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Somatic genomic tests continued

We explore another more somatic genomic tests that are important to know about in the final instalment of this two-part step.
© National Genomics Education, NHS England

In the previous step, we identified some sequencing-based tests that are particularly important to know about. Now, let’s look at some that don’t use sequencing.

Fluorescence in situ hybridization (FISH)

DNA sequence analysis is not an ideal first-line test for analysing chromosome structural anomalies at the current time. For cytogenetic analysis, we can use another technology called fluorescence in situ hybridisation, or FISH.
FISH uses molecular DNA probes that detect complementary sequences along a chromosome. The DNA specific probes are tagged with a coloured fluorochrome allowing them to be visualised using a fluorescence microscope. These probes can provide information on both the copy number of a chromosome region, whether a region is deleted or duplicated, but also the location or position of that region to look for structural rearrangements within the genome. FISH can therefore provide information about gene overexpression, identify rearrangements known to lead to fusion proteins, and other information about genomic variants contributing to oncogenesis. Figure 1 shows an example of FISH being used in non-small cell lung carcinoma.
Two images: A and B, each containing red and green probes. Image B shows more red probes than green probes.
Figure 1: Non-small cell lung cancer tissue with chromosomes stained to assess CCDC6 gene (green probe) and RET gene (red probe) location and position. Image A shows equal amounts of each probe (green and red). Image B shows more red than green probes (see arrow), indicating that there has been an increase in copy number of the RET gene, resulting in increased expression of the gene.

Molecular tumour profiling

As well as looking at the genomic DNA, it can also be helpful to look at the expression profiles within a cell – the transcripts that are over or under expressed by the genes. This can provide helpful diagnostic or prognostic information in certain settings and can be used to risk stratify patients, allowing for personalised treatment. For example, RNA expression assays are used as a tool to profile breast cancers. In early breast cancer this can help to identify those who may benefit from adjuvant chemotherapy, and spare those who will not.
There are currently several commercial gene expression assays available that analyse the expression of sets of genes as a surrogate marker for metastatic potential, and identify tumours with more aggressive biology. All of the assays currently in routine use analyse RNA extracted from formalin-fixed, paraffin-embedded (FFPE) breast tumour samples.

Homologous recombination deficiency (HRD) test

Another important commercial assay available for use is the homologous recombination deficiency, or HRD test. Homologous recombination is a DNA repair mechanism for double-stranded breaks. If this mechanism is impaired, this leads to increased DNA errors, genomic instability and carcinogenesis. The HRD test looks at the presence of variants in the BRCA1 and BRCA2 genes, which play a key role in homologous recombination, and other features of genomic instability, including loss of heterozygosity, telomeric allelic imbalance and large-scale state transitions. Patients with tumours that are HRD-positive, regardless of BRCA status, may derive benefit from PARP inhibitor therapy, as per the PAOLA-1 study. This test is particularly relevant in high-grade serous ovarian carcinoma.
There is ongoing research looking at HRD testing and its clinical utility in other cancer types, potentially widening the use of PARP inhibitor therapy in other cancers.

Immunohistochemistry (IHC)

In addition to looking at genomic DNA and RNA expression, we can also look at protein expression within tumours.
Immunohistochemistry, or IHC, is a diagnostic test that detects protein expression within tumour tissue, which may provide evidence towards certain genes driving oncogenesis. In IHC, pathologists use different antibodies to look at the expression of different proteins in order to characterise the type of cancer.
IHC tells us whether the protein is expressed within the tumour, but not if the genetic change that led to loss of expression was somatically acquired (and is therefore present in the tumour only) or if it is present constitutionally. Therefore, further genomic testing of both the somatic and the constitutional DNA may be required to clarify.
For example, in colorectal cancer, IHC Is used to identify loss of expression of mismatch repair (MMR) proteins, as seen in figure 2. Absence of MMR proteins suggests that the gene which would normally produce that protein may be non-functional.
Figure 2: Expression of four mismatch repair proteins in a colorectal cancer. At the top panel of this image, we can see loss of expression of the MLH1 and PMS2 proteins, which is shown as reduced brown staining. On the bottom panel, we can see normal expression of the MSH2 and MSH6 proteins. This is suggestive of a defective mismatch repair system in this cancer.
When tumours are MMR-deficient, constitutional MMR testing may be indicated depending on the pattern of loss and results of any additional somatic testing undertaken. Further testing would be undertaken in the tumour, however, to look for acquired mechanisms for this, with constitutional testing only being required if no somatic mechanism was identified.

Microsatellite instability analysis (MSI)

Another technique to identify MMR deficiency is microsatellite analysis. Microsatellites are short, repeating sections of DNA, which can expand and vary in length. In MMR deficiency, errors in DNA base pairing are not routinely corrected and can lead to this variation in the number of repeats occurring within the cell, known as microsatellite instability.

MSI testing is used to examine the repeat sizes of select microsatellite markers using massively parallel sequencing technologies. Tumours may be identified as MSI high, indicating instability across multiple markers, or MSI low, indicating instability at one marker only. More commonly, you may hear the term MSI-S, which stands for MSI stable and is often used interchangeably with MSI-L. An MSI-H result is important as it is the molecular fingerprint of an MMR-deficient tumour.

Testing MMR-deficient tumours

There is international data which suggests that immunotherapy may be of benefit in a tumour-agnostic fashion in MMR-deficient tumours. However, in clinical practice, which tumours would be tested and subsequently treated with immunotherapy will also depend on other factors, such as regulatory approvals and national guidelines. When an MMR-deficient tumour is identified, a family history should be taken, and constitutional MMR gene testing should be considered to identify potential Lynch syndrome. This depends on the pattern of loss and results of any additional somatic testing required.

What have we learned?

In summary, the most appropriate test for a cancer patient will depend on the cancer type, the type of variants known to cause oncogenesis in that tumour type, and the most efficient way of detecting this, and the clinical information we wish to identify, whether that is related to diagnosis, prognosis, or therapeutic intent. These steps haven’t covered all possible tumour tests relevant for clinical management. Additional tests include MLH1 promoter hypermethylation, microarray and multiplex ligation-dependent probe amplification (MLPA). You can find out more about these on the GeNotes Knowledge Hub.

The National Genomic Test Directory for cancer will enable you to identify the different tests which are available for each cancer type, as well as the technologies used to identify the relevant underlying genomic variation or molecular mechanism. We’ll be learning more about the test directory later this week, including how to navigate it.

Remember, your local laboratory can also support you in choosing the right test for your patient, so reach out to them if you need support.

Talking point

  • Have you ever encountered a situation in which you had to weigh the advantages and disadvantages of different somatic genomic tests when requesting one for a patient? What helped you to decide?
© National Genomics Education, NHS England
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Genomics in the NHS: A Clinician's Guide to Genomic Testing for Cancer (Solid Tumours)

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