Chapter 11: Tissue and Molecular Diagnosis

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. Immunohistochemistry is conventionally separate from this category.

Basic methods in diagnostic molecular pathology

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). It is worth noting that real-time PCR (RTPCR) is a different method, typically used for quantifi cation, 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 numerous applications in oncology (see Detection of clinically relevant abnormalities in genes on page 193 of the textbook), including mutational analysis (Figure 11.29 on page 192 of textbook and Figure S11.1 below), testing for clonality (Figure S11.2), detection of fusion transcripts resulting from cytogenetic changes, 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.

Genomic changes in tumours

In normal circumstances, there is precise control of the division 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 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. Activation of proto-oncogenes by genetic changes may induce or accelerate cell proliferation, while inhibition of tumour suppressor 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.

Abnormalities occur at various points during tumorigenesis. The classical model for this process is the ‘adenoma-carcinoma sequence’ of Fearon and Vogelstein, whereby the accumulation of mutations such as APC, KRAS and TP53 in the colorectal mucosa corresponds broadly to the transformation of non-neoplastic mucosa into a colorectal adenoma and subsequently a carcinoma. Current models show that the picture is often very complex and differs 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 copy number changes. Point mutations are single changes in the sequence of nucleotides in DNA and can be germline, i.e. inherited from a parent and accordingly present in every cell in the body, or somatic, i.e. acquired at some point during life and affecting only the tumour cells. Deletions and insertions (indels) of nucleotides result in a frameshift mutation. Examples include TP53 tumour suppressor 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 immunoglobulin 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 replication, chromosomal structure 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 (Figure S11.1) are among the most common. Genetic changes can disrupt various pathways, 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, RB), 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 microsatellites, resulting in MSI. Tumours with this characteristic 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, from sporadic methylation of the MLH1 gene (see Mismatch repair gene abnormalities in tumours on page 195 of the textbook).