dmch presentation.ppt

Cancer Tissue Biomarker

Immunohistochemistry

Opportunity and Pitfalls of CTB biomarker & Advantages Mutation study in Cancer Prediction, Confirmation and Prognosis

Kinds of Biomarkers

Prognostic biomarkersMeasured before treatment to indicate long-term outcome for patients untreated or receiving standard treatmentMay reflect both disease aggressiveness and effect of standard treatmentUsed to determine who needs more intensive treatmentPredictive biomarkersMeasured before treatment to identify who will benefit from a particular treatment

Early detection biomarkersMGMT

Endpoint biomarkersMeasured before, during and after treatment to monitor pace of disease and treatment effect

Most cancer treatments benefit only a minority of patients to whom they are administered

Being able to predict who requires intensive treatment and who is likely to benefit from which treatments could save patients from unnecessary debilitating adverse effects of treatments that they dont need or benefit from enhance their chance of receiving a treatment that helps themHelp control medical costs Improve the success rate of clinical drug development

Reliance Life Sciences, Mumbai, India.

Method for IHC Direct MethodIndirect Method

Bangladesh OfficeLab Science Diagnostics .Dhaka-153/1 Green Road, Panthapath,.Chittagong 106/ A, KB Fazlul Kader Road, In front of CMCH Main Gate.01742 383381 - 01612383383 - 01612383386

Introduction to IHCA one step staining method.

Process involves a labeled antibody directly reacting with the antigen in tissue sections.

Simple and rapid process.

Suffers problems with sensitivity due to little signal amplification.

Less commonly used.Two-step staining method.

Process involves two antibodies, one against the antigen being probed and second labeled antibody against the first antibody.Relatively complex but quite efficient process.Highly sensitive process with the advantage of relatively lesser labeled antibodies being used.More commonly preferred.

The process of localization of proteins in cells of a tissue section by binding specific antibodies to the antigens of cancerous cells is known as Immunohistochemistry (IHC).There are two methods in IHC technique. Considering the advantages and superiority of the method we employIndirect Immunohistochemistry in our investigations.

INDIRECT METHOD

DIRECT METHOD

An enzyme-labeled primary antibody binds to the tissue antigen

A substrate-chromogen solution is added producing a colored end-product

One Step Direct Method

Secondary Ab(rabbit anti-mouse)Primary Ab(mouse)substrate-chromogenAn unlabeled primary antibody binds to the tissue antigen

An enzyme-labeled secondary antibody binds to the primary antibody

A substrate-chromogen solution is added producing a colored end- product

Several secondary antibodies are likely to bind with various epitopes on the primary antibody, thus increasing the signal

Two-Step Indirect Method - Procedure

A biotinylated secondary antibody binds to the primary antibodyEach secondary antibody contains multiple biotin molecules; several secondary antibodies can bind to the primary antibody

An enzyme-labeled streptavidin is added and binds to the secondary antibody

A substrate-chromogen solution is added producing a coloured end-productHRP-streptavidin

LSAB Procedure

At RLS we do IHC by using the :- Labeled Streptavidin Biotin Detection System or LSABD

Single paraffin block of representative tumour (50%-70%) tissue fixed in 10 % neutral buffered formalin is ideally suited for biomarker testing.

Stringency of ER, PgR Her-2 testing requirement : tissue should fixed for 6-48 hrs for results to be valid.

Unstained slides containing tumour are accepted only if these are coated with poly-l-lysine or silane.

The paraffin embedded tissue block must be sent to the laboratory in a sturdy sterile container.

In case only tissue sections are sent for CTB,it should be on unstained Poly-L-Lysine coated slides sent in a bubble padded packing.

Optimal tissue for testing

To give clear picture of cancer invasion & metastasis

To decide appropriate line of therapy

In prognosis and response to treatmentThe identification of disseminated neoplastically transformed malignant cells and proteins by IHC helps...

Clinical Utility

Diagnostic Confirmation & Identification

Service NameMarker StudiedClinical Utility

Breast Cancer

ER, PgR,Her-2/neu Ki-67,p53,WT1, E-Cadherin, GCDFPTumour response to hormonal therapy.

Tumour response to targeted therapies like anti-Her- 2/neu monoclonal antibodies .Differentiation between lobular carcinoma in Situ & Ductal carcinoma in situ in case of Needle Biopses.Aggressive Nature & prognosis of Disease.

Response to Chemotherapy.

Differentiation between Ovarian adenocarcinoma metastatic to pleural cavity from a metastatic Breast carcinoma.



Carcinoma Of Unknown Origin

CK7/20, TTF-1, PSA, HepPar-1, CEA , ER, CA 125, WT1After zeroing in on the neoplasm as being a carcinoma, the source of metastasis can be deduced using appropriate markers, i.e.,

Provides multi-system differential diagnosis and categorization of various cancers: Urothelial / Pancreatic / Ovarian mucinous carcinoma Hepatocellular carcinoma / Prostatic carcinoma / Squamous cell carcinoma Breast cancer / NSCLC / Ovarian serous cancer / Colorectal adenocarcinoma

The source of metastases is either Prostate gland, Liver, Breast, ovary or female genital tract



Colorectal Cancer

MLH-1 / MSH-2,CK7, CK20 Ascertains the presence of colorectal cancer, based on CK7/CK20 + coordinate staining pattern.

Along with molecular testing, MLH-1 / MSH-2 helps to predict the response to chemotherapy in individuals with



Hodgkin's Lymphoma Vs.Non-Hodgkin's Lymphoma

CD3, CD15, CD20, CD30, CD45, Differentiates Hodgkin's Lymphoma from Non-Hodgkin's Lymphoma.

Characterizes the malignancy as either of the following..Hodgkin's LymphomaAnaplastic Large Cell LymphomaPrimary Mediastinal B-cell LymphomaT-Cell Rich B-Cell Lymphoma.

Melanoma Markers

S-100, HMB-45Helps in diagnosis of undifferentiated malignant tumours of unknown primary origin Also helps in identification and confirmation of Malignant Melanomas of different types



Neuroendocrine Malignancy

Chromogranin A, Synaptophysin Helpful in identification of neuroendocrine differentiation of carcinomas.

Corroborates the diagnosis of Neuronal tumours (viz. Ganglioneuroblastoma, Ganglioneuroma, Ganglioglioma, Phaeochromocytoma, etc.)

Poorly Differentiated Malignancy

Pan-Cytokeratin (AE1/AE3), EMA,LCA, S-100, PLAP, Vimentin In case of poorly differentiated malignancy, the panel will help to provide differential diagnosis for following malignant conditions

Carcinoma Melanoma Lymphoma Germ Cell Seminoma Non-Seminomatous Germ Cell Tumours



Thyroid Cancer

TTF-1, Calcitonin, CA 19-9 Ascertains the origin of primary tumour viz. thyroid, lung, etc. Helpful in confirmation of Medullary Thyroid Carcinoma . Useful in differentiation of

Primary non-mucinous Lung Adenocarcinoma from metastatic non-pulmonary AdenocarcinomaPulmonary Adenocarcinoma from Breast Carcinoma.Papillary Thyroid Carcinoma from Follicular Thyroid Carcinoma

Undifferentiated Gastric MalignancyLow Molecular Weight CK18, EMA, LCA, CD3, CD20, Vimentin, CEA Helpful in distinguishing Undifferentiated / Poorly Differentiated Gastric Carcinoma from Gastric Non-Hodgkin's Lymphoma and Sarcomatoid Carcinoma.



Testicular / Germ Cell Tumour

PLAP, FP, CA 125 Helps in identification of Germ Cell Tumours and Intra-tubular Germ Cell Neoplasia Provides differential diagnosis forUncharacterized tumours of mediastinum including Desmoplastic Small Round Cell Tumour, Rhabdomyosarcoma, ChoriocarcinomaNon-Seminomatous Tumours Vs. Pure Classic Seminomas Ovarian Clear Cell Carcinoma Vs. Clear Cell Renal Cell CarcinomaEpitheloid Sarcoma and Desmoplastic Small Round Cell Tumour Vs. Mesenchymal TumoursLymphoma MarkersLCA (CD 45) A Primary screening panel to identify haemato-lymphoid proliferations. Lymphoblastic Lymphoma/LeukaemiaTdT, CD43, LCA (CD45 Helps in identification of pre-B and pre-T Acute Lymphoblastic Leukaemia / Lymphoblastic Lymphoma (ALL/LBL).Helps in establishing diagnosis for Lymphoblastic Lymphoma (B and T cell type).


Marker StudiedClinical Utility

CD 30CD30 antigen is part of the tumour necrosis factor receptor superfamily. By definition, CD30 is present inAnaplastic Large Cell Lymphoma and Lymphomatoid Papulosis and is seen in some Reed Sternberg(RS cells) in 95% of classic Hodgkins Diseas

CD 20CD20 epitope is acquired late in the pre-B cell stage of maturation.CD20 is strongly positive in one-half of Lymphoblastic lymphoma/leukemias, almost all mature B celllymphomas (excluding plasma cell lesions), on Reed Sternberg (RS) cells in a quarter of classic HodgkinsDisease and on almost no T-cell lymphomas

CD 45 ( LCA )CD45( Leucocyte Common Antigen) is a transmembrane glycoprotein antigen expressed on all leucocytes in anumber of isoforms. It is a useful marker for identifying most lymphomas, except roughly 30% of AnaplasticLarge Cell Lymphomas (ALCL) and most Hodgkins Disease.

Ki-67 is a cell cycle proliferation marker that correlates strongly with S-phase fraction of the tumour.

High labeling index ( i.e.,> 13.5% for breast cancer) is strongly correlated with adverse prognostic factors such as poor histology , high cytologic grade and negative steroid receptor status.

Low Ki-67 labeling index would indicate slow proliferating growth and slower rate of recurrence, regardless of lymph node status.

Prognostic Biomarker

Biomarker Ki-67 in Breast Cancer

p53 is a tumour suppressor protein mutant in 50% of human cancers, including 30% of breast cancers.

In health, p53 is present in an inactive form and at very low levels escaping detection by IHC.

P53 immunostaining correlates with increasing tumor size and stage, low estrogen/ progesterone receptor content and resistance to paclitaxel based chemotherapy.

Tumours containing p53 mutations, regardless of nodal status, recur more quickly and frequently

Utility of Biomarker p53 in Breast cancer

Revolutionized the use of prognostic-therapeutic immunohistochemistry (IHC) in the practical management of patientsIdentified in 10-20% of breast cancerPatients with positive expression have significantly shorter disease-free survival and overall survivalIdentification is critical as such tumors require targeted therapy such as trastuzumabOnly two methods are currently approved and recommended for its detection: IHC and FISH

Her2/neu also called cerb2 biomarker

Her2/neu

Loss of E-Cadherin expression is a very early change in Lobular breast carcinogenesis and can differentiate between Lobular Carcinoma In Situ (LCIS)/ Lobular Carcinoma and Ductal Carcinoma In Situ (DCIS) /Infiltrating Duct Carcinoma : positive staining denoting the presence of DCIS or infiltrating duct carcinoma.

E-Cadherin loss may play a role in progression, development of distant metastasis and recurrence in invasive non lobular carcinomas of the breast.

Biomarker E Cadherin in Breast Cancer

Caspases which belong to the family of cysteine proteases, play an essential role in the initiation/regulation of apoptosis (to make sure that the body rids itself of damaged cells that could otherwise be mutated into cancer cells).

Aberrant apoptotic regulation has been associated with carcinogenesis and therapeutic resistance.

According to published reports, a down-regulation of Caspase 8 in breast cancer cell lines resulted in resistance to TNF-alpha induced apoptosis.

Thus Caspase 8 expression indicates better prognosis.

Biomarker Caspase 8 in Breast Cancer

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor involved in the proliferation and differentiation of normal cells. EGFR is often overexpressed on malignant cells, including 40 to 80 percent of patients with Non-Small Cell Lung Cancer (NSCLC). Overexpression of EGFR in tumours leads to uncontrolled signal transduction through the receptor, which leads to increased proliferation, tumor growth, and metastasis.

This has led to the emergence of targeted therapy using EGFR inhibitor drugs Gefitinib ( Iressa) and Erlotinib (Tarceva) both of which may be used to treat patients with advanced Non Small Cell Lung Cancer (NSCLC).We Do Both EGFR Mutation test for Exon 18,19,20,21, and Biomarker

Biomarker EGFR in NSCLC

Plays a significant role in lung and colon carcinogenesis

High expression is observed in squamous cell carcinomas, large cell and adenocarcinomas

Adverse prognostic factor in non-small cell lung carcinoma

Demonstration of positive expression with IHC often required before starting treatment with Gefitinib or Erlotinib in lung or colon cancer patients

Biomarker EGFR in NSCLC & COLON Cancer

Indication of Specific Therapy

Biomarker

Mutation Study

Indication of Specific Therapy

Marker / Mutation StudiedUtility / Specific Therapy

EGFR Biomarker

Lung/Bladder/Overian cancer. The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor involved in the proliferation and differentiation of normal cells. EGFR is often overexpressed on malignant cells, including 40 to 80 percent of patients with Non-Small Cell Lung Cancer (NSCLC). Overexpression of EGFR in tumours leads to uncontrolled signal transduction through the receptor, which leads to increased proliferation, tumor growth, and metastasis.

This has led to the emergence of targeted therapy using EGFR inhibitor drugs Gefitinib ( Iressa) and Erlotinib (Tarceva) both of which may be used to treat patients with advanced Non Small Cell Lung Cancer (NSCLC).

MGMT( O6-Methyl Guanine-DNA Methyl Transferase)MGMT (O6-methylguanine-DNA methyltransferase) is an important DNA repair protein and tumour suppressor protein expressed in normal human tissues but overexpressed in all types of tumours.MGMT is involved in tumor cell resistance to the cytostatic activity of chemotherapeutic alkylating agents with there being a strong correlation present between MGMT activity and tumour drug resistance.

EGFR Mutation Analysisfor exon 18,19,20,&21A Predictive Test for Gefitinib & Erlotinib drug responseIn NSCLC cases, EGFR Mutation in exon 18-21 of Tyrosine Kinase region are comonly known.EGFR Mutation are predominant in patients of Asian ethnicity, adenocarcinoma, females, and non-smokers.Mutation screening provides the most direct and valuable guidance for decision on EGFR-TKIs therapy.Sequence analysis is considered the gold standered for Mutation detection.NSCLC patients with somatic mutations in EGFR, if treated with TKI are associated withResponse to therapyProlonged overall survival (OS)

At Reliance life sciences we do EGFR Mutation analysis & EGFR Biomarker study

MGMT MS-PCR( O6-Methyl Guanine-DNA Methyl Transferas)A Predictive test for response to Alkylating agent therapy MGMT methylation correlates with improved survival in cancers treated with alkylating agents.In cancer patients, MGMT promoter methylation status helps in taking decision on alkylating agent therapies.Using Methylation Specific PCR, MGMT gene methylation status helps to Predict response to alkylating therapiesTake decision on alternate regimensAvoid unnecessary administration of alkylating drugs in non-responders.

At Reliance life sciences, we do MGMT MS-PCR & MGMT Biomarker.

KRAS Mutation AnalysisA Predictive test for anti-EGFR Drug responseMutation in codon 12, 13 & 61 of KRAS gene are significantly responsible for malignant transformationKRAS Mutation in colon tumours80% in codon 1210% in codon 135% in codon 61 KRAS Mutation predict the response to Cetuximab therapyHigher probability for no response in mutated tumours(91.3%)10 fold higher chances to have relative response in non mutated tumours.Associated with wrose prognosis Performing KRAS Mutation analysis..Detecting around 95% of total KRAS gene mutations in colon cancer.

Camptosar Label Revised and FDA Approved UGT TestUGT1A1 Genotyping test

Indication for Specific Therapy

a) MGMT MS PCR. Chemo Response test for Temozolamide.EGFR Hotspot Mutation for Exon 18,19,20 & 21. Drug Response test for Gefitinib or Earlotinib for NSCLC & Colon Cancer.c) HER-2/neu Amplification by FISH. (Trustozumab Responsive). UGT1A1 Genotyping test for Irinotican Dose determination for colorectal.K RAS Mutation analysis Exon 2 and Exon 3 . Drug response test for Cetuximub &Panitumumab in colorectal cancer.BRCA1 & BRCA2 Mutation. Predictive test for Genetic Mutation in Breast cancer with genetic councelling. RET Gene Mutation. Predictive test for Medulary Thiroidcarcinoma. h) CD 20 Biomarker for Rituximab

We do--Largest range of Cancer Tissue Biomarkers/PanelIndirect IHC advantagHigh sensitivity upto 95%Studying 14 different cancer typesMUTATION StudyMGMT MS-PCR,EGFR Hot spot Mutation,KRAS Mutation,UGT1A1 Genotyping test,HER-2/neu by FISH,BCR/ABL Mutation

At Reliance Life Sciences

Our Accreditations / Affiliations

Service Provider for

Reliance Life Sciences Pvt Ltd.Navi Mumbai, India

Bangladesh Collection officeLab Science Diagnostic Ltd. 153/1 Green Road, 1st Floor.Cell 01922107402, 01742383381-83.Ph. 02-9145049

UGT1A1 Genotyping - IrinotecanIndications for TestingPatient who will receive irinotecan(in doses >150 mg/m2), who has a history of irinotecan sensitivity, or who experiences neutropenia while receiving irinotecanPatient suspected to have Gilbert syndromeLaboratory TestingUGT1A1(TA) polymorphism testRisk of irinotecan toxicity by genotypeUGT1A1genotype does not significantly influence risk of irinotecan toxicity when low dose therapy with irinotecan (eg, 15-75 mg/m2daily for five days for two consecutive weeks) is employed

Colorectal CancerLaboratory TestingColonoscopy all polyps should be removed and sent for histology examinationMolecular testing (on tissue)MSI testing for suspectedLynch syndromeMSI also found in 15-20% of sporadic colon cancerMMR mutation testing (MLH1,MSH2) should be guided by immunohistochemistryKRASmutation testingAdvanced (metastatic) colon cancer patients may benefit from anti-EGFR therapy as monotherapy or combined with chemotherapyAll candidates for anti-EGFR therapy should have testing forKRASmutation (American Society of Clinical Oncology [ASCO] National Comprehensive Cancer Network [NCCN] guideline recommendation)IfKRASmutation testing is positive, patients may have an inhibited response to anti-EGFR therapy and should not receive this therapy

UGT1A1 Genotyping - IrinotecanIndications for TestingPatient who will receive irinotecan(in doses >150 mg/m2), who has a history of irinotecan sensitivity, or who experiences neutropenia while receiving irinotecanPatient suspected to have Gilbert syndromeLaboratory TestingUGT1A1(TA) polymorphism testRisk of irinotecan toxicity by genotypeUGT1A1genotype does not significantly influence risk of irinotecan toxicity when low dose therapy with irinotecan (eg, 15-75 mg/m2daily for five days for two consecutive weeks) is employed

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