Transcript lung cancer egfr mutation at 18,19,20 21exons, ras

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- 400026,
India.
Email - [email protected] Web site -www.jaslokhospital.net
National Institute of Environmental Health Sciences (NIEHS), NIH, USA (1995- 1996)
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
Keystone Symposia 1999
Molecular Basis of Cancer
Taos, New Mexico, USA
Visited James Watson Institute at Cold Spring Harbor, New York, USA,
(1999)
University of Texas, M. D. Anderson Cancer Centre, Houston Texas, USA (2002-2004)
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
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
Translational Research
Bench side promises To Bedside Reality
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)
TYPES OF MOLECULAR DIAGNOSTICS ASSAYS DONE
PCR
REAL TIME PCR
AUTOMATED DNA SEQUENCING
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
HUMAN GENOME PROJECT
NIH Director Francis Collins
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.
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.
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.
•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.
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
GENE EXPRESSION PROFILING
MICROARRAY TECHNOLOGY
RNA SEQUENCING
NEXT GENERATION SEQUENCING
MOLECULAR BIOMARKERS FOR BREAST CANCER
BREAST CANCER
Breast cancer is one of the most common and leading causes of cancer death
in women all over the world.
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.
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.
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
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
can be used as a marker for proliferating cells.
•Jones et al. 2009 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 .
•
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.
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
and grade of the tumor.
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.
MOLECULAR BIOMARKERS IN LUNG CANCER
Lung Cancer
•Lung Cancer is the most common cancer in the world and has high mortality rate due
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
stage and are difficult to cure due to metastasis of these tumor cells to distant sites.
•Passive smokers have about 20–30% increase risk in developing lung cancer and it is
more dangerous than direct smoking.
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
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
Gefitinib or Erlotinib. Recently Erlotinib in 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
point mutations in exons 18, 20, and 21 [103].
•Overall, it appears that EGFR mutation detection is one of the best prognostic and
predictive biomarker for NSCLC.
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
mutations are missense mutations at 12, 13, or 61 codons of Ras gene.
•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
high number of copies of mutant Ras gene.
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.
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.
COLORECTAL CANCER (CRC)
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.
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) gene have been found in CRC.
• 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
TARGETED THERAPIES FOR COLORECTAL CANCER
• Bevacizumab targets Vascular Endothelial Growth Factor (VEGF), while
Cetuximab and Panitumumab can target the epidermal growth factor receptor
(EGFR), are now approved as the first and second line therapies for CRC.
•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.
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.
ACKNOWLEDGEMENT
•MANAGEMENT JASLOK HOSPITAL & RESEARCH
CENTRE, MUMBAI
•STAFF & STUDENTS OF DEPT OF MOLECULAR
MEDICINE & BIOLOGY
THANK YOU