Molecular Diagnostics

Molecular testing for DNA repair gene alterations

Molecular testing for pathogenic alterations in DNA damage response (DDR) genes is not yet routine practice in the clinic for prostate cancer. There is increasing evidence that pathogenic DDR alterations can predict personal risk of developing secondary cancers, and familial risk of cancers such as prostate, urothelial, pancreas, small bowel, gastric, breast and ovarian cancer^1–8. There is also growing evidence that pathogenic DDR alterations could serve as predictive biomarkers for prostate cancer responses to some investigative treatments^9–11.

Dr Alexander Wyatt of the Vancouver Prostate Centre points out why molecular testing is important in prostate cancer.

The V2.2020 NCCN Clinical Practice Guidelines In Oncology (NCCN Guidelines^®) for Prostate Cancer already recommends molecular testing for pathogenic germline mutations in DNA damage repair genes for prostate cancer patients with high-risk, very high-risk, regional or metastatic prostate cancer or a family history of cancer¹². These genes include BRCA2, BRCA1, ATM, PALB2, and CHEK2 of the homologous recombination repair (HRR) pathway and MLH1, MSH2, MSH6, and PMS2 of the mismatch repair pathway (MMR)¹². Furthermore, the V2.2020 NCCN Guidelines^® for Prostate Cancer suggests to consider multigene molecular testing of tumours in patients with low, intermediate and high-risk prostate cancer, and a life expectancy of 10 years or more¹². It also recommends tumour testing for HRR gene mutations in patients with metastatic prostate cancer, and suggests to consider tumour testing for MSI or defective MMR in these patients¹². Tumour testing for HRR gene mutations, and MSI or defective MMR should also be considered in patients with regional disease¹². It is therefore likely that molecular testing will soon become routine practice in the clinic.

The ESMO Clinical Practice Guidelines for Prostate Cancer (2020) also recommends germline testing for BRCA2 and other DNA damage response (DDR) genes associated with predisposition syndromes in prostate cancer patients who have a family history of cancer¹³. It also suggests to consider germline testing for these genes in patients with metastatic prostate cancer¹³. In terms of tumour testing, the ESMO Guidelines suggest to consider tumour testing for homologous recombination repair genes and mismatch repair defects in patients with metastatic castration-resistant prostate cancer (mCRPC)¹³.

The EAU guidelines (2020) do not currently provide any recommendations on molecular testing for DNA repair gene alterations in prostate cancer; however, it recommends offering early PSA testing to men who have an elevated risk of prostate cancer, including men >40 years old who carry BRCA2 mutations¹⁴.

Dr Alexander Wyatt of the Vancouver Prostate Centre comments on how well molecular testing guidelines are being adopted in prostate cancer clinical practice.

Germline mutation
A genetic mutation that is inherited from a parent and is found in all cells¹⁵ 

Somatic mutation
A mutation that arises spontaneously and is found in tumour cells¹⁵

Currently, international and local guidelines are limited in their inclusion of molecular diagnostics for assessing DDR alterations in prostate cancer. However, molecular diagnostics that assess DDR gene alteration using techniques such as next generation sequencing (NGS) are already widely available and used in clinical laboratories¹⁶. This is because BRCA1 and BRCA2 mutations already identified in breast and ovarian cancer patients serve as clinical markers for familial and personal cancer risk, and as predictive biomarkers for treatment responses to PARP inhibitors, targeted therapy options approved for the treatment of breast and ovarian cancer^17,18. These tests vary in their capabilities. Germline testing aims to identify heritable pathologic mutations in genes of interest that provide information on personal and familial risk of cancer¹⁶. Tumour testing identifies both germline and somatic mutations in tumour-derived samples and is better suited to identify predictive markers of treatment response¹⁶.

Dr Alexander Wyatt, a prostate cancer genomics expert at the Vancouver Prostate Centre explains why molecular testing is important, and how it will affect clinical practice for oncologists and urologists.

It is likely that existing molecular diagnostic tests and companion diagnostics will be used and modified to assess mutations in prostate cancer patients¹⁹.

For more information, please visit our pages on HRR and MMR and on the clinical impact of DDR mutations in prostate cancer.

Germline mutations versus somatic mutations

Up to 27% of metastatic castration-resistant prostate cancer (mCRPC) patients have a germline or somatic mutation in DNA damage response genes²⁰. Approximately half of these patients (or 12–15% of all mCRPC patients) have germline (heritable) mutations in these genes (Figure 1)^21,22.

Germline mutations

A germline (heritable) mutation is a mutation that is inherited from one or both parents and is subsequently present in all cells of the body, including germ cells¹⁵. If a patient has a pathogenic germline mutation, it will be present in both normal and tumour cells and they may pass it on to their own children.

Somatic mutations

A somatic mutation is a mutation that arises spontaneously in the body’s cells and cannot be passed on to offspring¹⁵. Tumour-associated somatic mutations often arise due to high levels of tumour-related genome instability and are therefore usually detectable in tumour tissue, but not in normal cells²³.

Figure 1. Pathways for germline and somatic BRCA mutations in a patient. Adapted from Knudson, 2001; Simon & Zhang, 2007^24,25. Most patients who inherit a germline BRCA mutation possess a single affected BRCA allele (monoallelic loss). A single pathogenic somatic mutation in the second functional allele is required to create biallelic loss of BRCA. For non-carriers, at least two pathogenic somatic mutations, one in each BRCA allele, is necessary to create biallelic loss of BRCA. Loss of function can also result from epigenetic and other non-genomic mechanisms²⁶. gBRCA2m, germline breast cancer gene mutation.

Germline testing versus tumour testing

Germline testing is used to identify germline mutations only, whereas tumour testing assesses any mutation (identifies both germline and somatic mutations) present within the tumour (Figure 2)¹⁶.

Figure 2. Patient samples used for molecular testing of germline and somatic mutations. Adapted from Cheng et al., 2019; Slavin et al., 2018^16,27. Germline testing is carried out on normal non-tumour samples such as DNA derived from lymphocytes in blood. Somatic mutations can be identified in tumour-derived samples such as tumour tissue or circulating tumour DNA (ctDNA) present in blood. Note that testing of tumour-derived samples may identify a mixture of germline and somatic mutations; however, it is not always easy to distinguish between the two alteration types.

Dr Alexander Wyatt, a research group leader on prostate cancer molecular diagnostics, introduces the three main testing methods for interrogating DNA repair gene alterations in prostate cancer and some of the challenges associated with each one.

Germline testing provides information that helps the clinician to understand personal and familial risk of cancer¹⁶. Though germline mutations are present in both normal and tumour tissue, only normal tissue is used to carry out germline testing. This is because tumour testing will also identify somatic mutations that are not easy to distinguish from germline mutations¹⁶. The most used normal (non-tumour) sample is whole blood, from which lymphocyte DNA is extracted for sequencing (whole blood testing) (Figure 2, Table 1). In cases where tumour testing is not possible, germline testing can give an indication of gene alterations present in the tumour based on the presence of germline mutations; however, it will not provide any information on somatic mutations present within the tumour.

Tumour testing provides information that helps the clinician to assess tumour features that may inform treatment decisions¹⁶. It is carried out on DNA extracted from tumour-derived samples that may carry both germline and somatic mutations (Figure 2, Table 1). The most commonly used sample for tumour testing is tumour tissue taken from either the primary or metastatic tumour site (tumour tissue testing)¹⁶. Another tumour-derived sample is circulating tumour DNA (plasma ctDNA testing) that is released into the blood stream from the tumour site and requires a simple blood draw for retrieval¹⁶. It is important to note that further germline testing is required to confirm that a tumour-associated mutation is indeed of somatic origin¹⁶. Table 1 summarises some of the advantages and disadvantages of the three types of testing.

Table 1. A summary of the advantages and disadvantages of different molecular testing approaches^16,28–31. ctDNA, circulating tumour DNA; DDR, DNA damage response

Whole blood testing

Whole blood testing is typically used to detect germline mutations and therefore is the technique used to assess personal and familial risk of cancer¹⁶. Sampling involves a simple blood draw from which normal lymphocytes are isolated for DNA extraction and sequencing. Note that the presence of a haematological malignancy may lead to the identification of a somatic mutation that is not of germline origin³². Patients with prior allogeneic bone marrow transplant may also receive a false germline test result³².

If tumour-derived samples are not available for tumour testing, whole blood testing can provide some indication of gene alterations (germline only) present within the tumour. When doing this, it is important to consider that approximately 50% of prostate cancers with a DNA repair gene alteration harbour somatic alterations only, meaning that an actionable alteration may be missed when using germline testing (Figure 3)²¹. This is true for both primary tumours (4.6% germline versus 10–19% tumour-associated) and metastatic prostate cancer (8–11.8% germline versus 23–27% tumour-associated), as well as for the BRCA2 gene, the most commonly altered DNA repair gene in prostate cancer (Figure 3)^{20,21,31–33}.

Figure 3. Frequency of germline and somatic DNA repair gene alterations in prostate cancer. Adapted from Abida et al., 2017²¹. Data represent alterations identified in archival and new biopsy samples from n = 50 (11%) locoregional, 53 (12%) biochemically recurrent and 348 (77%) metastatic samples. ATM, ataxia telangiectasia mutated; BRCA1/2, breast cancer gene 1/2; CHEK2, checkpoint kinase 2.

Since identification of a pathogenic germline mutation may have consequences on both a patient and their family, it is important to discuss the possible outcomes of germline testing prior to gaining patient consent to test.

Tumour tissue testing

Tumour tissue testing is the gold standard test for identifying tumour-associated alterations in prostate cancer³⁰. Since this test will identify both germline and somatic alterations present within the tumour, it is the typically preferred test to inform on actionable alterations present within the tumour. This feature is particularly relevant in prostate cancer since approximately 50% of prostate tumours with a DNA repair gene alteration harbour only somatic mutations²¹. Furthermore, the identification of a pathogenic mutation from tumour tissue testing should trigger counselling and dedicated germline testing if a germline mutation is suspected, to better understand the origin of the identified alteration (germline or somatic)^12,13. Tumour testing can therefore indirectly identify alterations that are potentially associated with personal and familial risk of cancer.

Tumour tissue testing is a well validated and sensitive test carried out on stored tumour tissue samples retrieved primarily through diagnostic biopsy or from surgical procedures (for example, formalin-fixed paraffin-embedded [FFPE] primary tumour tissue cores). It therefore does not typically require additional invasive sample collection. In the absence of a diagnostic or resected sample, the decision may be taken to collect a new tissue sample using invasive techniques, which may be challenging in advanced prostate cancer patients who lack soft tissue metastases, such as those with bone metastases²⁸. When using stored tumour tissue samples, it is important to note that the age of the sample may impact on the quality of the DNA extracted from the sample, especially if it has been exposed to air (oxidation damage)^36,37. Additionally, somatic mutations acquired during disease progression may be missed when assessing early diagnostic samples for patients who have progressed. However, this is less likely to impact on the identification of DNA repair gene alterations as they tend to be early events that are also present in primary tissue³⁸.

Dr Alexander Wyatt recommends carrying out early requests for stored prostate tumour samples, not exposing samples to air and to involve pathologists early on to assess sample suitability (quality and quantity) for molecular testing

In this video clip, Dr Alexander Wyatt, a research group leader on prostate cancer genomics and molecular diagnostics, gives advice on how to improve molecular testing success rates for prostate cancer.

Limitations for prostate tumour tissue testing can include poor tumour tissue quality and problems encountered during sample processing^37,39,40. Spatial inter- and intra-tumour heterogeneity, variance in tumour content (tumour versus stroma material) and inaccuracies in biopsy retrieval can impact on both the quantity and quality of tumour tissue acquired^{16,29,39,41,42}. Issues encountered during sample processing include technical difficulties in DNA extraction, for instance on samples with poor fixation or for bone metastasis samples from metastatic prostate cancer that require an additional decalcification step^28,37.

Plasma ctDNA testing

Plasma circulating tumour DNA (ctDNA) testing is a new technique that is currently under development in prostate cancer and not yet routinely available. ctDNA is DNA that has been ‘shed’ into the bloodstream from dying tumour cells (Figure 4)⁴³. Putative ctDNA is identified by the presence of somatic alterations in the total cell-free DNA (cfDNA) pool that is derived from both dying tumour and normal cells. The ratio of ctDNA to cfDNA (ctDNA/cfDNA fraction) is relatively high in metastatic castrate-resistant prostate cancer (mCRPC), with approximately 60–70% of progressing mCRPC patients displaying an abundance of ctDNA^30,44. Despite some patient-to-patient variability, patient tumours with high proliferation, visceral spread and high tumour volume tend to have the highest ctDNA fractions^31,44.

Figure 4. The origin of circulating tumour DNA (ctDNA). Adapted from NIH, accessed 14 June 2020⁴³. ctDNA is released into the bloodstream from apoptotic tumour cells and is identifiable in plasma prepared from a blood draw. Tumour testing assesses tumour tissue whereas whole blood testing analyses lymphocytes enriched from a blood draw.

Retrieval of ctDNA from a patient involves plasma preparation from a blood sample and is therefore a minimally invasive alternative to a tumour tissue biopsy (Figure 4)^29,30. Plasma ctDNA testing may therefore be useful in the future for prostate cancer patients who have advanced disease that is too difficult to biopsy. Indeed, a recent study on mCRPC samples identified high concordance between genomic alterations identified in tumour tissue and those identified from liquid biopsies (Figure 5)³⁰.

Figure 5. High concordance for gene alterations identified in mCRPC by ctDNA testing (liquid biopsy) and tumour tissue testing (solid biopsy). Adapted from Wyatt et al., 2017³⁰. AF, allele frequency. 

One major challenge for plasma ctDNA testing relates to the variability in shedding of DNA into the bloodstream. The rate of shedding of ctDNA is dependent on tumour location, tumour size and tumour vascularity, features that may vary between patients, disease sites and disease states. For instance, ctDNA levels are generally undetectable in patients who have localised prostate cancer; however, is generally detectable for advanced prostate cancer⁴⁵. ctDNA levels may also be reduced to below threshold levels in patients who are responding to treatments³⁹. For tumours with very low rates of shedding or a low tumour burden, ctDNA may account for as little as 0.01% of total cell-free DNA²⁹. In such cases, liquid biopsies may not provide enough ctDNA material for sequencing and can lead to the recording of false negative results.

ctDNA thresholds for identifying different alteration types also vary. Dependent on the technology used to sequencing, a ctDNA level above 0.1% will usually allow for the detection of a mutation, whereas higher ctDNA levels are required to detect amplifications and deletions^30,44. Since dying cells release fragments of DNA, ctDNA analysis is additionally not able to identify large scale rearrangements. This is also important when deciding whether the results attained are exhaustive, or likely due to insufficient ctDNA (false negative) which may be an issue in approximately 30–40% patients who present with low ctDNA levels^30,44.

It is important to note that plasma ctDNA testing is still being explored as a tool to identify DNA repair gene alterations in prostate cancer; however, it may in the future be a suitable alternative to tumour tissue testing in cases where tumour tissue sample is unavailable, or of low quality. For now, tumour tissue testing provides a more sensitive and accurate readout when compared to plasma ctDNA testing, particularly in patients with early stage or low-volume disease.

Genetic counselling

The aim of molecular testing is to identify pathogenic mutations that may positively impact on risk monitoring and guide treatment decisions. Whilst local guidelines vary in implementation, genetic counsellors play an important role as part of a multi-disciplinary team in managing cancer patients who require germline testing (Figure 6)¹⁶.

Figure 6. Prostate cancer patient management requires a multi-disciplinary approach. Adapted from Murphy et al., 2019⁴⁶.

Identification of a pathogenic germline mutation may have consequences on both a patient and their family. It is therefore important to discuss the possible outcomes of germline testing with a patient prior to testing and before the patient consents to testing. This is most often done through a trained genetic counsellor who explains the medical, psychological, legal and ethical consequences of genetic testing to the patient^16,47.

Typical discussion points between the genetic counsellor and the patient include¹⁶:

• The impact of identifying a pathogenic germline mutation on personal and family risk of cancer
• Cascade testing for family members if a pathogenic germline mutation is identified
• The potential impact on treatment decisions and symptom monitoring
• The possibility of identifying a variant of unknown significance (VUS)

Typically, a patient is referred for genetic counselling when¹⁶:

• there is a family history of cancer
• family cascade testing is needed
• when tumour tissue testing uncovers a pathogenic mutation that is known or suspected to be germline in nature
• when the patient is distressed or has unanswered questions.

Dr Alexander Wyatt of the Vancouver Prostate Centre highlights some of the challenges of implementing cascade testing for family members of prostate cancer patients.

It is becoming clear that emerging genetic risk markers for various cancer types will impact on the availability of trained genetic counsellors. It is expected that at least some of the roles of a genetic counsellor will be integrated into the normal practice of oncologists and urologists, a method that has been tested and proven to be successful in managing breast and ovarian cancer patients⁴⁷. 

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