of
segments of chromosomal material, loss of whole chromosomes (e.g.
in renal cell carcinoma) and translocations with associated fusion
genes (e.g. EWSR1-FLI1 in Ewing’s sarcoma).
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 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.
Genomic changes in tumours
▪️Tumours require loss of control of cell proliferation.
▪️Abnormalities of numerous genes can affect
proliferation and facilitate tumour development.
▪️The relevant genes fall into 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.
Genes and carcinogenesis
Genes
● (Proto-) oncogenes
● KRAS
● BRAF
● EGFR
● BCL2
Tumour suppressor genes
● TP53
● BRCA1/2
Pathways
● Proliferation and signal transduction
● Cell cycle control
● DNA repair
● Apoptosis
Indications for molecular analysis of tumour tissue
● Diagnosis and classifcation
● Selection of therapy
● Prognosis
● Staging
● Monitoring disease burden
● Screening for germline mutations
● Confirmation of neoplasia (e.g. clonality)
Detection methods for main molecular changes
● Point mutations and small insertions and deletions: NGS, PCR
● Fusions: FISH, NGS, PCR
● Amplifications: FISH, NGS
● Tumour mutation burden: NGS
● Immunohistochemistry may be a very useful initial test, and is
often sufficient
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.
Translocations
-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.
Tumour types that may respond to immune checkpoint
inhibitor drugs
● Breast carcinoma
● Urothelial carcinoma
●Non-small cell lung cancer
●Small cell lung cancer
●Hepatocellular carcinoma
● Malignant melanoma
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).
Microsatellite instability and mismatch repair genes
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 classification, 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-fuorouracil
● Screening for germline mutation, i.e. Lynch syndrome
Molecular analysis in colorectal carcinoma
Mismatch repair gene abnormalities
● Multiple considerations (see Summary box 11.17)
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
● Specific therapy available: tyrosine kinase inhibitors
Molecular and related changes in non-small cell lung
carcinoma
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)
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.
• Advantages include more fexible 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.
