Cancer Flashcards
Pharmacogenetics
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).
Pharmacogenomics
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).
A patient’s response to a drug may depend on:
• Pharmacokinetic factors • Pharmacodynamic factors • Non-pharmacogenomic factors
Pharmacokinetic factors
- Absorption - Distribution - Metabolism - Elimination
Pharmacodynamic factors
- Target proteins - Downstream messengers
Non-pharmacogenomic factors
- Age and gender - Lifestyle habits, such as smoking and drinking alcohol - Other diseases - Medications taken for other conditions
Financial implications of adverse drug reactions in UK
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
Health implications of adverse drug reactions in UK
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
Benefits of Pharmacogenetics
• 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
Pharmacogenetics examples
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
Warfarin - PGx
• 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.
Pharmacogenetics in Cancer - examples
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.
Pharmacogenetics in Cancer - challenges
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.
What is the fuction of EGFR and which cancers overexpress EGFR
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.
Percentage of NSCLC ptients with EGFR mutation
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.
Name a first-line small molecule tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) used in NSCLC
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).
What is difference between erlotinib and gefitinib?
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
What is the origin of breast cancer and risks?
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.
What causes resistance to erlotinib and gefitinib?
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.
Name a drug for pwtients with EGFRm(+) sensitizing and T790M variant-mediated resistance to EGFR inhibitors in lung cancer.
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.
Function of KRAS and prognosis of patients with KRAS mutations?
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
Can EGFR and KRAS mutations appear together in one tumour and significance?
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.
Echinoderm microtubule associated protein-like 4 (EML4)–ALK oncogenic gene fusion
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
What is the most common fusion partner seen with ALK (Anapalastic Lymphoma Kinase, is a receptor tyrosine kinase)?
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).
Do patients develop resistance to ALK TKIs?
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.
How do we test for ALK NSCLC mutations?
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.
Frequency of NTRK gene fusions in lung cancer and significance
~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.
Name other biomirkers in NSCLC (other than EGFR, KRAS, ALK, NTRK)
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)
What is PD-L1 and what treatment can be used for PD-LI positive tumours?
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.
Name four majority aetiology/pathogenesis groups of CRC
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).
What proportion of patients have metastatic disease at the time of diagnosis?
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).
What proportion of tumours are positive for KRAS activating mutations and what is the significance?
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).
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.
Name monclonal antibodies that block the human EGFR
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.
What is the proportion of BRAF and NRAS mutations in CRC and their significance?
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.
What are BRAF mutations associated with in CRC?
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.
What are NRAS mutations associated with in CRC?
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.
What are PIK3a mutations associated with in CRC?
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.
What is the incidence of Melanoma in UK?
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.
What is the CRUK Stratified Medicine Programme 1?
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
What is the pre-sreen testing strategy for Lynch Syndrome?
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
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?
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.
What is HBOC syndrome?
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)
Give examples of Rasopathies
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.
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.
