Dr. Pravin D. Potdar. M.Sc, Ph.D, D.M.L.T.,DHE, DMS

Transcription

1 Molecular Profiling of Cancer Cells Strategies for Developing Biomarkers for Targeted Therapies of Cancer Dr. Pravin D. Potdar. M.Sc, Ph.D, D.M.L.T.,DHE, DMS Head, Department of Molecular Medicine and Biology, Jaslok Hospital and Research Centre, Mumbai , India. - ppotdar@jaslokhospital.net Web site -

2 National Institute of Environmental Health Sciences (NIEHS), NIH, USA ( ) Short term Scientist Exchange program Award from National Cancer Institute (NCI), USA - Identified 3 Novel genes presently on NCBI data base i.e. GGT-rel gene,kpl1 gene KPL2 gene Dr. Paul Nettesheim M.D. Research Director, NIEHS(NIH) Differential Display study to identify Novel genes involved in mucocilliary differentiation of Rat & human Tracheobranchial epithelia cells

3 Keystone Symposia 1999 Molecular Basis of Cancer Taos, New Mexico, USA Visited James Watson Institute at Cold Spring Harbor, New York, USA, (1999)

4 University of Texas, M. D. Anderson Cancer Centre, Houston Texas, USA ( ) Research Scientist (Faculty position for 2 &1/2 years) Developed several clones having SSTR receptors in Pancreatic cell line as a targeted therapy for pancreatic cancer Molecular Targeted therapy in prostate and pancreatic cancer

5 Department of Molecular Medicine & Biology JASLOK HOSPITAL & RESEARCH CENTRE, MUMBAI Join in April 2005 JASLOK HOSPITAL & RESEARCH CENTRE Multi Specialty hospital, with 350 beds and almost 300 consultants

6 Translational Research Bench side promises To Bedside Reality

7 ESTABLISHMENT OF DEPARTMENT OF MOLECULAR MEDICINE & BIOLOGY Molecular Diagnostics (40 PCR, Real Time PCR and sequence bases tests) Research & Development (Breast Cancer, CML,, Neurological disorders,hematological disorders, Infectious diseases Metabolic syndrome, and cardiovascular diseases ) Education (15 days and 6 months Research training program), 4 funding grants from Jaslok Hospital & Research centre, Mumbai)

8 TYPES OF MOLECULAR DIAGNOSTICS ASSAYS DONE PCR REAL TIME PCR AUTOMATED DNA SEQUENCING

9 BIOMARKERS ESTABLISHED AS RESEARCH PROGRAMS (4 GRANTS ARE OBTAINED FROM JASLOK HOSPITAL & RESEARCH CENTRE, MUMBAI) BREAST CANCER BRCA1&2, EGFR, ER, PR,EGFR MUTATION AT 18,19,20 21EXONS, RAS MUTATION AT EXON 2 CHRONIC MYELOID LUEKEMIA BCR-ABL, FACTOR V LEIDEN ACUTE MYELOID LUEKEMIA BCR-ABL, JAK2, FLT3, NPM1 FACTOR V LEIDEN Beta- Thalassemia FIVE INDIAN MUTATIONS LUNG CANCER EGFR MUTATION AT 18,19,20 21EXONS, RAS MUTATION AT EXON 2 GALL BLADDER CANCER D310 MITOCHONDRAIL DNA MUTATION STEM CELLS & CANCER STEM CELLS IN VITRO STUDY TO UNDERSTAND MECHANISM OF CANCER

10 NIH Director Francis Collins HUMAN GENOME PROJECT April 14, 2003 Implementing New Discipline NIH Director Francis Collins, then-director of the U.S. National Human Genome Research Institute, announces that a six-country consortium has successfully drawn up a complete map of the human genome, completing one of the most ambitious scientific projects ever and offering a major opportunity for medical advances, April 14, 2003, at a press conference at the National Institute of Health in Bethesda, Md.

11 MOLECULAR DIAGNOSTICS After completion of Human Genome Project, the major challenge in oncology now is to translate available genetic information by advancement of current and/or novel gene based technologies for improved diagnosis and management of various types of cancers.

12 BIOMARKERS OF CANCER One of the main goals of cancer research is to identify molecules which are deregulated in the process of cancer development which can be used for an early diagnosis and therapies for cancers. These molecules are called BIOMARKERS. Biomarkers can include nucleic acids, proteins, lipids, secondary metabolites, cytokines and chemokines that are aberrantly present in body fluids or cancer tissues. The molecular biomarkers are mainly identified by using Genomics, Proteomics or Imaging technologies such as PCR, microarrays, FISH, etc. technologies.

13 Several molecular biomarkers have been identified and molecular profiling of tumor may be useful in identifying specific types of malignancy. However, there is still a great need to identify and characterize more cancers biomarkers to further improve early diagnosis and therapy regimes. This presentation will provide a brief account of various current prognostic and predictive molecular biomarkers for breast, lung& colon cancer in human. Most of these cancers are fatal due to late diagnosis and unavailability of specific therapies.

14 MECHANISM OF CANCER DEVELOPEMENT Cancer is a multistep process which involves major three stages: initiation, promotion and progression to form a malignant tumor An activation of oncogenes and/or deletion of tumor suppressor genes Epigenetic gene silencing arising from aberrantly methylated CpG Islands in promoter regions of genes An impairment of microrna-mediated gene regulation pathway

15 GENE EXPRESSION PROFILING MICROARRAY TECHNOLOGY RNA SEQUENCING NEXT GENERATION SEQUENCING

16 MOLECULAR BIOMARKERS FOR BREAST CANCER

17 BREAST CANCER Breastcancerisoneofthemostcommonandleadingcausesofcancerdeath inwomenallovertheworld. Breast cancer mortality can be reduced by developing technologies for early diagnosis coupled with appropriate targeted therapies and proper patient follow-ups. Several biomarkers for breast cancer have been described. But there is still need for new biomarkers which can predict early onset, recurrence, as well as response to current therapies. However, the major obstacle in developing useful biomarkers of breast cancer is due to a lack of sensitivity and specificity of many identified biomarkers which limit their use in clinical practice.

18 Prognostic and Predictive Marker for Breast Cancer Estrogen Receptor(ER) as a Prognostic and Predictive Marker Tamoxifen is a direct targeted endocrine therapy that is being used in the treatment of ER-positive breast cancer patients and has significantly reduced mortality in these patients Recently, American Society of Clinical Oncology [ASCO] and the College of American Pathologists [CAP] have recommended ER testing in all newly diagnosed cases as well as in any local or distant recurrence breast cancer patients.

19 HER 2 as a Prognostic and Predictive Marker for Breast Cancer Over expression of HER2 is associated with a more aggressive tumor phenotype and have poorer prognosis. Trastuzumab is the first US Food and Drug Administration approved HER2-directed therapy for the management of HER2-positive metastatic breast cancer (MBC) and is usually prescribed in combination with chemotherapy HER2 amplification with benefit from adjuvant doxorubicin based chemotherapy Doxorubicin plus cyclophosphamide followed by paclitaxel therapy in HER2 Positive breast cancer HER2-positive breast cancer patients can also benefit from Anthracyclines therapy. In 2007, American Society of Clinical Oncology/College of American Pathologists has recommended routine HER2 testing in breast cancer patients

20 Ki67 as a prognostic and predictive marker in breast cancer marker Ki67 has been used as prognostic marker in breast cancer for routine use Ki67 is expressed in proliferating cells and absent in quiescent cells and thus Ki67 canbeusedasamarkerforproliferatingcells. Jones et al has indicated that Ki67 is a strong predictor for recurrence-free and overall survival of the patient in post neoadjuvant chemotherapy assessment and achieve a pathological Complete Response[pCR] in these patients. Higher level of Ki67 after 2 weeks of endocrine treatment has been linked to shorter recurrence-free survival[39-41]. In new approaches, combining established markers such as ER, Progesterone Receptor(PgR), HER2 with Ki67 are currently been under investigation. In a recent 2012 review, the authors have recommended that Ki67 be used in practice as prognostic and predictive marker in breast cancer management.

21 Cyclin D1 as a prognostic Marker for Breast Cancer Cyclin D1 is an important regulator of cell cycle progression and is over expressed in 45% of breast cancer. Over expression of cyclin D1 is confined to specific phenotypes, i.e. in Lobular carcinoma, whereas, in ductal carcinomas, they are almost exclusively ER positive. Over expression of cyclin D1 is a prognostic factor in invasive breast cancer among ER-positive patients and its over expression is associated with early relapse and poor prognosis. Cyclin D1 has a potential role in resistance to endocrine therapy. Direct targeting of the cyclin D1 gene may be a potential therapy target for breast cancer management. Cyline D1 could potentially be a prognostic maker in Tamoxifen treatment for breast Cancer.

22 Cyclin E as a prognostic Marker for Breast Cancer Dysregulation of cyclin E occurs in almost 18-22% of the breast cancers Low molecular weight cyclin E isoforms have been shown to have prognostic importance for breast cancer. Alteration of Cyclin E expression increase with the increasing stage andgradeofthetumor. Recent studies have shown that cyclin E over expression has caused Trastuzumab resistance in HER2+ breast cancer patients, therefore co-treatment of trastuzumab with CDK2 inhibitors may be a valid strategy for therapy of breast cancer.

23 MOLECULAR BIOMARKERS IN LUNG CANCER

24 Lung Cancer LungCanceristhemostcommoncancerintheworldandhashighmortalityratedue to late diagnosis of this disease. Smoking is one of the major cause of this cancer and 80 to 90% of lung cancers are due to long-term exposure to tobacco smoke. However, 10 15% of lung cancer is also developed in nonsmokers. There are three main subtypes of Non Small Cell Lung Cancer (NSCLC) such as adenocarcinoma, squamous-cell lung carcinoma, and large-cell lung carcinoma. Almost 40% of lung cancers are adenocarcinoma, which are associated with smoking habits. In Small-Cell Lung Carcinoma (SCLC), 60-70% patients are diagnosed at metastatic stageandaredifficulttocureduetometastasisofthesetumorcellstodistantsites. Passive smokers have about 20 30% increase risk in developing lung cancer and it is more dangerous than direct smoking.

25 MECHANISM OF LUNG CANCER DEVELOPMENT Lung cancer is initiated by activation of oncogenes or inactivation of tumor suppressor genes. Nearly 10 30% of lung adenocarcinomas have K-ras mutation. Mutations or amplification in EGFR gene is common in non-small-cell lung cancer and can be treated with EGFR-inhibitors.. Studies have also shown that p53, a tumor suppressor gene, located on chromosome 17p, is also affected in almost 60-75% of lung cancer patients

26 EGFR Mutation as a prognostic & predictive Marker in Lung Cancer Epidermal growth factor receptor (EGFR) mutations have been found in several series of NSCLC tumors from surgically resected patients and in patients treated with Gefitinibor Erlotinib. Recently Erlotinibin combination with bevacizumab as a first line therapy for NSCLC having EGFR mutation. The EGFR mutation frequency varies with different patient characteristics i.e. the EGFR mutation frequency in East Asian females is 58% compared with only 20% in women of other ethnicities. Cigarette smoking status is the best predictor of the presence of TKI-sensitive EGFR mutations. The highest frequency of EGFR mutation is observed in nonsmokers. Nearly, 60% of all EGFR mutations have deletions at exon 19, 25% of mutations are missense mutations in exon 21 i.e.l858r and the remaining 15% mutations are rare pointmutationsinexons18,20,and21[103]. Overall, it appears that EGFR mutation detection is one of the best prognostic and predictive biomarker for NSCLC.

27 RAS Mutation as a prognostic & Predictive Marker in Lung Cancer The Kirsten rat sarcoma virus (K-RAS) is an oncogenes and almost 20% of adenocarcinomas of the lung are associated with KRAS mutation. However, KRAS mutations are uncommon in lung squamous cell carcinoma. KRAS is constitutively activated in lung cancer and majority of cases, these mutationsaremissensemutationsat12,13,or61codonsofrasgene. KRAS mutations are found in tumors from both former/current smokers but they are rarer in never smokers. The role of KRAS mutation as a prognostic or predictive marker in NSCLC is not yet known although it has been shown that KRAS gene mutations are negative predictors of radiographic response to Erlotinib and Gefitinib and are strongly associated with tobacco smoking. Recently Sarah Bacus have shown that patients having KRAS gene mutation respond to antifolate treatment whereas, this treatment is not effective in patients having highnumberofcopiesofmutantrasgene.

28 EML4-ALK FUSION GENE AS A PROGNOSTIC& PREDICTIVE MARKER IN LUNG CANCER The EML4-ALK fusion gene is one of several new molecular markers identified in patients with NSCLC. The ALK-EML4 fusion gene has recently been detected in 6.7% of Japanese non-small cell lung cancers. The EML4-ALK translocation can be easily visualized on paraffin embedded tumor tissue using a technique called break-apart FISH where a translocation will result in a separation of fluorescent probes flanking the break-point. The EML4-ALK translocation is a very exciting new target and potential predictive marker for NSCLC hence inhibitors of ALK kinase have been developed and examined in preclinical models with some success. Recently FDA has approved ALK inhibitors include Crizotinib and ALIMTA. Crizotinib is the standard treatment for the patients with ALK positive lung cancer while ALIMTA is used as maintenance therapy with Cisplatin for locally advanced metastatic NSCLC patients.

29 DNA REPAIR (ERCC1 AND RRM1) MARKER IN LUNG CANCER Platinum-based drugs (cisplatin and carboplatin) are standard chemotherapy treatment for NSCLC. Excision Repair Cross-Complementation group 1 (ERCC1) marker is useful in assessing the short-term survival benefit of chemotherapy in lung cancer patients. Patients with low ERCC1 expression are benefited from chemotherapy, whereas, those with high ERCC1 expression did not, suggested the potential use to the expression of DNA repair genes to tailor the use of adjuvant chemotherapy in lung cancer. The molecular marker, Ribonucleotide Reductase-M1(RRM1) was also introduced Low expression of RRM1, lung cancer cells are more sensitive to Gemcitabine, whereas, high expression of RRM1 indicates the drug resistance. ERCC1, & RRMI markers can be used in future trials for predicting treatment response in NSCLC.

30 COLORECTAL CANCER (CRC)

31 COLORECTAL CANCER(CRC) Colorectal Cancer is mainly occurs due to lifestyle and increasing age whereas; very few cases are associated with genetic disorders. CRC can be prevented by screening at a frequent interval after the age of 50. Almost 75-95% of CRC occurs in people with little or no genetic risk. CRC can also be hereditary as it has threefold greater risk of this cancer if there is a family history in two or more first-degree relatives. The most common colorectal cancer is Hereditary Nonpolyposis Colorectal Cancer (HNPCC or Lynch syndrome) which is present in around 3% of people.

32 MOLECULAR BIOMARKERS FOR COLORECTAL CANCER (CRC) CRC is a disease originating from the epithelial cells and most frequently occurs due to mutations in the Wnt signaling pathway. The APC gene is a most commonly mutated gene in CRC Beside the defects in the Wnt-APC-beta-catenin signaling pathway. The mutated or deactivated of TP53, BAX, TGF-β and Deleted in Colorectal Cancer (DCC)genehavebeenfound incrc. The DCC gene with Loss of Heterozygosity (LOH) at the chromosome 18q21 locus has shown important prognostic information about the clinical staging of CRC. KRAS, RAF, and PI3K oncogenes are over expressed in colorectal cancer, whereas PTEN, a tumor suppressor gene is sometimes mutated and deactivated in CRC

33 TARGETED THERAPIES FOR COLORECTAL CANCER Bevacizumab targets Vascular Endothelial Growth Factor (VEGF), while Cetuximab and Panitumumab can target the epidermal growth factor receptor (EGFR), arenowapprovedasthefirstandsecondlinetherapiesforcrc. KRAS mutation is usually associated with the aggressiveness of malignant diseases and has been associated with tumor relapse in CRC patients. Overall survival of patients with wild type KRAS (WTKRAS) in their tumor is significantly higher, compared with those patients with a mutated KRAS. American Society of Clinical Oncology 2008, have suggested that BRAF mutations can be predictive of poor cetuximab response and of shorter patient s overall survival. it seems that more basic research is needed for better understanding of available technologies to identify biomarkers which are used for both accurate prognosis and prediction of therapeutic response for the cure of CRC.

34 CONCLUSIONS & FUTURE DIRECTIONS Since completion of Human Genome Project in 1993, PCR based tests are implemented for diagnosis of various types of cancers due to their high sensitivity. Many of these tests can be performed on non-invasive sources such as blood cells or body fluids at very minimal quantity. The cancers described here are frequently detected only at later stages of development hence are difficult to treat and patient prognosis is poor. So, there is a great need to identify more biomarkers of cancer, which are prognostic& predictive markers to be use in diagnosis and therapy of cancers. careful validation studies are needed to identify specific molecular cancer biomarkers that can be used for both early diagnostics and therapeutic management of cancers.

35 ACKNOWLEDGEMENT MANAGEMENT JASLOK HOSPITAL & RESEARCH CENTRE, MUMBAI STAFF & STUDENTS OF DEPT OF MOLECULAR MEDICINE & BIOLOGY

36 THANK YOU