Role of Genomics and Liquid Biopsy in Breast Cancers Dr. Mithua Ghosh Director- R&D Head- Department of Molecular & Clinical Genomics

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1 Role of Genomics and Liquid Biopsy in Breast Cancers Dr. Mithua Ghosh Director- R&D Head- Department of Molecular & Clinical Genomics Triesta Genomics and Translational Research HCG 1

2 Breast Cancers- A Perspective Major Challenges Early Detection Right Diagnosis Subtyping Staging and Grading Breast cancers Prognosis Right Therapy Monitoring and Surveillance Tumor heterogeneity Multi step and multifactorial Prognosis Organ specific metastasis Metastases differ from their primary Tumors can evolve, are clonal And become resistant Drug Resistance Genetic Multiple tests needed Right Drug,right time, right dose Best Clinical Outcome 2

3 Comprehensive Diagnostics An unmeet need of the day in Molecular Profiling Genomics Proteomics metabolomics Predictive markers of Drug sensitivity/resistance Patient stratification of responders and non responders Predictive markers of adverse effects/ toxicity of drugs Right.. Drug Patient Dose Time

4 Comprehensive Diagnostics An unmeet need of the day Technology Morphology - IHC Diag Prog Minimal Residual Disease Personnel expertise Infrastructure / Cost of test Turn around low +++ Flowcytometry high +++ Cytogenetics/ FISH /- + high ++ RT:PCR medium ++ Q:PCR +/ medium ++ NGS High

5 TRIESTA GENOMICS & TRANSLATIONAL RESEARCH State of the art molecular and clinical genomics/ genetic counselling centre offering comprehensive Diagnostics and Research services. Supports Genomic Medicine - that involves usage of genomic data of patients for better diagnosis, making effective therapeutic decision and to achieve overall improved health outcomes. High level Tumor boards involving clinicians, pathologists, Molecular pathologists, clinical geneticists and scientists A Multidisciplinary approach of evaluating treatment decision.

6 Actionable Mutation - What are we talking about? Mutation Somatic/ Sporadic/Acquired Driver Actionable mutation Passenger A U.S. FDA-approved targeted therapy in the tumor type A U.S. FDA-approved targeted therapy in another tumor type- of drugs Or A clinical trial for which the observed alteration is linked by mechanism of action to the therapy under study 6

7 Tests Offered A. Somatic Mutation Test SOMATIC MUTATION TEST(48 gene panel) SOMATIC MUTATION TEST(151 gene panel) Tissue specific cancer panel- Breast Tissue specific cancer panel- Lung Tissue specific cancer panel- Colorectal B. Germline Mutation Test Germline Mutation Test( Cancer specific gene panel) Single Gene mutation for family screening 7

8 Genomic Data from HCG Triesta has profiled Cancer patents in last two years Pancreas 2% Lung 5% Clinical Utility of the Mutations Others 33% Breast 40% No Mutation 20% Prognostic Value 35% Clinical Utility 45% ectal % Gynae 10% No Mutation is also a significant result 8

9 Genomics Data in Breast Cancer Fig 1&2: Somatic variants were detected in 75% of cases with direct impact on therapy or prognosis No Mutation 33% Clinical Utility, 44% BC patients screened and reported 600 Prognostic Value, 35% Median age 50.5yrs Hormone +ive 38% Her2 +ive 21% Her2 -ive 27% TNBC 24% No Utility Utility Fig 1: Mutation Spectrum in Breast Cancer

10 Genomics Data in Breast Cancer Fig.4 Mutations were identified in PI3K/AKT/mTOR pathway in substantial fraction (28%) of BC. PI3K /AKT PTEN ER+ive /Pr+ive PI3K 53% (34%) PI3K AKT PTEN 28%(30%) 18%(26%) 2.5% (4%) 6.5%(7%) Fig.5 Aberration in this pathway was more prevalent in HR+ve and HER2-ve including TNBC.. Her2 +ive 10% (22%) Her2 -ive TNBC 40% 24% (8%) Fig.6 NGS has identified nine PI3K (H1047R) exon 20 mutation and five exon 9 mutations(e545k) 67% cases with PI3K mutation had co mutation with p53 80% of BC with mutant PIK3CA presented with liver metastasis. 10% of PI3K/PTEN/mTOR pathway altered BC showed no/partial pcr post NACT/adjuvant chemotherapy and poor outcome.

11 HR +ve BEZ-235 GDC-0941 Treatment regimen involving aromatase inhibitors reported to be more beneficial than tamoxifen therapy. Ramirez-Ardila et al. 20I3. Approved for advanced HR+ve, HER2-ve BC with Exemestane. HER2 +ve Lack of response to anti-her-2 agents like trastuzumab in HER +ve BC patients. Bern's et al. 2007; Cizkova et al. 2011; Wang et al Resistance can be overcome by using PI3K or mtor inhibitors+ anti-her2 treatment. Andre et al NCT Everolimus for women with trastuzumab-resistant,her2+ve, advanced breast cancer(bolero-3): a randomised, double-blind, placebo-controlled phase 3 trial. NCT Everolimus +Trastuzumab and Paclitaxel in the Treatment of HER2 Positive Locally Advanced or metastatic BC (BOLERO-1) HER2 ve/tnbc Everolimus is FDA approved for HR +ve/ Her-ve and TNBC. NCT Phase Ib/II Trials of RAD001 in TNBC. NCT (NECTAR) Everolimus Plus Cisplatin in TNBC.

12 Case Study Patient: Female 29yrs Advanced metastatic carcinoma breast (Mets to liver, bone, lung, brain). Mol Path/pathology : ER +ve, PR +ve, HER-2/NEU +ve, KI-67 35% 3 cycles of chemotherapy with FAC regime and 6 cycles of chemotherapy with Paclitaxel.Herceptin. Post Chemotherapy. Received Palliative Radiotherapy to whole brain 10 fractions Gene : FDA Approved Therapy for the tumor type other tumor type Clinical Trial PIK3CA May render sensitivity to mtor pathway drugs May render resistance to anti-her2. Aromatase inhibitors more effective than Tamoxifen in ER positive breast cancer May render sensitivity to Everolimus/Te msirolimus NCTO NCTO NCTO NCTO P53 Mutation Aggressive disease with worst survival rate in Her2 + breast cancer 12

13 Case Study Patient Demographics Medical History Mol Path/ Path Female 38years Breast cancer with multiple liver and bone mets and CT Thorax on revealed multiple nodular lesion suggestive of metastases Patient received 12 week of chemotherapy with Paclitaxel + Herceptin upto and 1 dose of zoledroinic acid on ER+ive. PR+ive Her2+ve Test Findings FBXW7 c.1353dela p.glu452fs KRAS c.43g>a p.gly15ser PTEN c.202t>a p. Tyr68Asn PTEN c.775c>g p.his259asp SMAD4 c.1363c>t p.gln455* TP53 c.742c>t p.arg248trp ERBB2 >25 fold amplification 13

14 Data Analysis Her2 amplification Herceptin lapatinib PTEN loss and p53 Herceptin lapatinib Resistance to anti-tubulin chemotherapy FBXW7 mtor Inhibitor like Rpamycin KRAS SMAD4 Partially responsive to mtor inhibitor Resistance anti EGFR therapy Sensitizes diverse cancers to lapatinib p53 Lapatinib+ mtor inhibitor?? 14

15 Tackling Triple Negative Breast Cancer 15

16 Data Analysis TNBCs constitute 10% 20% of all breast cancers, more frequently affect younger patients, and are more prevalent in African-American women TNBC tumors are generally larger in size, are of higher grade, have lymph node involvement at diagnosis, and are biologically more aggressive. Heterogeneity of the disease and the absence of well-defined molecular targets. TNBC have higher rates of clinical response to presurgical (neoadjuvant) chemotherapy TNBC patients have a higher rate of distant recurrence and a poorer prognosi than women with other breast cancer subtypes.

17 Tackling Triple Negative Breast Cancer : Total Number of Ca breast cases who have undergone genetic testing: 400 Number of TNBC: 101 Number of TNBC cases for Somatic Testing: 46 Number of TNBC cases for Germline Testing: 55 Number of TNBC cases for both Somatic and Germline Testing: 26 Mutations Number of cases TP53 31 PIK3CA/PTEN 2 TP53 + PIK3CA/PTEN 6 Mutations Number of cases BRCA1 13 BRCA2 4 No Mutation 21 TP53 + Other mutations No mutation 9 4 BRCA1/2 + other mutations Other mutations 14 3 Other mutations include AKT1, KRAS, CDH1, RB1 TP53+BRCA1/2 6 PIK3CA+BRCA1/2 Nil Other mutations include BRIP1, RAD51, ATM, CDH1, CHEK2, MLH2, MSH2, TP53, MSH6

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19 Yes!!..But not all cancers are inherited. Only 5 to 10% of all cancers results directly from the gene defects inherited from the parents. Individuals who inherit a mutation in a cancer susceptibility gene have a much greater chance for developing cancer. This risk varies according to the gene that is being mutated. Cancer risk attributable to the various genes can be classified into high, moderate and low risk genes However, not everyone with a cancer susceptible gene will develop cancer. High Risk >4 fold, generally >50% Moderate Risk Low Risk 2-4 folds, generally between 20-50% Yet to be determined, generally up to 20%

20 Age-Young age of onset Family history Multiple primary cancers in one individual Recurrence of the same/different cancer Bilateral tumour/both breast and ovarian cancers Rare tumours Two or more primary types of BRCA1- or BRCA2-related cancers in a single family member The addition of pathological features of cancer such as medullary carcinoma and triple negative in women younger than Genetic testing using Next Generation sequencing. Analysis and interpretation Risk assessment Pathogenic Likely Pathogenic Variant of Unknown significance Average risk Moderately increased risk High risk Single Mutation Confirmation Testsanger Sequencing

21 Hereditary Breast and/or Ovarian Cancer (HBOC)Syndrome Hereditary Breast and Ovarian Cancer (HB)C)Syndrome is the cancer syndrome which increases the risk of developing breast/ovarian cancer in genetically related families. There are two genes that are commonly associated with hereditary breast and ovarian cancer, BRCA1 and BRCA2. Genes associated with HBOC are classified into two groups: -- High penetrance genes (includes BRCA1, BRCA2, TP53, CDH1, STK11, PTEN) -- Moderate/Low penetrance genes (includes CHEK2, PALB2, ATM, BRIP1, RAD51C, XRCC2/ RAD51, MRN protein complex,, RAD51D ) About 70 to 85% of HBOC cases are caused by mutations in either the BRCA1 or BRCA2 gene(autosomal dominant)

22 Genetic Triesta Genomics Genetic Counselling at Triesta Genomics and Translational Research Triesta Genomics & Translational Research, offers genetic counseling session by the trained professional Genetic Counselors to assess the patient s or individual s personal cancer risk. Our genetic counseling sessions include the Pre-Test & Post-Test Genetic Counseling. Pre-test counselling Apprehend your medical & family history Draw your family tree (pedigree) Assess your risk of inherited cancer syndrome Discuss the various aspects of genetic testing Discuss the different options of genetic testing Post-test counselling Discuss the results of genetic testing empathically to family member Provide family counselling Talk about the various cancer screening programms Discuss the different disease management options related to the particular syndrome If you are positive for a BRCA mutation there are several options that you and your physician could consider to reduce the risk for developing cancer-4 You options may include: Increased or Enhanced screening for cancer Prophylactic (risk-reducing) surgery Drug therapy or Chemoprevention Evaluation of your family members risk. Each first degree (parents, siblings and children) of the affected individual has 50% chance of having BRCA mutation. We recommend Single Mutation Confirmation Testing for the family members for the identified BRCA variation. Early detection of pathogenic variations is useful in effective surveillance of disease. It is important to discuss this option with your doctor and/or genetic counselor to understand which option is best for you. Problems always come with Solutions

23 HCG Data- Germline Mutation n= 400 Fig: 1 Percentage of patients screened for Fig: 2 Percentage of patients with/without Germline Mutations Fig: 3 Distribution of BRCA1, BRCA2 and other GRm 55% 45% 71% 29% 47% 32% % of patients screened for GRm % of patients not screened for Grm % of Patients with GRm % of Patients without GRm 21% % of BRCA1 % of BRCA2 Other GRm Gene % Age at dx (in yrs) Family History (Hx) BRCA (n=15) (n=8) BRCA (n=7) (n=9) Yes (n=16) No (n=4 ) UK(n=3) Yes (n=10) No (n=4) UK=2 AACR,2015 San Antonio,2014 Cancer Reserch, % 0% Her2(-ve), 48% Her2(+ve) 16% Screening 14% Unknown, 22%

24 Let the Ribbon be both Pink and Blue Men Get Breast Cancer Too... 24

25 TRIESTA GENOMICS & TRANSLATIONAL RESEARCH Male breast cancer (MBC) is a rare disease that accounts for 1% of the total breast cancer cases. Heterogeneous disease Age, ER,PR,family history, radiation Molecular and histological profile of MBC are quite distinct from MBC (Venkateshwari A et al. 2017). About 10% of MBC have been reported to harbour germline mutation in BRCA and other risk predisposition genes Clinical significance of the mutations No mutation 31% Prognosis 47% Clinical utility 22% Sl No Results Good response Poor response 1 PTEN mtor inhibitors 2 PIK3CA 3 AKT1 mtor inhibitors mtor inhibitors 4 ATM PARP inhibitors 5 RB1 Methotrexate 6 TP53 -- Chemo-radiation therapy 5-FU & cisplatin

26 TRIESTA GENOMICS & TRANSLATIONAL RESEARCH Patient : 49yrs Medical history: a. Diagnosed with CA right Breast in 2011, IDC, Grade III, Stage IIIC (T2N3aM0) b. Hormonal status: ER/PR+ve, Her2/neu -ve c. Treatment: Chemotherapy + Radiation Diagnosed with CA Thyroid in 2015 (second primary) Family history: Sister: Renal Cell Carcinoma (Both Kidneys) Recommended for Genetic testing-somatic and Germline Mutation Analysis Test Results of Somatic Mutation Analysis: PTEN Everolimus PI3KCA Everolimus and aromatase inhibitors Germline A heterozygous variant of NBN gene Final Diagnosis: Cowden Syndrome (1 in 200,000 individuals) - Autosomal Dominant

27 LIQUID BIOPSY 27

28 What is Liquid biopsy Liquid biopsy is technology to detect cancer biomarker from body fluids such as blood, saliva, urine, ascites, or pleural effusion. Like a tissue biopsy, it is a representative of the tissue from which it has spread. Liquid biopsies have become more clinically useful in recent years due to the ability to pair tests on circulating tumor cells / DNA with genomic tests. Diaz, Jr., L.A. and Bardelli, A.(2014) Am. Soc. Clin. Oncol. 32, 579.

29 Liquid biopsy as a complement to tissue biopsy Tissue biopsy is currently the standard of care for the molecular diagnosis of cancer. The conventional approach involves examination of tumor tissue by removing cells through a small needle or histological examination of a biopsy or surgical excision specimen. FFPE Samples Problems with quality of DNA due to fixation Some patients have no tumour sample available and Biopsies are invasive and risky, and difficult for inaccessible The sample represents the tumour at one fixed time point Evolution of tumor heterogeneity often difficult, risky, costly and often times unattainable. ctdna Samples ctdna shed directly from tumour Taking blood is simple in comparison; Serial samples can be taken at various time points during the patient s treatment Novel approaches that could efficiently detect tumor heterogeneity in the course of cancer system treatments

30 OncQuer Scout LIQUID BIOPSY Test OncQuer TM Scout Liquid Test is a quick and minimally invasive test that uses droplet digital PCR (ddpcr) based technology to detect genetic mutations present at very low frequencies in a wild type background, as in the case of circulating tumor DNA in the plasma of cancer patients.

31 Clinical application Technology Person with symptom or diagnosed Doctors consultation and prescription Collection and Transportation Blood sample taken in streck tubes/edta tubes cfdna is extracted from plasma using the QIAamp Circulating Nucleic Acid kit on the QIAVac system Single gene Mutation Multi gene Mutation Quantitation of Mutational load qpcr Real Time PCR Droplet Digital PCR Report Generation and Prescription Data analysis & Clicical report Generation Personalized treatment recommendation and prescription by the doctor Regular Monitoring of the patient using OncQuer Scout LB test Tumor Genotyping Real Time detection and monitoring of genetic mutation Minimal Residual Disease Determine mechanism of resistance, disease progression and identification of newer treatments Early Intervention Diagnose the cancer earlier through screening Surrogate of Drug Response Monitoring tumor burden as surrogates of treatment response. Resistance Emergence of genetic alteration leading to drug resistance

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