Dr Reeba Mary Issac External Part Time Ph.D candidate Department of Pathology, YMC

1. BRCA GENE MUTATION ANALYSIS AND HISTOPATHOLOGICAL CORRELATION IN FEMALE breast CANCER patients attending a tertiary care centre in kerala • Co-Guide • Dr Jessy M.M • Professor • Department of Pathology,Pushpagiri Medical College, Tiruvalla, Kerala. Research Student • Guide • Dr PremaSaldhana • Professor • Department of Pathology,YMC. • Dr Reeba Mary Issac • External Part Time Ph.D candidate • Department of Pathology, YMC

2. CONTENTS • Introduction • Literature Review • Lacunae in the literature • Relevance of my study • Aim and Objectives • Methodology • Statistical analysis • Budget plan • Timeline • References Pre PhD Synopsis

3. INTRODUCTION • Cancer is a leading cause of disease worldwide. • In females, breast cancer was the most common cancer worldwide in women contributing to more than 25% of the total number of new cases diagnosed in 2012 (Ferlay J et al, GLOBOCAN,2012) • In breast cancer, both genetic and environmental factors play a role, with family history being the most important factor for determining the breast cancer risk. Pre PhD Synopsis

4. 5–10 % of breast cancers are caused by the presence of inherited mutations (Tung N, Batelli C et al, 2015). • About 25% of these cases can be attributed to specific inherited mutation in the tumor suppressor genes BRCA1 or BRCA2 (Kim H, Choi DH, 2013). • BRCA gene mutation association with other cancers (Mersch J, Jackson MA et al, 2015). Pre PhD Synopsis

5. Review Literature Pre PhD Synopsis

6. Review Literature Pre PhD Synopsis

7. LACUNAE IN THE LITERATURE • Predictive genetic testing for geneticmutations is increasingly becoming a part of clinical practice. • However the limited knowledge about the prevalence of these genes in the general population has limited the availability of predictive genetic testing in many countries in Asia. • Identification of BRCA mutation pattern would represent an essential prerequisite for a prevention program based on DNA analysis. Pre PhD Synopsis

8. RELEVANCE OF THE STUDY • Individuals with cancer having mutated BRCA gene can opt for targeted therapies. Poly(ADP-ribose) polymerase inhibitors are a novel therapeutic option for the treatment of breast and ovarian cancers which preferentially target BRCA defective cells and spare those with normal function. • Early identification of individuals carrying a predisposing gene mutation in BRCA genes is of utmost importance. • Several options are available for managing cancer risk in such individuals. These include modifying nutritional and lifestyle factors, enhanced screening and prophylactic(risk-reducing) surgery. Pre PhD Synopsis

9. AIM • To find out the frequencies and patterns of BRCA 1 and BRCA2 gene mutations in female breast cancer patients attending a tertiary care centre in Kerala and to relate the histopathological features with such gene mutations. Pre PhD Synopsis

10. OBJECTIVES 1. To identify the frequencies and patterns of germline mutations of BRCA 1 and BRCA2 inbreast cancer. 2. To do primary screening for BRCA1 and BRCA2 mutation by immunohistochemistry on tissue samples. 3. To identify the type of mutation by doing real time PCR and sequencing on the positive samples obtained by immunohistochemistry. Pre PhD Synopsis

11. 4. To characterize the histopathological features of BRCA1/2 gene mutated breast cancers. 5. To compare the histopathological characteristics of BRCA gene mutated cancers and non BRCA gene mutated cancers . 6. To identify the presence of other cancers in those found to have mutated gene. Pre PhD Synopsis

12. Methodology • The study will be initiated after getting clearance from Institutional Ethics Committee. • The proposed study would be carried out in the Department of Pathology and Research Centre of Pushpagiri Institute of Medical Sciences Thiruvalla, Pathanamthitta, Kerala. Pre PhD Synopsis

13. Study population: All patients diagnosed with breast cancer on histopathological examination will be included in the study • Sample size: 124 • Duration of study: 3 years Pre PhD Synopsis

14. INCLUSION CRITERIA • Females who are consenting for genetic analysis . • All females who have undergone breast lump excision/trucut biopsy or modified radical mastectomy. Pre PhD Synopsis

15. Exclusion criteria • Females who are not consenting for genetic analysis • If mastectomy specimen is available, then trucut biopsy/lumpectomy will be excluded . • Any patient with a prior history of treatment elsewhere and those with recurrence will be excluded from the analysis. Pre PhD Synopsis

16. Biopsy confirmed cancer cases will be selected for the study • Epidemiological data will be collected from all the cancer patients using a pre-structured questionnaire • The questionnaire will include personal particulars such as age, religion, marital status, socio-demographic information, education, occupation, diet, selected lifestyle factors • Women will also be enquired about their reproductive history including details of menstrual status, age at first child birth, duration of breast feeding, and detailed information on previous surgeries, family history of cancer and cancer treatment Pre PhD Synopsis

17. 1-2 ml of blood sample will be collected in EDTA tubes from which DNA will be isolated and stored at -80˚C • Immunohistochemistry will be done on the tissue samples using BRCA1 and BRCA 2 antibodies • DNA analysis for BRCA gene mutation will be done on positive blood samples by real time polymerase chain reaction and sequencing • In addition, histopathological features of BRCA gene mutated breast cancers will be studied Pre PhD Synopsis

18. Histopathological features of breast cancer will be classified as follows:- Tumor subtype Histological grade (Bloom Richardson’s grading) Confluent necrosis Presence of lymphocytic infiltration • Histopathological features of BRCA gene mutated and non BRCA gene mutated cancers will be studied and compared. Pre PhD Synopsis

19. Patient Informed consent Form • Patient identification Number: Title: BRCA gene mutation analysis and histopathological correlation in female breast cancer patients. • Name of Principle Investigator: Phone No: The nature and purpose of the study , titled above is explained in detail to me in a language that I understand and I confirm that I have had the opportunity to clarify doubts. The potential benefits and other relevant details related to the expected duration of the study have been explained to me in detail. I understand that my participation in the study is voluntary and I am free to withdraw at any time without giving any reason, without my medical care or legal rights being affected. I understand that the information collected about my participation in this study and details of my medical note may be looked at by responsible individuals . I give permission for the investigator to have access of my records. I agree to take part in the above study. Signature / left thumb impression Place Date Name of the participant: Son/ Daughter/ Spouse of: Complete postal address with phone number: This is to certify that the above consent has been obtained in my presence. Signature of the chief examiner Place Date

20. STATISTICAL ANALYSIS • Data will be analyzed using EPI-INFO • The summarized data will be interpreted accordingly and then be presented in the form of tables • Prevalence of BRCA gene mutation in breast cancers will be calculated • Ratio of BRCA 1/BRCA 2 will also be calculated • Fischers/Chi-square test will be applied to find out the statistical significance • Measurable variables will be compared between the two groups using Mann- Whitney U test. Pre PhD Synopsis

21. Timeline Pre PhD Synopsis

22. Budget plan Pre PhD Synopsis

23. References • Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. International journal of cancer. 2015 Mar 1;136(5). • Tung N, Battelli C, Allen B, Kaldate R, Bhatnagar S, Bowles K, Timms K, Garber JE, Herold C, Ellisen L, Krejdovsky J. Frequency of mutations in individuals with breast cancer referred for BRCA1 and BRCA2 testing using next‐generation sequencing with a 25‐gene panel. Cancer. 2015 Jan 1;121(1):25-33. • Kim H, Choi DH. Distribution of BRCA1 and BRCA2 mutations in Asian patients with breast cancer. Journal of breast cancer. 2013 Dec 1;16(4):357-65. • Mersch J, Jackson MA, Park M, Nebgen D, Peterson SK, Singletary C, Arun BK, Litton JK. Cancers associated with BRCA1 and BRCA2 mutations other than breast and ovarian. Cancer. 2015 Jan 15;121(2):269-75. 5. Kobayashi H, Ohno S, Sasaki Y, Matsuura M. Hereditary breast and ovarian cancer susceptibility genes (Review). Oncology reports. 2013 Sep 1;30(3):1019-29. 6. Vaidyanathan K, Lakhotia S, Ravishankar HM, et al . , BRCA1 and BRCA2 germline mutation analysis among Indian women from South India: identification of four novel mutations and high frequency occurrence of 185delAG mutation . J Biosci . 2009Sep ;34(3): 415-22. • Vani S, Leelakumar S, Volga S. Et al.,Novel germline mutations in BRCA2 gene among 96 hereditary breast and breast–ovarian cancer families from Kerala,South India. J Cancer Res ClinOncol 2007 May; 133:867–74

24. References 8. Hamilton R. Original Research: Being Young, Female, and BRCA Positive. AJN The American Journal of Nursing. 2012 Oct 1;112(10):26-31. 9. Gan A, Green AR, Nolan CC, Martin S, Deen S. Poly (adenosine diphosphate-ribose) polymerase expression in BRCA-proficient ovarian high-grade serous carcinoma; association with patient survival. Human pathology. 2013 Aug 31;44(8):1638-47. 10. Guinan EM, Hussey J, McGarrigle SA, Healy LA, O’Sullivan JN, Bennett K et al. A prospective investigation of predictive and modifiable risk factors for breast cancer in unaffected BRCA1 and BRCA2 gene carriers. BMC cancer. 2013 Mar 21;13(1):1. Pre PhD Synopsis