CANCER. Clinical Validation of Breast Cancer Predictive Markers

Transcription

1 Clinical Validation of Breast Cancer Predictive Markers David Hicks, MD Loralee McMahon, MS, HTL(ASCP) CANCER The human body is composed of billions of highly regulated cells Cancer cells no longer respond to normal regulatory mechanisms - Autonomous growth Cancer cells - overrule normal cell cycle checkpoints and start to divide & grow in uncontrolled manner Become immortal Arrange blood (nutrient) supply (angiogenesis) Invade surrounding tissue Spread to distant sites (metastasis) Cancer may be hereditary or caused by environmental factors - But all cancer is initiated by errors in the DNA DNA and Cancer Cancer may be hereditary or caused by environmental factors - But all cancer is initiated by errors in the DNA The errors can be divided into three categories; 1. Mutations In which very few bases are substituted, removed or added 2. Translocations In which large fragments of DNA strands exchange locations and thereby end up at wrong places in the DNA Nucleus DNA (chromosomes) 3. Changes in the gene copy number In a normal cell we have two copies of each gene but in some cancer cells -Extra copies (amplification) -Gene loss (deletion)

2 Breast Cancer Patients Breast Cancer Diversity Known Breast Cancer Biomarkers ER Various Disease Subtypes Clinical Diversity Accurate identification - subsets for therapy Tamoxifen Herceptin HER2 Assessment of tumor biology and molecular drivers of disease progression Potential targets for therapy HER2+ Breast Cancer: HER2 Gene Amplification Responsible for HER2 Receptor Over-expression in Most Cases HER2 Gene HER2 mrna HER2 Protein Normal copy # Physiologic expression Normal amount Normal amount ~20,000 receptors/cell Amplified copy # Pathologic alteration Chromosome 17 Increased amount HER2 gene amplification 15-20% of early breast cancers HER2 alteration occurs early in disease course (stable genetic change) Gene amplification drives overexpression of the HER2 protein Over-expression > 1,000,000 receptors/cell Receptor signaling proliferation angiogenesis apoptosis invasion = Aggressive disease Potential Consequences of HER2 Dysregulation Normal: ~20,000-50,000 HER2 receptors per cell 1 Normal HER2 expression regulated growth & development, is associated with 2 :!Angiogenesis!Cell proliferation!apoptosis 1. Pegram M et al. Cancer Treat Res. 2000;103: Rowinsky EK. Annu Rev Med. 2004;55:

3 Potential Consequences of HER2 Dysregulation (cont d) HER2-positive: ~2 million HER2 receptors per cell 1 1. Pegram M et al. Cancer Treat Res. 2000;103: Rowinsky EK. Annu Rev Med. 2004;55: HER2 overexpression is associated with 2 : Angiogenesis Cell proliferation Invasion Metastasis Tumor cell survival Apoptosis HER2 Over-Expression: Ideal Target for Breast Cancer Treatment Correlates with tumor development Significant adverse prognostic factor Found in both primary & metastatic lesions Trastuzumab developed as a targeted therapy against HER2 (+) breast cancer Trastuzumab effective in HER2 (+) breast cancer in adjuvant and metastatic settings Only HER2 (+) patients benefit from treatment 8 Proposed Trastuzumab Mechanism of Action Herceptin HER2 receptor Humanized monoclonal antibody Herceptin demonstrates a high specificity and affinity for an extracellular epitope of the HER2 receptor protein Blocks receptor signaling May promote immune mediated cell death Synergistic when combined with chemotherapy Pre-clinical studies & clinical trials suggested Herceptin only effective against tumors that over-express HER2 1. Sliwkowski MX et al. Semin Oncol. 1999;26(suppl 12):60-70; 2. Yakes FM et al. Cancer Res. 2002;62: ; 3. Arnould L et al. Br J Cancer. 2006;94: ; 4. Bianco AR. J Chemother. 2004;16:52-54; 5. Pegram MD et al. J Natl Cancer Inst. 2004;96: ; 6. Baselga J. Cancer Res. 1998;58: ; 7. Herceptin Prescribing Information. Genentech, Inc. October 29, 2010.

4 Rationale for HER2 Testing in Every New Breast Cancer Patient Testing helps identify which patients will be most appropriate for treatment & likely to respond HER2 testing has significant implications for therapeutic decisions Quality assurance & accuracy, high priority to ensure optimal patient selection! Wolff et al. J Clin Oncol. 2007;15:118; Konecny et al. J Natl Cancer Inst. 2003;95:142; Menard et al. J Clin Oncol. 2001;19:329; Konecny et al. J Natl Cancer Inst. 2004;96:1141; Hayes et al. J Clin Oncol. 2006;24:5S. Abstract 510; Slamon et al. N Engl J Med. 2001;344:783; Cobleigh et al. J Clin Oncol. 1999;17:2639; Vogel et al. J Clin Oncol. 2002;20: Determining HER2 Status in Breast Cancer in Clinical Practice Biological event Testing Method Gene amplification Southern blot/fish*/pcr mrna synthesis Northern blot/rtpcr/rish Protein synthesis Western blot Cell membrane protein IHC* Protein shed from cell surface (ECD) Serum ELISA *Both FISH and IHC have been clinically validated and FDA approved for patient selection for HER2-targeted therapy HER2 IHC Interpretation: Breast Cancer HER2 IHC Negative HER2 IHC Equivocal HER2 IHC Positive Not candidate Tx Reflex FISH testing Candidate Tx

5 Chicken-wire pattern, intense staining & uniformity score HER2 (3+) ASCO/CAP guidelines > 95% gene amplified by FISH Complete HER2 membrane staining, non-uniform, thin rims, score IHC (2+), HER2 equivocal, reflex to FISH FISH, HER2 and CEP17 FISH morphologically driven, direct visualization of tumor cells (smaller quantities of tissue) HER2 gene is located at 17q12.1 on the long arm of chromosome 17 Normally a cell has 2 copies of chromosome 17 and therefore 2 copies of the HER2 gene Chromosome 17 Centromere HER2 gene G T A T C A T G A C C C Most HER2 assays Count HER2 & CEP17 T A C C A HER2/CEP17 Probe hybridized with target Fluorescence in Situ Hybridization Principles of the FISH Assay Freshly cut tissue sections Pre-treat tissue & cells to remove their cytoplasmic and nuclear proteins (barrier to probe) Denature target DNA by heating in formamide solution Hybridize labeled probe to denatured DNA Perform stringent wash to eliminate non-specific binding of probe Visualize results with a fluorescent microscope 15

6 Breast cancer area of infiltrating tumor cells HER2 status Non-amplified (dual colored) Good hybridization signals, nuclei are of uniform diameter 16 Non-overlapping, with intact nuclear borders 16 Breast cancer area of infiltrating tumor cells HER2 status Amplified (dual colored) HER2 Testing: IHC versus FISH: Complementary Approaches IHC & FISH: complementary methods Examine different aspects of biology of HER2driven breast cancer1 Gene amplification drives protein over-expression Original studies of HER2: gene amplification & protein overexpression highly correlated HER2 expression by IHC (FS) / gene amplification Results were highly correlated FFPE tissues used in clinical practice ASCO/CAP task force IHC & FISH are clinically validated and equally efficient in identifying patients likely to respond to Herceptin2 1. Hicks DG et al. Hum Pathol. 2005;36: Herceptin (trastuzumab) PI. May 2008, Wolff AC et al. J Clin Oncol. 2007;25:

7 Standardization of HER2 Testing: Sources of Variability HER2 testing inaccuracy remains an issue with both IHC and FISH assays Various factors can explain the variability observed in clinical practice Performance Challenges HER2 Test Results Depend on Standardization Pre-analytical variables Time to fixation Type of fixative Time in fixative Method of tissue processing Analytical variables Assay validation Equipment calibration Type of antigen retrieval Test reagents Use of standardized control materials Training of staff Use of automated methods Post-analytical variables Interpretation criteria Use of image analysis Quality assurance Laboratory accreditation Proficiency testing Pathologist certification Assay validation helps ensure that the results are reliable Wolff AC et al. J Clin Oncol. 2007;25(1): Validation Development and Implementation WHY?? 1. Patients 2. Lawyers

8 Key Players CAP Accreditation Program CMS Governing State - NYS Joint Commission More informed patients Pharmaceutical Companies FDA NYS Clinical Laboratory Standards of Practice There are several standards that deal with validation of laboratory procedures. Don t deviate from the manufacturer s instructions Validate using an alternate test/platform Documentation should include validation procedure, data and results. Principles of Analytic Validation of Immunohistochemistry Assays Developed by the CAP to provide some guidance to the lab 14 total recommendations Includes all IHC testing for the complete list

9 CAP Checklist ANP ANP ANP ANP ANP ANP ANP ANP ANP ANP All deal with testing, validation, and reporting of ER/ PR/HER2 How do I Start? Training and Optimization Get some training from the vendor or another lab on how to perform the test Optimize the test How does it perform in your hands and on your tissue samples? Write this down this will become your SOP

10 Do your homework What is the goal? How will you reach it? What method should we compare our method against? Ask your Director and/or other pathologists to assist Write it down!! Method Comparison What validated methods are out there? Is it feasible? Cost Time Steps to a Validation Plan Collect Perform Document Summarize

11 Summarize your plan in a document How are you going to validate? What are the methods you are using? What method is being used to validate the test against? Outline the time frame (try to stick to it) Write the SOP s Call in a favor Plan Collect Collect data reference papers Collect cases pull together the cases that you are using for validation Your case set should include a range of positive, negative, and equivocal Perform Do what your plan says Try to keep to the time line Ask your vendor for assistance

12 Documentation Keep the documents in a binder for the inspectors. Have the slides available for reference or to show an inspector As procedures are added or changed, validate and document Make Documentation Simple Make it easy to understand Antibody Lab 1 Lab 2 Sign off/date ER Positive Positive Dr. ABC Her Dr. ABC Calculate your Concordance rate 100% Data for HER2 IHC Validation (Compare IHC to FISH) IHC FISH Ratio < >2.2 Total

13 Data for HER2 FISH Validation (Compare FISH at lab A to FISH at lab B) Reference URMC < > 2.2 < > Summarize Take your data and analyze it Did everything come out as expected? If not, investigate those cases that didn t correlate Write up a simple summary. Example Summary 100 cases of Breast Cancer were selected from archival material and stained for ER, PR, and Her2. The tests were performed using the ER, PR and Her2 kit from Acme Antibody and performed according to the package insert. Serial sections of the same cases were sent to ABC Labs and stained with ER, PR, and Her2 from XYZ Antibody Company. The concordance rate was 96.9%.

14 How to Make Validation Easy Ask for help vendor or another lab Document everything Make sure you have staff available to perform the validation Use Tissue Micro Arrays Tissue Micro Array (TMA) Saves time, money, and reagents! Validation on a TMA CD 20 T cells PSA- prostate CA

15 Questions?