Cancer

Question 1

Pharmacogenetics

Answer 1

Pharmacogenetics is the study of genetic causes of individual variations in drug response. Pharmacogenetics represents a study of individual gene-drug interactions, usually one or two genes that have dominant effect on a drug response (SIMPLE relationship).

Question 2

Pharmacogenomics

Answer 2

Pharmacogenomics is genome wide analysis of genetic determinants of drug efficacy and toxicity. Pharmacogenomics represents a study of genomic influence on drug response, often using high-throughput data - sequencing, SNP array, expression, proteomics (COMPLEX interactions).

Question 3

A patient’s response to a drug may depend on:

Answer 3

• Pharmacokinetic factors • Pharmacodynamic factors • Non-pharmacogenomic factors

Question 4

Pharmacokinetic factors

Answer 4

• Absorption - Distribution - Metabolism - Elimination

Question 5

Pharmacodynamic factors

Answer 5

• Target proteins - Downstream messengers

Question 6

Non-pharmacogenomic factors

Answer 6

• Age and gender - Lifestyle habits, such as smoking and drinking alcohol - Other diseases - Medications taken for other conditions

Question 7

Financial implications of adverse drug reactions in UK

Answer 7

The health and financial implications of ADRs are significant: about 6–7% of hospital admissions in the UK are due to ADRs. ADR cost a minimum of £98.5 million (1 billion) every year

Question 8

Health implications of adverse drug reactions in UK

Answer 8

About 6–7% of hospital admissions in the UK are due to ADRs. ADRs are thought to occur in 10–20% of hospital in-patients and over 2% of people admitted with an ADR died. Primary care 25% develop ADR. Avoidable adverse drug reactions (ADR) cause 712 deaths per year The most common ADRs were mental and behavioural disorders due to drugs and the drugs most commonly implicated in ADRs were systemic agents such as anticancer drugs and immunosuppressants

Question 9

Benefits of Pharmacogenetics

Answer 9

• Safer dosing options – right dose, right time, right patient • Avoid drug toxicity and adverse side effects • Ensure maximal efficacy • Improve drug development/choices • Explain variable response to drugs • Reduce Healthcare costs

Question 10

Pharmacogenetics examples

Answer 10

Rheumatoid Arthritis - Etanercept (Enbrel®) is a TNFα inhibitor, an immune modulator often used in combination with methotrexate.Polymorphisms in TNFα modulate drug response. Neonatal Diabetes (NDM) - caused by activating mutations in KCNJ11 or ABCC8 genes. Mutated K-ATP channel is insensitive to ATP levels and cannot release insulin in response to hyperglycaemia. Most patients with K-ATP mutations respond to sulphonylurea. Sulphonylurea is metabolised by the cytochrome P450 CYP2C9 enzyme. Polymorphisms in this enzyme can result in slow or rapid metabolism of the drug. Warfarin, CYP2C9 and VKORC1

Question 11

Warfarin - PGx

Answer 11

• Warfarin - the most common oral anti-coagulant in the world used in patients who are at risk of embolism or thrombosis. • Acts by reducing vitamin K availability (required for activation of clotting factors II, VII, IX and X). • During activation vitamin K is converted to an inactive form • During these reactions, vitamin K is converted to the inactive vitamin K epoxide, and this is recycled by the action of vitamin K epoxide reductase (VKOR). Warfarin inhibits VKOR. • Risks associated with incorrect Warfarin dosage include excessive bleeding; can be reversed by administering vitamin K, or in cases of severe bleeding a prothrombin complex concentrate. CYP2C9 and VKORC1 SNPs contribute 15% and 25% to variation in warfarin response. CYP2C9*2 and CYP2C9*3 confer a reduced rate of warfarin metabolism and a higher risk of bleeding complications. VKORC1 SNPs (1173C>T and -1639G>A) are associated with increased warfarin sensitivity and require a reduced maintenance dose.

Question 12

Pharmacogenetics in Cancer - examples

Answer 12

The application of pharmacogenetics in oncology is of a great significance because of the narrow therapeutic index of chemotherapeutic drugs and the risk for life-threatening adverse effects.

Genetic polymorphisms can be found in the genes that code for the metabolic enzymes and cellular targets for most of the chemotherapy drugs.

5-Fluorouracil - uracil analog - DPYD

Irinotecan - a topoisomerase I inhibitor - UGT1A1

Platinum agents - cisplatin, carboplatin and oxaliplatin - inhibiting cell replication as a result of formation of DNA adducts - Glutathione-S-transferases (GSTs), catalyze the conjugation of GSH to platinum agents, forming less toxic and more water-soluble conjugates.

Question 13

Pharmacogenetics in Cancer - challenges

Answer 13

Difficult to conduct studies in humans as chemotherapies are too toxic to give the healthy individuals

Need to screen large no. of patients to identify rarer mutations

Cancer patients are often treated with different combinations and dosages of drugs so it is hard to find large samples of patients who have been treated the same way.

Drug efficacy and toxicity are likely multigenic traits making it difficult to distinguish between functional driver mutations versus random, non-functional mutations.

Question 14

What is the fuction of EGFR and which cancers overexpress EGFR

Answer 14

The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is the transmembrane glycoprotein receptor and a member of the epidermal growth factor family (EGF-family) of extracellular protein ligands with essential roles in regulating cell proliferation, survival, differentiation and migration. EGFR plays a key role in controlling two major cellular signalling pathways, one stimulating cell proliferation and growth, and the other controlling the survival pathway.

EGFR gain of function mutations lead to constitutive activation of downstream signalling pathways, which is critical for tumour growth.

EGFR is frequently overexpressed in breast, lung, ovarian and brain tumours.

Question 15

Percentage of NSCLC ptients with EGFR mutation

Answer 15

Approximately 10-30% of NSCLC patients have an EGFR mutation; ~90% of EGFR-activating mutations are either short in-frame deletions involving a conserved Leu Arg Glu Ala (LREA) motif in exon 19 (45%) or a missense point mutation (Leu858Arg) in exon 21 (40%). Mutations are more frequent in Asian ethnicity, females > males, adenocarcinoma histology, and never smokers. Patients with EGFR mutations have a better prognosis compared to those who have wild-type tumours.

Question 16

Name a first-line small molecule tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) used in NSCLC

Answer 16

Gefitinib (Iressa) is a selective small molecule inhibitor of the epidermal growth factor receptor tyrosine kinase and is an effective treatment for patients with tumours with activating mutations of the EGFR tyrosine kinase domain regardless of line of therapy. It functions by blocking the ATP binding site.

Gefitinib is a treatment for non small cell lung cancer (NSCLC) that has spread into the surrounding tissues (locally advanced) or to other parts of the body. Gefitinib is also used in clinical trials for other cancer types.

Only available if patients test positive for an EGFR tyrosine kinase domain mutation (exons 18-21).

Question 17

What is difference between erlotinib and gefitinib?

Answer 17

Gefitinib has been approved only for EGFR mutation bearing patients regardless the line of treatment, while erlotinib (Tarceva) is also indicated in patients without EGFR mutation who undergo second- or third-line treatment.

Gefitinib is recommended as an option for the first-line treatment of people with locally advanced or metastatic non-small-cell lung cancer (NSCLC) if: they test positive for the epidermal growth factor receptor tyrosine kinase (EGFR-TK) mutation and the manufacturer provides gefitinib at the fixed price agreed under the patient access scheme.

Erlotinib is recommended as an option for treating locally advanced or metastatic non‑small‑cell lung cancer that has progressed in people who have had non‑targeted chemotherapy because of delayed confirmation that their tumour is epidermal growth factor receptor tyrosine kinase (EGFR‑TK) mutation‑positive

Question 18

What is the origin of breast cancer and risks?

Answer 18

Heterogeneous group of neoplasms the most common form originating from the epithelial cells lining the milk ducts.

Most common cancer among women in the UK (about 30% of new cancer cases in females).

Estimated lifetime risk for women in the UK is 1 in 8.

Female BC incidence has been increasing for many years in developed countries.

Risk factors: age, genetics and exposure to risk factors (including some possibly preventable lifestyle factors e.g. weight, alcohol consumption and the use of HRT).

Strongest risk factor is family history - individual risk increases with increasing number. of affected relatives and a decreasing age of diagnosis

15-20% of BC cases are familial i.e. there is significant family history. However, hereditary BC i.e. predisposition inherited in an autosomal dominant manner, only accounts for 5-10% of all breast cancer cases.

Question 19

What causes resistance to erlotinib and gefitinib?

Answer 19

Exon 20 mutations trigger EGFR activation, and typically confer resistance to erlotinib and gefitnib.

Despite the initial remarkable activity of targeted treatment in these patients, most present with disease progression during the first 12 to 24 (median 10) months. Secondary resistance mutations have proven to be the most frequent cause of acquired resistance. Among them, the EGFR mutation T790M in exon 20 seems to be the leading mutation responsible for that resistance. The finding of the p.T790M variant is most commonly associated with relapse following initial therapy with EGFR TKI, which is a known mechanism of resistance. If this variant is identified prior to TKI exposure, genetic counselling should be considered, because germline p.T790M is associated with familial lung cancer predisposition and additional testing is warranted.

The EGFR exon 20 insertions confer a lack of responsiveness to EGFR TKI therapy.

· Osimertinib (AZD9291), is a novel oral, potent and selective third-generation irreversible (EGFR TKI) inhibitor of both EGFRm(+) sensitizing and T790M variant-mediated resistance to EGFR inhibitors in lung cancer.

Question 20

Name a drug for pwtients with EGFRm(+) sensitizing and T790M variant-mediated resistance to EGFR inhibitors in lung cancer.

Answer 20

Osimertinib (AZD9291), is a novel oral, potent and selective third-generation irreversible (EGFR TKI) inhibitor of both EGFRm(+) sensitizing and T790M variant-mediated resistance to EGFR inhibitors in lung cancer.

Question 21

Function of KRAS and prognosis of patients with KRAS mutations?

Answer 21

KRAS (KRAS proto-oncogene), is a G-protein with intrinsic GTPase activity, and activating mutaitons result in unregulated signaling through the MAP/ERK pathway. The presence of a KRAS mutation is a prognostic predictor of poor survival when compared to KRAS mutation negative patients. Found in ~22% of NSCLC, with highest prevalence in smokers

Question 22

Can EGFR and KRAS mutations appear together in one tumour and significance?

Answer 22

KRAS and EFGR mutations are mutually exclusive and rarely, if ever, occur in the same tumour.

Owing to the low probability of overlapping targetable alterations, the presence of a known activating mutation in KRAS identifies patients who are unlikely to benefit from further molecular testing. Mutations in KRAS have been associated with reduced responsiveness to EGFR TKI therapy. An activating mutations confer a worse prognosis and is associated with poor survival.

Targeted therapy is not currently available for patients with KRAS mutations, although immune checkpoint inhibitors appear to be effective.

Question 23

Echinoderm microtubule associated protein-like 4 (EML4)–ALK oncogenic gene fusion

Answer 23

ALK rearrangements are enriched in younger and never smoker cohorts

~4%-6% of lung adenocarcinomas

The presence of an ALK rearrangement is associated with responsiveness to ALK TKIs, with recent studies demonstrating improved efficacy of alectinib over crizotinib as a 1st line therapy.

Most commonly occur independent of KRAS and EGFR mutations

Question 24

What is the most common fusion partner seen with ALK (Anapalastic Lymphoma Kinase, is a receptor tyrosine kinase)?

Answer 24

EML4-ALK fusion (including those who had progressed despite at least 1 prior line of therapy) showed response rates of ~50-60% to Crizotinib, a TKI.

Crizotinib is now recommended as an option for untreated ALK-positive advanced NSCLC in adults (if the company provides it with the discount agreed in the patient access scheme).

Question 25

Do patients develop resistance to ALK TKIs?

Answer 25

Most patients develop resistance to therapy via ALK kinase domain mutations (e.g. L1196M, F1174L, C1156Y), which usually develop 12 months from initiation of therapy. Given this resistance, novel 2nd generation ALK inhibitors are in development.

Question 26

How do we test for ALK NSCLC mutations?

Answer 26

FISH analysis is considered the gold standard for ALK NSCLC mutation testing. FFPE tumour tissue is currently tested by FISH using an ALK break-apart probe. RT-PCR can detect the EML4-ALK fusion transcripts if all appropriate primer sets are included. ALK over-expression can also be detected using IHC RNA-next generation sequencing - Fusion-agnostic strategy: Using this method, it’s possible to identify and characterize novel and known fusions agnostic of the fusion partner, using hybrid-capture strategy. Amplicon-based approach uses primer pairs to target a specific gene, or sequence, of interest. However, amplicon assays can be prone to dropouts when mutations occur at primer binding sites, yielding unreliable results and often requiring confirmatory testing.

Question 27

Frequency of NTRK gene fusions in lung cancer and significance

Answer 27

~0.2% of all lung adenocarcinomas (very uncommon) Numerous fusion partners identified; NTRK gene fusions encode tropomyosin receptor kinase (TRK) fusion proteins (e.g. TRKA, TRKB, TRKC) that act as oncogenic drivers for solid tumours—they do not typically overlap with other oncogenic drivers such as EGFR, ALK, or ROS1. DNA based NGS may not detect some NTRK1 and NTRK3 fusions; RNA-based NGS be considered to assess for fusions.

Question 28

Name other biomirkers in NSCLC (other than EGFR, KRAS, ALK, NTRK)

Answer 28

Other biomarkers in NSCLC include ROS1 rearrangements (associated with responsiveness to ROS1 TKIs), RET rearrangements (selpercatinib), MET exon 14 skipping (capmatinib), BRAF mutations including V600E (associated with responsiveness to combined therapy with oral inhibitors of BRAF and MEK such as dabrafenib plus trametinib), and PD-L1 (Programmed Death Ligand 1)

Question 29

What is PD-L1 and what treatment can be used for PD-LI positive tumours?

Answer 29

PD-L1, a current hot topic in oncology, is a co-regulatory molecule that can be expressed on tumour cells and inhibits T-cell mediated cell death; in the presence of PD-L1, T-cell activity is suppressed. Checkpoint inhibitor antibodies block the PD-1 and PD-L1 interaction, thereby improving the antitumor effects of endogenous T cells. IHC testing for PD-LI can be utilized to identify disease most likely to respond to a 1st line anti PD-1/PD-L1. Currently, the FDA-approved companion diagnostic for PD-L1 guides the utilization of pembrolizumab in patients with NSCLC.

Question 30

Name four majority aetiology/pathogenesis groups of CRC

Answer 30

Chromosomal Instability Pathway (most commonly associated with KRAS, BRAF, NRAS, and PIK3CA mutations) Sessile Serrated Pathway (most commonly associated with BRAF mutations) Microsatellite Instability Pathway (MMR protein deficient) Hereditary CRC (Lynch Syndrome associated germline variants).

Question 31

What proportion of patients have metastatic disease at the time of diagnosis?

Answer 31

At the time of diagnosis, an estimated 20–55% of people with CRC already have metastatic disease. In addition, of the people who have undergone surgery for early-stage colorectal cancer, approximately 50–60% will eventually develop metastatic disease, most commonly in the liver. All patients with metastatic CRC should have the tumour tissue typed for RAS (KRAS and NRAS) and BRAF mutations as a single test or as part of an NGS panel (NCCN Guidelines, 2020).

Question 32

What proportion of tumours are positive for KRAS activating mutations and what is the significance?

Answer 32

50-70% of metastatic CRC (i.e. 30-50% KRAS wild-type) with 85-90% occurring in codons 12 and 13 (but codons 61 and 146 are less common). ## Footnote Patients with metastatic CRC carrying activating KRAS mutations (exon 2,3,4) do not benefit from receiving targeted EGFR inhibition therapies (negative biomarker of response) and should not be treated with cetuximab or panitumumab.

Question 33

Name monclonal antibodies that block the human EGFR

Answer 33

Panitumumab and cetuximab are recombinant monoclonal antibodies that block the human EGFR, inhibiting the growth of tumours expressing EGFR. NICE recommend Cetuximab and panitumumab for previously untreated metastatic colorectal cancer) in combination with chemotherapy (f-fluorouracil, folinic acid and oxaliplatin (FOLFOX) or 5-fluorouracil, folinic acid and irinotecan (FOLFIRI)) both cetuximab and panitumumab are approved as 1st-line treatment for patients with metastatic (advanced) CRC in patients with ‘wild-type’ RAS (KRASor NRAS) tumours only.

Question 34

What is the proportion of BRAF and NRAS mutations in CRC and their significance?

Answer 34

combined account for 10% BRAF mutations are mutually exclusive in CRC with KRAS mutations. BRAF V600E mutation makes response to panitumumab or cetuximab highly unlikely unless given with a BRAF inhibitor.

Question 35

What are BRAF mutations associated with in CRC?

Answer 35

All BRAF mutations are associated with reduced response to anti-EGFR therapy. If found in association with microsatellite stable (MSS) or Microsatellite-instability Low (MSI-L CRC), BRAF mutations are associated with a poor prognosis (no impact on MSI-H tumors). Note - the presence of V600E BRAF mutations in CRC is apparently not predictive of response to BRAF inhibitors such as Vemurafenib as for malignant melanoma (see below). Preclinical CRC models demonstrate EGFR inhibition also required to overcome feedback resistance.

Question 36

What are NRAS mutations associated with in CRC?

Answer 36

Activating NRAS mutations occur in ~1-6% of CRC, with the majority in codons 12 and 13. NRAS mutations(exon 2, 3, 4) should not be treated with cetuximab or panitumumab, as these mutations are associated with a reduced response to EGFR blockade.

Question 37

What are PIK3a mutations associated with in CRC?

Answer 37

Activating mutations in PIK3CA occur in 10-30% of all CRC, and are most likely to occur in exons 1, 2, 9, and 20 (with 9 and 20 the most common). Currently remains unclear whether mutations are of prognostic significance but do note these are not mutually exclusive of KRAS or BRAF mutations. Currently, the clinical effect of PIK3CA mutations on response to anti-EGFR therapy has shown conflicting results.

Question 38

What is the incidence of Melanoma in UK?

Answer 38

Around 15,400 people are diagnosed with melanoma in the UK each year. The incidence of malignant melanoma in Britain has risen faster than any other common cancer. Over the last decade, the number of people diagnosed with melanoma in the UK has increased by almost half. Melanoma is the 5th most common cancer in the UK.

Question 39

What is the function of BRAF?

Answer 39

BRAF is a member of the Raf kinase family of growth signal transduction protein kinases, specifically, it is a serine threonine kinase. This protein plays a role in regulating the MAP kinase/ERKs signalling pathway, which affects cell division, differentiation, and secretion. It acts downstream of EGFR and KRAS, and mutations lead to unrestrained cell growth and proliferation

Question 40

Frequency od acquired BRAF mutations and tumour types

Answer 40

Acquired mutations of BRAF commonly occur in melanoma, colorectal and lung tumours. The mutations are activating, leading to uncontrolled signalling. A single activating mutation of the BRAF gene, V600E is most common (80-90%) which leads to constitutive activation of Raf, V600K is the next most common (up to 20% of cases), and V600R/M/D/G (account for 5%).

Question 41

Do BRAF mutations occur alongside KRAS mutations?

Answer 41

BRAF mutations rarely occur, if ever alongside KRAS mutations. As the effect of an activating mutation in either gene is similar, it is usually beneficial to test for both mutations simultaneously however, cascade testing is also in use.

Question 42

What is the most common BRAF mutation and it's significance?

Answer 42

The V600E mutation has been shown to make colorectal cancers resistant to antibody-based anti-EGFR therapies (cetuximab and panitumumab, as outline above). Patients with this mutation in their tumours are therefore considered unlikely to benefit from anti-EGFR therapies. However, cutaneous melanoma BRAF V600E mutations are associated with sensitivity to MEK inhibitors, but clinical trials have shown a combination of BRAF and MEK inhibitors are superior to either agent alone in patients with V600E.

Question 43

How do we test for BRAF in melanoma?

Answer 43

IHC may be used to screen for BRAF V600E, as an indirect test that detects the mutated protein with confirmatory molecular testing. Testing for the BRAF V600E mutation is usually by Real-Time PCR, pyrosequencing or using small multigene panels

Question 44

Name BRAF inhibitors in melanoma

Answer 44

Vemurafenib (http://guidance.nice.org.uk/TA269) and Dabrafenib are BRAF gene mutation inhibitors for melanoma patients. In BRAF V600E gene mutation +ve patients it resulted in tumour regression and increased survival varying by a few months and had complete and partial responses, however, although approved as a single agent therapy for metastatic or unresectable melanoma, these agents are almost never given without concomitant MEK inhibition therapy (NCCN Guidelines, 2020).

Question 45

Function of c-KIT (proto-oncogene c-KIT) mutations and significance in cancer

Answer 45

KIT is a receptor tyrosine kinase that promotes cell growth and proliferation. KIT mutations may occur in multiple “hotspots” across the gene and differ in the sensitivity to KIT inhibitors (e.g. imatinib, sunitinib, nilotinib). Imatinib is a selective TKI designed to target the BCR-ABL1 aberrant protein in patients with CML. Imatinib was later used to treat advanced stage GastroIntestinal Stromal Tumours (GIST) carrying activating mutations in KIT and PDGFRA.

Question 46

What is the frequency of NTRK fusions?

Answer 46

NTRK gene fusions have been estimated to occur in up to 1% of all solid tumours and are pathognomonic in certain rare paediatric and adult cancers (Amatu et al., 2019).

Question 47

How many partner fusions have been identified NTRK

Answer 47

To date over 80 different partner fusion genes have been identified in a wide range of tumours.

Question 48

Apart from solid tumours, what other cancer types are NTRK gene fusions detected in?

Answer 48

In addition to being present in solid tumours, NTRK gene fusions are also detected in acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia at a frequency of \<5%.

Question 49

What is the function of NTRK gene family?

Answer 49

The neurotrophic tyrosine kinase (NTRK) gene family is responsible for the normal development and function of both the central and peripheral nervous system, regulating neuronal growth, proliferation and survival as well as during embryonic development (Amatu et al., 2019). The genes NTRK1 (1q21-q22), NTRK2 (9q22.1) and NTRK3 (15q25), encode for the tropomyosin receptor kinase (TRK) proteins TRKA, TRKB, and TRKC. The fusion of the NTRK gene (3’ NTRK with 5’ fusion gene) can be expressed and result in ligand-independent activation promoting cell proliferation and survival and driving tumour growth via the TRK pathway.

Question 50

What is the therapy for tumours with NTRK fusions?

Answer 50

NICE recently published guidance in April and August 2020 recommending the TRK-inhibitors larotrectinib and entrectinib respecitively as treatment options for adult and paediatric patients with NTRK fusion-positive solid tumours. These drugs are histology-independent (tumour-agnostic) treatments that target the NTRK gene fusion, regardless of where the cancer originally started within the body. ## Footnote These TKIs are only recommended if all of the following conditions are met: There is a histologically proven diagnosis of a solid tumour (not including myeloma, leukaemia or lymphoma) The disease is locally advanced or metastatic or surgery could cause severe health problems They have not had an NTRK inhibitor before They have no satisfactory treatment options

Question 51

What is the The Cancer Research UK (CRUK) Stratified Medicine Programme?

Question 52

What is the CRUK Stratified Medicine Programme 1?

Answer 52

Pilot study to test the feasibility of running a genetic pre-screening programme within existing NHS infrastructure - July 2011 to July 2013) - demonstrate on a small scale how the NHS can provide molecular diagnosis with solid tumour types routinely. In phase one of the programme (in collaboration with Astra Zeneca, Pfizer, Illumina, and Innovate UK), clinicians in eight Experimental Cancer Medicine Centres (known as clinical hubs) recruited people with the following cancer types: breast, colorectal, lung, prostate, ovarian and melanoma. Patients undergoing cancer care at these hospitals were asked for permission to use surplus tissue taken during routine surgery. \>9000 samples in total (10,000 patients consented) were sent to one of three centralised, quality assured laboratories (known as technology hubs) where DNA was extracted, stored and tested for a set of specific genetic variations that had been selected based on previous research showing they would provide useful information

Question 53

CRUK benefits

Answer 53

Aside from successfully demonstrating the feasibility of such a programme, the pilot study yielded various other benefits through: Establishing a multiprofessional, collaborative network comprising representatives from the fields of oncology, histopathology, surgery, biomedical science, clinical informatics, molecular genetics and cytogenetics. Screening and recruitment of patients to international, commercially sponsored clinical studies. Contributing to enhanced bio-banking and informatics facilities at NHS sites through the use of electronic test requesting and reporting using structured XML (extensible mark-up language) format and transfer via a dedicated, secure FTP (file transfer protocol). Creating a tumour type-based external quality assurance scheme administered through UK NEQAS (National External Quality Assurance Scheme). Embedding targeted Next Generation Sequencing (NGS) technologies into clinical practice

Question 54

CRUK recommendetations

Answer 54

PATIENTS & CONSENT - Alleviating Patient Concerns - education campaign, Standardised research consent PATHOLOGY - National pathology standards * Optimum sample requirements * Tissue fixation and processing factors * Nomenclature and reporting terminology * Configuration of efficient workflows in NHS histopathology departments Guidelines on macrodissection - tissue macrodissection is required prior to mutation analysis. Communicating clinical results - Integration of molecular data with histopathological data, provided as a timely, comprehensive report for clinical decision making. DATA - Interoperability of data systems - Develop an integrated electronic patient record with full inter- operability between the systems in use in different clinical areas. Consistent data recording, Up-to-date coding systems, UK-wide data solutions, Modernisation of data exchange TESTING - Overcoming poor DNA quality, Clinically relevant turnaround times, Validating new technology, Improving interpretation, Improving patient treatment, Budgeting for the future, Maintaining quality

Question 55

What is the Stratified Medicine Programme 2?

Answer 55

Part two of the CRUK programme known as SMP2, has the goal of creating a national genetic pre-screening programme and advancing treatment for people with late stage non-small cell lung cancer (NSCLC). The focus for SMP2 is the development of a clinical trial, called the National Lung Matrix Trial, which is part of the next generation of flexible clinical trials, testing multiple drugs in multiple groups of patients. It is a collaboration between Cancer Research UK, the NHS and two drug companies—AstraZeneca and Pfizer. Biopsy samples will be sent to hubs for genetic analysis and then the results will be returned to the treating clinician, so that patients can be enrolled in the arm of the trial offering the drug that may best benefit them. The trial will include drugs in development from both companies. Screen up to 2,000 non-small cell lung cancer patients a year to identify the key genetic faults driving the growth of their cancer. Continue to pioneer the use of next-generation sequencing (NGS) technology in the NHS to prove large scale genetic testing works within the NHS. Use this information to match patients to the best treatment option from the multiple “arms” of The National Lung Matrix Trial.

Question 56

What is the trial design of Stratified Medicine Programme 2?

Answer 56

The Stratified Medicine Programme 2 is an observational pre-screening study led by Professor Peter Johnson. The pre-screening network is made up of all 18 Experimental Cancer Medicine Centres (ECMCs)(link is external), as well as selected feeder hospitals across the UK. Aims of the trial To determine which patients benefit most from treatments based on the genetic signature of their tumour, including measurements of tumour shrinkage and drug safety. To assess changes in amounts of ctDNA present in a patient’s blood before, during and after treatment to help identify which changes are linked to drug resistance.

Question 57

Key outcomes uf CRUK

Answer 57

Key outcomes Between July 2011 and July 2013, over 10,000 people with melanoma, breast, ovarian, lung, colorectal, and prostate cancer had their tumours tested. Over 8,000 patient records are now securely stored in a research database, with access for researchers. We made the switch from individual gene tests to NGS, to test multiple genes at the same time, saving time and money. The programme established a dedicated, nationwide network of hospitals and individuals experienced in providing genetic testing, laying the foundations for the Stratified Medicine Programme 2.​​

Question 58

Incidence of CRC

Answer 58

The incidence of Colorectal cancer (CRC) varies significantly among different populations. Worldwide ~85% of cases are considered sporadic ~15% are associated with an established familial genetic syndrome 3-5% Lynch Syndrome \<1% FAP, Juvenile polyposis syndrome, Peutz-Jegher syndrome, PTEN harmatoma tumour syndrome \>10% unspecified familial cancer

Question 59

What is Lynch Syndrome and incidence?

Answer 59

Previously known as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) Autosomal dominant cancer susceptibility syndrome Germline mutations in mismatch repair genes (MLH1, MSH2, MSH6 and PMS2 (also EPCAM, see below)) Followed by a secondary somatic loss of the remaining copy of the gene (loss of heterozygosity (LOH)) Shows gene dependant, age related penetrance with variable expressivity Predicted that total prevalence of Lynch syndrome is up to 1/250 (Insight) = most common form of predisposition to cancer Accounts for 3-5% of CRC

Question 60

What are the cancer types associated with Lynch Syndrome?

Answer 60

Cancer types according to gene: * MLH1 mutations – greater tendency to GI cancers * MSH2 mutations – associated with greater variety of cancers * MSH6 and PMS2 – associated with reduced age-related penetrance Cancer spectrum: * Colorectal cancer * Endometrial carcinoma * Small intestine carcinoma (MSH2 & MLH1) * Hepato-biliary tract and pancreas cancer (MSH2 & MLH1) * Gastric cancer (MSH2 & MLH1) * Ovarian non-serous cancer (MSH2 and MLH1) * Renal pelvis and ureter carcinoma (MSH2 & MSH6) * Bladder carcinoma (MSH2 & MSH6) * Sebaceous gland carcinoma (and adenoma – see Muir-Torre Syndrome) * Prostate cancer (MSH2) * Breast cancer (MLH1) * Brain cancer

Question 61

What is the mean age onset of Lynch syndrome?

Answer 61

Mean age of onset – 45 years N.B. Mean age-of-onset is higher in cases with MSH6 and especially PMS2 mutations and are frequently non-penetrant - many cases are missed by applying stringent family history and age-of-onset based selection criteria. Therefore, the mutation frequencies for these genes are likely to be much higher (recent evidence suggests similar frequency of MSH6 to MLH1 & MSH2)

Question 62

What is the mutation spectrum of Lynch Syndrome?

Answer 62

* 80-90% mutations are in MLH1 and MSH2 * 7-10% in MSH6 * Less than 1% in PMS1 and PMS2 * 3% due to deletions in the 3’ end of EPCAM gene, upstream of MSH2 creating EPCAM-MSH2 fusion transcripts resulting in epigenetic hypermethylation of the MSH2 promoter and loss of MSH2 expression * A 10Mb inversion on chromosome arm 2p, which disrupts MSH2 has been reported as a frequent cause of unexplained Lynch syndrome. * A LINE-1 mediated retrotranspositional insertion in PMS2 has been identified as a novel mutation type for Lynch syndrome. Not detectable by MLPA and direct Sanger sequencing on genomic DNA. * Germline methylation of the MLH1 promoter region has also been reported as a heritable cause of Lynch syndrome

Question 63

What are the clinical criteria applied to select patient for LS testing?

Answer 63

Amsterdam criteria * Developed to identify LS for research studies and to distinguish between HNPCC from non HNPCC patients Bethesda guidelines * Developed to identify patients with CRC who should be tested for LS * More sensitive but less specific than Amsterdam criteria * Helps those with smaller families

Question 64

What is the pre-sreen testing strategy for Lynch Syndrome?

Answer 64

Pre-screen on FFPE samples It is now routine practice to request tumour samples for LS for immunohistochemistry (IHC) and microsatellite instability (MSI) studies prior to mutation screening. The results from this analysis indicate whether mutation screening should be performed, and which gene(s) should be targeted. Tumour blocks/slides assessed by histopathology laboratories for the presence/absence of the MLH1, MSH2, MSH6 and PMS2 proteins using commercially available antibodies IHC is ~95% sensitive for DNA MMR deficiency Often concurrent loss of MSH2 and MSH6, or MLH1 and PMS2 is observed as these proteins form heterodimers in the MMR pathway If a sample shows loss of protein expression on IHC we DO NOT go on to test this patient for microsatellite instability (MSI) as we would expect these samples to show MSI. For patients with loss of MLH1/PMS2, DNA is from the tumour for MLH1 methylation studies prior to mutation screening Patients with normal IHC should undergo MSI testing as there may be a missense mutation present that causes a nonfunctional immunoreactive protein (this has been reported in approximately 5% of cases). This is a potential drawback to IHC

Question 65

What is Microsatellite instability (MSI)?

Answer 65

Genetic instability characterised by length alterations to microsatellites – occurs in the majority of Lynch associated cancers, also in a small proportion of sporadic cancers. MSI can be assayed in the lab by extracting DNA from tumour samples (slides or FFPE). Tests a panel of 5 (most commonly) quasi-monomorphic mononucleotide markers (Bat25, Bat26, Mono27, NR21 and NR24) to assess microsatellite instability Three outcomes: * Tumours with two or more altered mononucleotide markers are high-level MSI (MSI-H). * Microsatellite stable (MSS). Not supportive of a MMR gene defect * If only one marker shows instability, this is not considered sufficient to be classed as instability associated with LS but may be of significance and warrant further investigation.

Question 66

What are MLH1 promoter methylation studies?

Answer 66

Hypermethylation of the MLH1 promoter has been shown in a high proportion of sporadic cancers (approx. 15%). Results in absence of MLH1 protein on IHC. Therefore – any cases with MLH1 loss seen on IHC should have promoter methylation testing (MS-MLPA kit available). This kit detects abnormal methylation at 5 sites in the hMLH1 promoter, 4 sites in the MSH2 promoter (used to confirm 3’ EPCAM deletions) and other MMR promoter regions. Hypermethylation of the hMLH1 promoter in tumour samples is associated with sporadic cancer. Samples with no abnormal methylation should be tested for a germline MLH1 mutation Hypermethylation is a somatic change in tumour, but has very rarely been seen in blood DNA as a heritable germline mutation. Blood DNA can be tested for germline hypermethylation at the same time as the tumour (if available).

Question 67

What is the significance of BRAF mutation (V600E)?

Answer 67

BRAF mutations are associated with MLH1 methylation, and indicate sporadic cancer. BRAF is a proto=oncogene V600E mutation commonly occurs in non MSI-high tumours Used as screening method to avoid unnecessary MMR gene screening BUT a study (Parsons et al, 2012) have demonstrated that the BRAF V600E mutations also occurs in MMR germline mutation carriers at a frequency of 1%. Because of this finding they suggest that BRAF analysis should be done in combination with MLH1 promoter methylation analysis in tumours as this combination is a stronger indication that the tumour is sporadic. Constitutional MLH1 promoter hypermethylation is not associated with BRAF p.Val600Glu mutation

Question 68

What is the strategy for gerline MMR gene testing?

Answer 68

Gene panel approach is used in multiple labs for sequence and dosage analysis of MMR genes. PMS2 is challenging due to highly homologous pseudogene, PMS2CL. The 3’ end of the gene is non-amenable to NGS analysis and required long-range nested PCR to amplify PMS2 only. Large rearrangements can be detected by MLPA, NGS copy number analysis (depending on NGS method/ analysis pipeline) or targeted array.

Question 69

What is the traditional testing strategy for Lynch Syndrome?

Answer 69

Stepwise strategy: * Evaluation of tumour tissue for MSI through molecular MSI testing and/or IHC of the four MMR proteins. The presence of MSI in the tumour alone is not sufficient to diagnosis Lynch syndrome because 10%-15% of sporadic colorectal cancers exhibit MSI. IHC testing helps identify the MMR gene that most likely harbours a germline mutation. * Molecular genetic testing of the tumour for methylation and/or somatic BRAF mutations to help identify those tumours more likely to be sporadic than hereditary. * Molecular genetic testing of the MMR genes to identify a germline mutation when findings are consistent with Lynch syndrome.

Question 70

What did the recent review (February 2017, NICE guidance) of the accuracy and cost-effectiveness of strategies to identify Lynch syndrome in newly diagnosed early-onset CRC patients recommend?

Answer 70

It recommends reflex testing for LS in newly diagnosed CRC patients \<50 years is cost-effective. Cascade testing of relatives should be employed where appropriate. In future, this may increase to diagnoses \<70yrs. Offer testing to all people with CRC, when first diagnosed, using IHC for MMR proteins or MSI testing to identify tumours with deficient DNA mismatch repair, and to guide further sequential testing for Lynch syndrome If the MLH1 IHC result is abnormal, use sequential BRAF V600E and MLH1 promoter hypermethylation testing to differentiate sporadic and LS-associated colorectal cancers. If the MSH2, MSH6 or PMS2 immunohistochemistry results are abnormal, confirm LS by genetic testing of germline DNA. If the BRAF V600E test is negative, do an MLH1 promoter hypermethylation test. If the MLH1 promoter hypermethylation test is negative, confirm Lynch syndrome by genetic testing of germline DNA.

Question 71

What do homozygous or compound heterozygous mutations in the MMR genes cause?

Answer 71

Homozygous or compound heterozygous mutations in the MMR genes results in mismatch repair cancer syndrome (MMRCS) – a rare childhood cancer predisposition syndrome with 4 main tumour types; haematological malignancies, brain/central nervous system tumours, colorectal tumours and multiple intestinal polyps.

Question 72

What is the treatment for Lynvh Syndrome?

Answer 72

* Treatment of manifestations: For colon cancer, full colectomy with ileorectal anastomosis is recommended. * Prevention of primary manifestations: Prophylactic removal of the colon prior to the development of colon cancer is generally not recommended for individuals known to have Lynch syndrome because routine colonoscopy is an effective preventive measure. Prophylactic removal of the uterus and ovaries (prior to the development of cancer) can be considered after childbearing is complete. * Surveillance: Colonoscopy with removal of precancerous polyps every one to two years beginning between ages 20 and 25 years or ten years before the earliest age of diagnosis in the family, whichever is earlier. The efficacy of surveillance for cancer of the endometrium, ovary, stomach, duodenum, and urinary tract is unknown. * Chemoprevention: 2 controlled randomised trials have evaluated the efficacy of aspirin in high-risk CRC patients – CAPP1 and CAPP2. CaPP3 trial ongoing and recruiting patients (http://www.capp3.org/). Aspirin is now recommended in people at high risk of colorectal cancer.

Question 73

What is FAP and what is the incidence and inheritance?

Answer 73

FAMILIAL ADENOMATOUS POLYPOSIS (FAP) * FAP is the most common polyposis syndrome with an incidence of 1:8500 births. * APC gene (component of WNT signalling pathway) * Autosomal dominant * 10% cases are de novo * Mosaicism has been reported (not uncommon) * Characterised by the development of hundreds to thousands of adenomatous colonic polyps during the second decade of life (range 7-36 years). By age 35yrs, 95% of individuals with FAP have polyps. * Children and adolescents tend to be asymptomatic until the adenomas cause rectal bleeding or anaemia. Non-specific symptoms including change in bowel habits, abdominal pain and weight loss. * There is almost a 100% risk of CRC if not treated (colectomy) at an early stage, with CRCs tending to develop approximately one decade after the polyps appear. Colectomy is advised when \>20-30 adenomas or multiples adenomas with advanced histology have occurred. * With current screening procedures, majority of patients are diagnosed before the development of CRC. * Colonoscopy screening begins at 10-12years for individuals with FAP or an APC mutation or individuals at risk of FAP but no genetic testing; annual colonoscopy once polyps are detected until colectomy. * Attenuated FAP (AFAP), when patients develop anywhere from 0 to 100+ adenomas, can also be due to particular mutations in APC. A similar phenotype can be due to mutations in other genes, including MUTYH and POLD1/POLE.

Question 74

What is the function of the APC gene?

Answer 74

* APC is a tumour suppressor located at 5q21 and consists of 15 exons spanning 108,353bp. * Loss of APC protein function is a known early event in the CR adenoma to carcinoma sequence and is associated with chromosomal instablility. * Normally APC functions in the Wnt signalling pathway by regulating β-catenin degradation (via regulating the phosphorylation of β-catenin, which marks it for destruction by the proteasome). In the absence of a Wnt signal or presence of wtAPC, β-catenin is degraded. * In the absence of APC (e.g. due to truncation mutation) (or in the presence of Wnt), mutant APC is unable to bind β-catenin, leading to accumulation of β-catenin, which is then available to activate transcription factors resulting in target gene expression. The target genes change the proliferation and differentiation state of cells. E.g. c-myc is one of these target genes and its expression leads to the expression of the proto-oncogene ornithine decarboxylase (ODC).

Question 75

What is attenuated FAP?

Answer 75

* Milder form of FAP * Fewer adenomas (\<100, right sided predominance and flat morphology) * Later onset (polyposis ~40years) * Later diagnosis but significant increased risk for CRC (~70% risk by age 80yrs). * CHRPE and desmoids tumours are rare in AFAP individuals. * AFAP is likely underdiagnosed given the lower no of colonic polyps and lower CRC risk, and phenotypic overlap with MAP and LS. * Associated with specific APC mutations.

Question 76

What is GARDNER SYNDROME?

Answer 76

FAP (i.e. multiple colorectal adenomas) with extracolonic manifestations of osteomas, and soft tissue tumours (i.e. desmoids, fibromas and epidermoid cysts).

Question 77

What is TURCOT SYNDROME?

Answer 77

FAP variant - colorectal neoplasia and brain tumours Is not an independent clinical entity. Most Turcot syndrome patients are \<30 years of age at the time of presentation Usual presenting symptoms are those of a primary CNS tumour (often brain or rarely a spinal tumour). Cafe-au-lait patches, sebaceous cysts & basal cell carcinomas are some of the skin manifestations that can occur in Turcot patients.

Question 78

What is MUTYH-ASSOCIATED POLYPOSIS (MAP)?

Answer 78

MAP is the most recently recognised CRC and polyposis syndrome and the first with a recessive inheritance pattern. Heterozygotes have a slight increased risk of adenoma and cancer but screening is not warranted. * \>500 MAP patients have been reported with the majority showing a presentation similar to AFAP patients with oligopolyposis (between 10 and a few hundred polyps). * 60% of patients with polyposis present with CRC with an average age of diagnosis of 48 years. * MAP affected patients have a 43-100% lifetime risk of CRC. They also have an increased risk of duodenal adenomas (4% lifetime risk) as well as ovary, bladder and skin cancers. * MAP is though to account for approximately 0.5% to 1% of CRCs with a penetrance of 19% and 43% by the ages of 50 and 60 respectively. * MAP CRC has been shown to have improved survival over sporadic cases, possibly due to an increased immune response. * Extracolonic symptoms tend to be similar to those reported in FAP/AFAP. * Histological and molecular features of MAP overlap with MSI-high and Lynch syndrome CRCs. **N.B. MUTYH and APC are good examples of "similar phenotype, different mechanism".**

Question 79

What is the function of MUTYH?

Answer 79

MUTYH (1p34.1) is a base excision repair (BER) gene, 11kb in size consisting of 16 exons. Germline biallelic mutations in MUTYH predispose individuals to MAP. MUTYH encodes a DNA glycosylase enzyme involved in the most frequent form of oxidative DNA damage repair, excising adenine bases from the DNA backbone where inappropriately paired with guanine, cytosine, or 8-oxo-7,8-dihydroguanine. Bi-allelic mutations are detected in 28% and 14% of APC negative patients with 10-100 polyps and 100-1000 polyps respectively. Effects on glycosylase function of MUTYH are likely to be mutation dependent with functional assays reporting inconsistent results from reduced to complete loss of glycosylase function. Bi-allelic germline mutations in MUTYH result in G:C to T:A transversions and these somatic mutations frequently occur in APC, KRAS and BRCA1/2. 64% of MAP CRCs have a KRAS c.34G\>T mutation in codon 12.

Question 80

What is the testing strategy for MUTYH?

Answer 80

Individuals suitable for genetic testing include those with 10-100 polyps and no APC mutation. A FH compatible with recessive inheritance usual & can assist in the discrimination between FAP and MAP. As 99% of MUTYH mutations are missense, sequencing analysis is effective for screening. The presence of 1 mutation in an individual with AR inheritance may indicate the presence of a rare mutation elsewhere in the MUTYH gene but may also be coincidental; full MUTYH gene mutation analysis can be offered in these cases. Failure to detect 2 germline MUTYH mutations by seq analysis may mean that a rare deletion is present, or a mutation in another gene is responsible for the polyposis, or the polyposis is the result of non-hereditary factors. MLPA analysis is available. Carrier testing of family members can be offered upon identification of a familial mutation. Carrier testing may also be offered to the partner of an affected individual in order to assess genetic risk to any offspring. In this circumstance, it is reasonable to just test for the 2 common mutations. Note: MUTYH mutations are present in 1-2% of the population. There is evidence to suggest that MUTYH germline mutation carriers (heterozygotes) have an increased incidence of CRC compared with the general population. In reality – many patients with polyposis are tested for APC and MUTYH simultaneously on a gene panel.

Question 81

What are the other causes of polyposis and CRC?

Answer 81

HMPS – hereditary mixed polyposis syndrome (GREM1 duplication) POLD1 and POLE variants – proofreading associated polymerase Peutz Jeghers syndrome – specific Peutz Jeghers polyps, identified by histology, STK11 gene Juvenile polyposis syndrome (SMAD4, BMPR1A genes), polyps are juvenile (not the age of the patient) PTEN harmatoma tumour syndrome – can show polyps but rarely isolated, often with macrocephaly, dev del, breast and thyroid cancers NTHL1 (base excision repair gene)– homozygous variants cause familial adenomatous polyposis-3

Question 82

When is hereditary breast/ovarian cancer due to BRCA1 or BRCA2 pathogenic variants is suspected?

Answer 82

There is early onset of disease (\<50 years) Two or more breast primaries Breast and ovarian cancer in a single individual Breast and ovarian cancers in close (first- second- and third-degree) relatives(s) from the same side of the family At risk populations (e.g., Ashkenazi Jewish) Family member with a confirmed BRCA1 or BRCA2 pathogenic variant Male breast cancer Ovarian cancer at any age

Question 83

Why is clinical diagnosis of familial breast cancer complicated?

Answer 83

Clinical diagnosis complicated by incomplete penetrance, high prevalence of sporadic breast cancer and different individuals in the same family can develop different types of cancer (phenocopies).

Question 84

Who should be tested for BRCA1 and 2?

Answer 84

NICE guidelines 2018 recommends that BRCA1 and BRCA2 testing is offered to all individuals under age 50 with triple negative breast cancer regardless of family history.

Question 85

What is HBOC syndrome?

Answer 85

Heterozygous germline BRCA1 and BRCA2 pathogenic variants result in autosomal dominant hereditary breast and ovarian cancer (HBOC) syndrome HBOC syndrome associated with increased risk of early-onset breast cancer, ovarian (including fallopian tube and primary peritoneal cancers), pancreatic and prostate cancer and melanoma (primarily in individuals with a BRCA2 pathogenic variant). accounts for ~5-7% of all breast cancer cases incomplete penetrance; individuals have a lifetime risk of 38-87% of breast cancer, 16.5-63% for ovarian cancer The risk of other malignancies is 8.6-20% for prostate cancer, 1-7% for pancreatic cancer but these are still significantly increased compared to relative population risk (prostate - 20-fold, pancreatic – 10-fold)

Question 86

What is the function of BRCA1 and BRCA2 proteins?

Answer 86

Both have roles in DNA repair including homologous recombination repair of double-stranded DNA breaks and nucleotide excision repair: BRCA1 forms complexes with a protein called BARD1, and co-localises with BRCA2 and RAD51 at sites of DNA damage. BRCA2/RAD51 mediate homologous recombination. BRCA1 also has roles in interacting with several proteins involved in cellular pathways controlling cell cycle progression and check-point control, gene transcription regulation and ubiquitination Loss of function of BRCA1 results in defects in DNA repair, defects in transcription, abnormal centrosome duplication, defective G2/M cell cycle checkpoint regulation, impaired spindle checkpoint, and chromosome damage. BRCA2 protein has no recognizable protein motifs and no apparent relation to the BRCA1 protein BRCA2 is transcribed in late G1 phase and remains elevated in S phase, indicating a role in DNA synthesis BRCA1 is expressed in most tissues and cell types analyzed, suggesting that it is not the gene expression pattern that leads to the tissue-restricted phenotype of breast and ovarian cancer.

Question 87

What is triple-negative breast cancer?

Answer 87

Triple-negative breast cancer is cancer that tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein.

Question 88

What is Li-Fraumeni syndrome?

Answer 88

Li-Fraumeni syndrome (LFS) - autosomal dominant, characterised by various neoplasms including soft tissue sarcoma, osteosarcoma, pre-menopausal breast cancer, brain tumours, adrenocortical carcinoma (ACC). 70% cases caused by pathogenic variants in TP53 (regulates genes involved in processes such as cell cycle arrest, apoptosis and DNA repair when cells are under stress). TP53 pathogenic variants: 50-60% risk of BC by age 45 in women (male breast cancer not reported in LFS)

Question 89

Which diseases is ATM associated with?

Answer 89

Ataxia telangiectasia mutated (ATM) – biallelic pathogenic variants cause ataxia telangiectasia: cerebellar ataxia, telangiectases, immune defects, predisposition to malignancy. Heterozygous pathogenic variants are associated with a 17-52% risk of breast cancer (although some specific pathogenic ATM variants may cause an even higher female breast cancer risk)

Question 90

What is the risk of breast cancer in PALB2 positive patients?

Answer 90

Partner and localiser of BRCA2 (PALB2) - biallelic pathogenic variants give FANCN. Heterozygous pathogenic variants are associated with a 33-58% risk of breast cancer. Although initially considered to be a moderate risk gene, the increase in risk of BC in PALB2 positive patients is now considered comparable to the risk of BC in BRCA2 positive patients (approx. 45%). PALB2 pathogenic variants have also been linked to an increased risk of pancreatic cancer,

Question 91

What genes would you include on a breast cancer gene panel?

Answer 91

In the UK, the UK Cancer Genetics Group (UK-CGG) published a consensus panel for familial breast cancer (Taylor A. et al) and included genes with sufficient evidence for clinical utility. The panel comprises BRCA1, BRCA2, TP53, PALB2, PTEN, STK11, CHEK2\* and ATM\* (\*recommended to report truncating variants only plus ATM c.7217T\>G p.(Val2424Gly)). It is important to consider that finding a pathogenic variant in a moderate risk gene in the context of a high-risk family history may not account for all the risk in the family. Inclusion of low penetrance genes on a panel may lead to difficulties where the clinical consequences of a variant are not clear and no clinical decisions can be based on their presence or absence.

Question 92

What is the cause of Neurofibromatosis Type 1, symptoms and incidence?

Answer 92

Neurofibromatosis type 1 (NF1; OMIM 162200) is the most common autosomal-dominant neurocutaneous disorder associated with tumour predisposition Affects 1 in 2,500 to 3,000 people worldwide Caused by defects in the tumour-suppressor gene NF1. Characterized mainly by cafe au lait spots and neurofibromas, present in more than 95% of adult patients.

Question 93

NF1 children have an increased risk of what?

Answer 93

Juvenile Myelomonocytic Leukaemia NF1 children have a 200-500 fold increased risk of suffering from a malignant myeloid disorder, particularly **JMML** (incidence of JMML in the UK as a whole is approx. 1 per million). 1 in 10 patients with JMML has NF1.

Question 94

Give examples of Rasopathies

Answer 94

RASopathies are a clinically defined group of genetic syndromes caused by germline mutations in genes that encode components or regulators of the Ras/mitogen-activated protein kinase (MAPK) pathway. ## Footnote Variants in this pathway cause NF1, Noonan syndrome, LEOPARD syndrome, Hereditary Gingival fibromatosis type1, Capillary malformation-AV syndrome, Legius syndrome, and Costello syndrome. NF1, LEOPARD, Noonan, Costello and cardifaciocutaneous syndrome overlap phenotypically and are referred to as neuro-cardio-facial cutaneous syndromes (NCFC). NF1-Noonan syndrome: Features of Noonan syndrome such as ocular hypertelorism, down-slanting palpebral fissures, low set ears, webbed neck and pulmonic stenosis occur in 12% of individuals with NF1.

Question 95

What causes TCS and incidence of TSC?

Answer 95

Incidence ~ 1 in 6000 – 10,000 Affects many organs with the development of benign tumours TSC is an autosomal dominant disorder characterised by a highly variable phenotype and the development of multiple hamartomas (benign tumour-like malformation) in multiple organs throughout the body. Incomplete penetrance and considerable interindividual phenotypic variability – can range from mild symptoms to severe multiorgan involvement Clinical manifestation can change over lifetime Often 1st diagnosed by skin manifestations, or secondary to epilepsy Autosomal dominant disease caused by pathogenic variants in TSC1 and TSC2 Tumour suppressor genes – TSC follows the classic Knudson 2 hit model – 1st hit germline pathogenic variant in one allele of TSC1 or TSC2, 2nd hit somatic loss of function of other allele Causes dramatic mTORC1 hyperacitivation and development of benign tumours Large number of cases de novo (~2/3rds) Testing also complicated by mosaicism No treatment – mTOR inhibitors in clinical trials show improvements in some symptoms

Question 96

Which cancer syndromes are RET and CDKN1B associated with?

Answer 96

Multiple Endocrine Neoplasia (MEN) Syndromes, AD, RET - germline activating mutations CDKN1B - germline inactivating mutations in p27

Question 97

Which syndrome is PTEN associated with?

Answer 97

Cowden syndrome (OMIM 158350) [Autosomal dominant] Clinical features A multiple hamartoma syndrome associated with high risk of developing benign and malignant tumours of the breast, thyroid and endometrium. Affected individuals usually have macrocephaly, trichilemmomas (benign tumour of hair follicle) and papillomatous papules; typically appear in the late 20s (major criteria). Lifetime risk of developing breast cancer is 85%, thyroid cancer (35%), and endometrial cancer (28%). Adult Lhermitte-Duclos disease (rare non-cancerous cerebellar tumour) is pathognomonic for CS. Minor criteria: intellectual disability (IQ under 75), Autism spectrum Disorder (ASD), colon cancer, lipomas, renal cell carcinoma, papillary or follicular variant of papillary thyroid cancer, and vascular anomalies.