Pathologic Diagnosis Of Tumours Notes

Pathologic Diagnosis Of Tumours

When a tumour is clinically detected, the first and foremost task is to confirm whether it is benign or malignant, and make a complete and refined diagnosis. The most certain and reliable method which has stood the test of time is the histological examination of biopsy, though recently many other methods to arrive at the correct diagnosis or confirm the histological diagnosis are available.

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Histological Methods

These methods are most valuable in arriving at an accurate diagnosis and are based on microscopic examination of excised tumour mass or open/needle biopsy from the mass, supported with complete clinical and data of other relevant clinical investigations.

The tissue must be fixed in 10% formalin for light microscopic examination and in glutaraldehyde for electron microscopic studies, while quick-frozen section and enzyme studies are carried out on fresh unfixed tissues. These methods are as under:

• Paraffin-embedding technique:  In this, 10% formalin-fixed tissue is used.
  • The representative tissue piece from a larger tumour mass or biopsy is processed through a tissue processor having an overnight cycle, embedded in molten paraffin wax for making tissue blocks.
  • These blocks are trimmed followed by fine-sectioning into 3-4 µm sections using rotary microtome for which either fixed knife or disposable blades are used for cutting.
  • These sections are then stained with haematoxylin and eosin (H & E) and examined microscopically.
• Frozen section:  In this technique, unfixed tissue is used and the procedure is generally carried out when the patient is undergoing surgery and is still under anaesthesia. Here, instead of an overnight tissue processing cycle and paraffin embedding, a cryostat machine is used and fresh unfixed tissue is submitted. The tissue biopsy is quickly frozen to ice at about –25°C acts as an embedding medium and then sectioned.
• Sections are then ready for rapid H & E or toluidine blue staining. Major indications for the frozen section are:
  • For rapid intraoperative diagnosis before proceeding to a major radical surgery
  • To know the extent of the presence of cancer at the surgical margin, and
  • To demonstrate certain cellular constituents which are lost in processing through chemicals (for example, Fat, enzymes etc).

The histological diagnosis by either of these methods is made on the basis of morphological features:

• Benign tumours reproduce features of normal tissue closely, they are localised, unable to invade and metastasise.
• Malignant tumours are identified by a lack of differentiation in cancer cells (anaplasia or cellular atypia) and may invade locally and metastasise to distant organs.

The light microscopic and ultrastructural characteristics of neoplastic cells have been described in the earlier part.

 Cytological Methods

Cytological methods for diagnosis consist of 2 types of methods:

Study of cells shed off into body cavities (exfoliative cytology) and study of cells by putting a fine needle introduced under vacuum into the lesion (fine needle aspiration cytology, FNAC).

1. The Exfoliative cytology: Cytologic smear (Papanicolaou or Pap smear) method was initially employed for detecting dysplasia, carcinoma in situ and invasive carcinoma of the uterine cervix.
   • However, its use has now been widely extended to include examination of sputum and bronchial washings; pleural, peritoneal and pericardial effusions; urine, gastric secretions, and CSF.
   • The method is based on microscopic identification of the characteristics of malignant cells which are non-cohesive and loose and are thus shed off or ‘exfoliated’ into the lumen.
   • However, a ‘negative diagnosis’ does not altogether rule out malignancy due to the possibility of sampling error.
2. Fine needle aspiration cytology (FNAC): Currently, cytopathology includes not only the study of exfoliated cells but also materials obtained from superficial and deep-seated lesions in the body which do not shed off cells freely.

• This method consists of study of cells obtained by a fine needle introduced under vacuum into the lesion, so-called fine needle aspiration cytology (FNAC).
• The superficial masses can be aspirated under direct vision while deep-seated masses such as intra-abdominal, pelvic organs and retroperitoneum.
• Are frequently investigated by ultrasound (US)-or computed tomography (CT)-guided fine needle aspirations.
• The smears are fixed in 95% ethanol by wet fixation or may be air-dried unfixed.
• While Papanicolaou’s method of staining is routinely employed in most laboratories for wet fixed smears, others prefer H and E due to similarity.
• In staining characteristics in the sections obtained by paraffin-embedding.
• Airdried smears are stained by May-Grünwald-Giemsa or Leishman stain.
• FNAC has a diagnostic reliability between 80 and 97% but it must not be substituted for clinical judgement or compete with an indicated histopathologic biopsy.

Cytological methods along with common examples are discussed in detail in Appendix I.

Histochemistry And Cytochemistry

Histochemistry and cytochemistry are additional diagnostic tools which help the pathologist in identifying the chemical composition of cells, their constituents and their products by special staining methods.

Though immunohistochemical techniques are more useful for tumour diagnosis (see below), many histochemical and cytochemical stains (also called as special stains) are still employed for this purpose. Some of the common examples.

Immunohistochemistry

With current technology, it is possible to use routinely processed paraffin-embedded tissue blocks for immunohistochemistry (IHC), thus making profound impact on diagnostic surgical pathology.

• Earlier, diagnostic surgical pathology used to be considered a subjective science with inter-observer variation, particularly in borderline lesions and lesions of undetermined origin, but use of IHC has added objectivity, specificity and reproducibility to the surgical pathologist’s diagnosis.
• IHC is an immunological method of recognising a cell by one or more of its specific components in the cell membrane, cytoplasm or nucleus and are accordingly interpreted
• These cell components (or antigens) combine with specific antibodies on the formalin-fixed paraffin sections or cytological smears.
• The complex of antigen-antibody on the slide is made visible for light microscopic identification by either fluorescent dyes (‘fluorochromes’) or by enzyme system (‘chromogens’).
• The specific antibody against a particular cellular antigen is obtained by the hybridoma technique for monoclonal antibody production.
• These monoclonal antibodies, besides being specific against antigens, are highly sensitive in the detection of antigenic component, and, therefore, impart objectivity to the subjective tumour diagnosis made by the surgical pathologist.

Common histochemical/cytochemical stains in tumour diagnosis:

Tumours of uncertain histogenesis:

IHC has brought about a revolution in the approach to the diagnosis of tumours of uncertain origin, primary as well as metastatic from an unknown primary tumour.

A panel of antibodies is chosen to resolve such problematic cases in diagnosis; the selection of antibodies is made on the basis of clinical history, morphologic features, and results of other relevant investigations.

Commonly, IHC stains for intermediate filaments (keratin, vimentin, desmin, neurofilaments, and glial fibrillary acidic proteins) expressed by the tumour cells are of immense value as first line of IHC stains, besides other common IHC stains listed in table.

A common panel of immunohistochemical stains for tumours of uncertain origin:

• Prognostic markers in cancer: Second important application of IHC is to predict the prognosis of tumours.
  • By detection of micrometastasis, occult metastasis, and by identification of certain features.
  • Acquired, or products elaborated, or genes overexpressed, by the malignant cells to predict the biologic behaviour of the tumour.
  • A few examples are: Proto-oncogenes (for example, HER-2/neu overexpression in carcinoma breast), tumour-suppressor genes (for example, RB gene, p53), growth factor receptors (for example, Epidermal growth factor receptor or EGFR), and tumour cell proliferation markers (for example,Ki67, proliferation cell nuclear antigen PCNA).
• Predicting response to therapy: IHC is widely used to predict therapeutic response in some tumours for examples, Carcinoma of the breast and prostate.
  • Both these tumours are under the growth regulation of hormones — oestrogen and androgen, respectively.
  • The specific receptors for these growth-regulating hormones are located on respective tumour cells.
  • Tumours expressing high level of receptor positivity would respond favourably to removal/ablation of the endogenous source of such hormones (oophorectomy in oestrogen-positive breast cancer and orchiectomy in androgen-positive prostatic carcinoma).
  • Alternatively, hormonal therapy is administered to counter their levels:
    • Oestrogen therapy in prostatic cancer and androgen therapy in breast cancer.
    • The results of oestrogen-receptors and progesterone receptors in breast cancer have a significant prognostic correlation.
    • Though the results of androgen-receptor studies in prostatic cancer have limited prognostic value.
• Microbial infections: IHC stains can be applied to confirm infectious agents in tissues by use of specific antibodies against microbial DNA or RNA e.g. detection of viruses (HBV, CMV, HPV, herpesviruses), bacteria (for example, Helicobacter pylori), and parasites (Pneumocystis jiroveci)

Electron Microscopy

Ultrastructural examination of tumour cells offers a selective role in diagnostic pathology. Electron microscopy may be helpful in confirming or substantiating a tumour diagnosis arrived at by light microscopy and immunohistochemistry.

A few general features of malignant tumour cells by EM examination can be appreciated:

• Cell junctions, their presence and type
• Cell surface, for example, the Presence of microvilli
• Cell shape and cytoplasmic extensions
• The shape of the nucleus and features of the nuclear membrane
• Nucleoli, their size and density
• Cytoplasmic organelles—their number is generally reduced
• Dense bodies in the cytoplasm
• Any other secretory product in the cytoplasm, for example, Melanosomes in melanoma and membrane-bound granules in endocrine tumours.

Serum Tumour Markers (Biochemical Assays)

In order to distinguish from the preceding techniques of tumour diagnosis in which ‘stains’ are imparted on the tumour cells in section or smear, tumour markers are biochemical assays of products elaborated by the tumour cells in blood or other body fluids.

It is, therefore, pertinent to keep in mind that many of these products are produced by normal body cells too, and thus the biochemical estimation of the product in blood or other fluid reflects the total substance and not by the tumour cells alone.

These methods, therefore, lack sensitivity as well as specificity and can only be employed for the following:

• Firstly, as an adjunct to the pathologic diagnosis arrived at by other methods and not for primary diagnosis of cancer.
• Secondly, it can be used for prognostic and therapeutic purposes.

Tumour markers include: Cell surface antigens (or oncofoetal antigens), cytoplasmic proteins, enzymes, hormones and cancer antigens; these are listed in.

However, two of the best-known examples of oncofoetal antigens secreted by foetal tissues as well as by tumours are alpha-foetoprotein (AFP) and carcinoembryonic antigens (CEA):

• Alpha-foetoprotein (AFP):  This is a glycoprotein synthesised normally by foetal liver cells.
  • Their serum levels are elevated in hepatocellular carcinoma and non-seminomatous germ cell tumours of the testis.
  • Certain non-neoplastic conditions also have increased serum levels of AFP for example,Hepatitis, cirrhosis, toxic liver injury and pregnancy.
• Carcino-embryonic antigen (CEA): CEA is also a glycoprotein normally synthesised in embryonic tissue of the gut, pancreas and liver.
  • Their serum levels are high in cancers of the gastrointestinal tract, pancreas and breast.
  • As in AFP, CEA levels are also elevated in certain non-neoplastic conditions e.g. in ulcerative colitis, Crohn’s disease, hepatitis and chronic bronchitis.

 Other Modern Aids In Pathologic Diagnosis Of Tumours

In addition to the methods described above, some other modern diagnostic techniques have emerged for tumour diagnostic pathology but their availability as well as applicability is limited.

Briefly, their role in tumour diagnosis is outlined below.

• Flow cytometry: This is a computerised technique by which the detailed characteristics of individual tumour cells are recognised and quantified and the data can be stored for subsequent comparison too.
  • Since for flow cytometry, single-cell suspensions are required to ‘flow’ through the ‘cytometer’, it can be employed on blood cells and their precursors in bone marrow aspirates and body fluids, and sometimes on fresh-frozen unfixed tissue.
  • The method employs either identification of cell surface antigen (for example, Classification of leukaemias and lymphomas), or by DNA content analysis (e.g. aneuploidy in various cancers).
• In situ hybridisation: This is a molecular technique by which nucleic acid sequences (cellular/viral DNA and RNA) can be localised by specifically-labelled nucleic acid probe directly in the intact cell (in situ) rather than by DNA extraction (see below).
  • A modification of in situ hybridisation technique is fluorescence in situ hybridisation (FISH) in which fluorescence dyes applied and is used to detect microdeletions, and subtelomere deletions and to look for alterations in chromosomal numbers.
  • In situ hybridisation may be used for the analysis of certain human tumours by the study of oncogenes aside from its use in diagnosis of viral infection.
• Cell proliferation analysis: Besides flow cytometry, the degree of proliferation of cells in tumours can be determined by various other methods as under:

 Important tumour markers:

• • Mitotic count:  This is the oldest but still widely used method in routine diagnostic pathology work. The number of cells in mitosis are counted per high power field for example, Categorising various types of smooth muscle tumours.
  • Radioautography:  In this method, the proliferating cells are labelled in vitro with thymidine and then the tissue is processed for paraffin-embedding. Thymidine-labelled cells (corresponding to S-phase) are then counted per 2000 tumour cell nuclei and expressed as a thymidine-labelling index. The method is employed as a prognostic marker in breast carcinoma.
  • Microspectrophotometric analysis:  The section is stained with Feulgen reaction which imparts staining to the DNA content of the cell and then DNA content is measured by microspectrophotometer. The method is tedious and has limited use.
  • IHC proliferation markers:  The nuclear antigen specific for cell growth and division is stained by immunohistochemical method and then positive cells are counted under the microscope or by an image analyzer for example, Ki-67, MIB-1, PCNA, and cyclins.
  • Nucleolar organiser region (NOR):  Nucleolus contains ribosomal components which are formed at chromosomal regions containing DNA called NORs. NORs have an affinity for silver.
    • This property is made used in staining the section with silver (AgNOR technique).
    • NORs appear as black intranuclear dots while the background is stained yellow-brown.

• Image analyser and morphometry: An image analyser is a software system in the computer attached to a microscope which is fitted with an image capture board.
• The system is used to perform measurement of architectural, cellular and nuclear features of tumour cells.
• Image analyser can be used for the following purposes: Morphometric study of tumour cells by measurement of architectural, cellular and nuclear features.
  • Quantitative nuclear DNA ploidy measurement.
  • Quantitative valuation of immunohistochemical staining.
• Molecular diagnostic techniques: Group of molecular cell biological methods in the tumour diagnostic laboratory are a variety of DNA/RNA-based molecular techniques.
  • In which the DNA/RNA are extracted (compared from in situ above) from the cell and then analysed.
  • These techniques are highly sensitive, specific and rapid and have revolutionised diagnostic pathology in neoplastic as well.
  • Non-neoplastic conditions  (for example, Infectious and inherited disorders, and in identity diagnosis). Molecular diagnostic techniques include: DNA analysis by Southern blot.
  • RNA analysis by northern blot, and polymerase chain reaction (PCR).
  • The following techniques of molecular methods in tumour diagnosis have applications in haematologic as well as nonhematologic malignancies:
    • Analysis of molecular cytogenetic abnormalities
    • Mutational analysis
    • Antigen receptor gene rearrangement
    • Study of oncogenic viruses at the molecular level.

Besides the application of these molecular techniques for the diagnosis of tumours, many of the newer molecular techniques are applied for predicting prognosis, biological behaviour of tumours, detection of minimal residual disease and for hereditary predisposition of other family members to develop a particular cancer.

• DNA microarray analysis of tumours: Currently, it is possible to perform molecular profiling of a tumour by use of gene chip technology which allows measurement of levels of expression of several thousand genes (up-regulation or down-regulation) simultaneously.
  • Fluorescent labels are used to code the cDNA synthesised by a trigger from mRNA.
  • The conventional DNA probes are substituted by a silicon chip which contains the entire range of genes and high-resolution scanners are used for the measurement.

Major Applications Of Molecular Methods In Cancer

Advances in molecular techniques have brought the former research tools to the laboratory for diagnosis, prognosis and for personalised and precise cancer treatment as under.

Detection Of Minimum Residual Disease (MRD)

In haematolymphoid malignancies, after remission has been induced, it is possible to detect and monitor the patient for the minimum residual disease for pre-empting relapse of malignant process for example, Chronic myeloid leukaemia, detection of BCR/ABL gene product by PCR-based amplification techniques.

Genome-Wide Mutational Analysis

With the completion of the Human Genome Project and the availability of sequencing technologies, genome-wide mutational profiling of cancer genomes has become possible.

• Whole genome sequencing has opened vistas to several newer possibilities in cancer management:
  • Knowledge of oncogenic viruses involved at the molecular level and their role in the pathogenesis of particular cancer in an individual.
  • Precise cancer diagnosis at the molecular level.
  • Potential for family screening for hereditary predisposition to cancer and for cancer prevention.

Personalised Cancer Treatment

Based on molecular profiling of cancer, it is possible to have an individualised knowledge of pathways of cancer and dysregulated genes in an individual and accordingly suggest tailor-made personalised therapy.

• Our current knowledge of molecular pathways and advances in technologies have revolutionised the field of cancer biology.
• These developments are already making steady progress towards their applications in individual tumours as ‘personalised genomics’ and for ‘precision medicine’ for individualised treatment.

Pathologic Diagnosis of Tumours:

Tissue diagnosis of a biopsy or excised specimen by histologic examination is of paramount importance. It includes a conventional paraffin-embedding technique and a rapid intraoperative frozen section method.

• Besides routine H & E staining, paraffin-embedded sections can be stained with special stains to demonstrate some cytoplasmic constituents.
• Immunohistochemical stains are employed as antibodies against cellular constituents acting as antigens which may be the cell membrane, nucleus or the cytoplasm.
• IHC stains help in the identification of the cell of origin of the tumour of uncertain histogenesis, as prognostic markers for tumours and for confirmation of microbial organisms.
• Serum tumour markers are biochemical assays of certain products elaborated by cancers which may help in prognostication of the case, for example, CEA, AFP, hCG, CA-125 etc.
• Besides, a few other modern ancillary techniques which have become available in diagnostic pathology are flow cytometry, in situ hybridisation, image analysers, cell proliferation analysis, molecular studies (for example, PCR) and DNA microarrays for molecular profiling of tumours.
• Major applications of molecular techniques are in the detection of minimum residual disease, in genome-wide mutational analysis, and suggest personalised cancer treatment