ONCOLOGY GENETICS & GENOMICS Kristi Wiggins MSN, RN,ANP-BC, AOCNP, CCRC Oncology Adult Nurse Practitioner Duke University Adult Stem Cell Transplant

Objectives Discuss basic concepts of genetics/genomics in oncology and how these impact personalized healthcare Distinguish between cancer genetic testing and genomic testing and name at least one of each Identify at least three components of a hereditary cancer risk assessment Name at least two types of molecular testing used to evaluate cancer genetic/genomic expression that provide diagnostic and prognostic information

All cancer is“genetic” What does this mean? The lay public tends to interpret this statement as meaning that all cancers are “inherited.” What this statement actually means is that cancers arise from normal cells that have had an alteration in one or more genes. Now, it is true that some types of cancers have a true hereditary component and the risk for those cancers is inherited, not the actual cancer itself. Most cancers are considered “sporadic” rather than inherited, in that causes are largely environmental. Even the cancers caused by exposure to environmental carcinogens are “genetic” because the carcinogen sufficiently damages or alters the genes in the exposed cells to make these cells forget how to be good citizens and instead take on (express) malignant characteristics.

Genetics: is the study of genes & heredity What is inherited from one’s predecessors Genetics focuses primarily on the likelihood of developing cancer Genetic tests find mutations, not disease

Cancer Genomics: The study of tumor biology & how genes interact and are expressed as a whole Genomics and gene expression profiling tools focus on the cancer itself and can help determine How aggressive is the cancer (prognosis) What is the likely benefit from treatment (prediction)

Identifying Patients at Risk Family History & Personal Medical History Identify diseases that appear to “run in the family” These can be considered genetic diseases. Taking a family history can provide important information about a patient’s risk of disease. http://www.hhs.gov/familyhistory/ http://www.geneticalliance.org/fhh Common diseases result from the combined effects of multiple genes and environmental factors. This makes it very difficult to predict whether or not an individual will inherit disease. One reason for this is that the number of genes contributing to so-called "polygenic" diseases is usually not known. The number of genes carried by parents or children that can increase risk is also not known. And, environmental factors can greatly vary an individual's risk of developing disease. Complex diseases that are influenced by multiple genes, risk is much more difficult to calculate.

Genetic Basis for Cancer Dysregulated cell signaling pathways Variations in gene expression These two characteristics control and influence genes to promote: Tumor initiation Tumor growth and spread While some cancers occur as a result of an inherited mutation in a cancer suppressor gene, more occur because of mutations acquired during a lifetime. In fact, it looks as if at least two “hits” or mutations must occur in a somatic cell (body cell other than the sex cells) in order for cancer to develop.

Metastasis Initiation Promotion Promotion Promotion Normal Cell Altered Cell Promotion Tumor Promotion Higher Grade Malignancy progression The start of cancer development and progression to the most malignant state, occurs through initiation, promotion, progression, and metastasis. All of these processes involve changes or influences at the gene level. (They also require time). At every point during this process, the body is making attempts to defeat the process. Promotion Metastasis

Stuck Accelerators & Faulty Brakes Proto-Oncogenes (normal role is “accelerator”) Tumor-Suppressors (normal role is “brake”)

Tumor Suppressor Genes: Two “hits” required Normal suppressor gene alleles, brakes function fine One suppressor gene allele mutated, accident waiting to happen This slide shows suppressor gene function as a “brake” analogy. When both sets of brakes in a car are in good repair and can perform their function, the car is prevented from hitting the brick wall (cancer development). If one set of brakes (one gene allele of a suppressor gene) is not functioning, the car can hit the brick wall more easily under certain conditions (such as wet or icy pavement). So the risk for cancer increases (but is not guaranteed) if one gene allele of a suppressor gene is mutated (either inherited that way or occurs as a result of carcinogenic exposure). If both sets of brakes (both gene alleles) aren’t functional (both have been mutated), the risk for hitting the wall or developing cancer is extremely high. Two suppressor gene alleles mutated, disaster

Genetic Testing: Heredity Genetics is the study of what is inherited from one’s predecessors (Germline). One allele, or one copy of genes, from each parent Genetics influence an individual’s genomics, it is responsible for only 5-10% of cancers Genetics focuses primarily on the likelihood of developing cancer Genetic tests find mutations, not disease Source: Understanding Cancer Series: Gene Testing, National Cancer Institute

Genetic Test Example BRCA1 and BRCA2 The genetic/hereditary make up of patients is tested for BRCA1 and BRCA2 mutations. Patients with those mutations have higher chances of developing breast cancer. Example of Genetic test: BRCA1 and BRCA2 look at specific gene mutations on the patient’s genetic make up that make them more susceptible to developing breast cancer in their lifetime. Example of Genomic test: Oncotype DX looks at the expression level of 21 different genes on the patient’s tumor tissue and correlates that information with the likelihood of distant recurrence in 10 years and the magnitude of chemotherapy benefit.

Genomic Test Example Oncotype DX® Breast Cancer Assay The expression level of 21 genes is measured in tumor tissue from patients that have already been diagnosed with breast cancer. This assay evaluates if a patient is going to recur (prognostic). And predicts benefit from chemotherapy and hormonal therapy (predictive). Example of Genetic test: BRCA1 and BRCA2 look at specific gene mutations on the patient’s genetic make up that make them more susceptible to developing breast cancer in their lifetime. Example of Genomic test: Oncotype DX looks at the expression level of 21 different genes on the patient’s tumor tissue and correlates that information with the likelihood of distant recurrence in 10 years and the magnitude of chemotherapy benefit.

Genetic Testing vs. Genomic Tumor Profiling Germline Heredity One allele, copy of genes, from each parent BRCA 1 & 2 Tumor DNA Malignant transformation Over-expression of normal genes Her2neu Mutated Suppressor genes p53 EGFR lung cancer – Tarceva Her2neu – Herceptin, Tykerb Focus on Tumor profiling and targeting tumor abnormalities to stop cancer growth.

Genomic Testing: Role in Cancer Diagnosis & Treatment The Presence, Absence, or Over-Abundance of Genes Can Infer 1. Prognosis 2. Treatment Outcomes Gene array to determine risk – i.e. Oncotype DX Chemo responsiveness – specific genes translate into response to specific drugs Correlate w/triple neg reponse to anthracyclines – usually? i.e. 4 sub-types of Breast cancer Luminal A – ER/PR+ HER2 neg = good risk Luminal B – variants with ER+ / PR- HER2 neg = moderate risk Basal-like – Triple negative = poor risk

TUMOR PROFILING Cancer gene expression Determine risk Determine prognosis Evaluate relationship to chemotherapy responsiveness Lead into MICROARRAY technology

Microarray Analysis Explain function, markers/probes, and application of Microarray technology “shows marker/protein pattern” Much of the excitement today centers on gene expression profiling that uses a technology called microarrays. A DNA microarray is a thin-sized chip that has been spotted at fixed locations with thousands of single-stranded DNA fragments corresponding to various genes of interest. A single microarray may contain 10,000 or more spots, each containing pieces of DNA from a different gene. Fluorescent-labeled probe DNA fragments are added to ask if there are any places on the microarray where the probe strands can match and bind. Complete patterns of gene activity can be captured with this technology. www.biomedcentral.com

Cancer Gene Expression Oncotype DX® 21-Gene Recurrence Score™ (RS) Assay 16 Cancer and 5 Reference Genes From 3 Studies PROLIFERATION Ki-67 STK15 Survivin Cyclin B1 MYBL2 ESTROGEN ER PR Bcl2 SCUBE2 INVASION Stromelysin 3 Cathepsin L2 HER2 GRB7 BAG1 GSTM1 REFERENCE Beta-actin GAPDH RPLPO GUS TFRC CD68 The final gene set used for the Oncotype DX assay includes the 16 cancer genes identified in the clinical trials: 5 genes are in the proliferation group, 2 in the HER2 group, 4 in the estrogen receptor group, 2 in the invasion group, and 3 are unaligned. Some of the genes are well known in the breast cancer literature; others are relatively new. The 5 reference genes are used for normalizing the expression of the cancer-related genes. The 16 genes presented in this slide were selected for the Oncotype DX™ assay based on the three clinical trials, which demonstrated a consistent statistical link between these genes and distant breast cancer recurrence and the most robust predictive power across the three studies. Paik et al. N England J Med. 2004;351: 2817-2826

Importance of Gene Expression Risk Prognosis Predict Chemosensitivity Pharmacogenomics

Pharmacogenomics How an individual's genetic make-up affects the body's response to drugs Holds the promise that drugs might one day be tailor-made for individuals, adapted to each person's unique genetic makeup. GOALS: Better, safer drugs More accurate dosing Better vaccines Reduced cost Advanced disease screening Environment, diet, age, lifestyle, and state of health all can influence a person's response to medicines http://www.ornl.gov/sci/techresources/Human_Genome/medicine/pharma.shtml httwww.ornl.gov/sci/techresources/Human_Genome/medicine/pharma.shtml

Definitions Pharmacokinetics process by which a drug is absorbed, distributed, metabolized, and eliminated by the body. What you do to a drug (i.e. your SNPs). Pharmacodynamics action of a drug in the body, including absorption, distribution, localization in the tissues, biotransformation, and excretion What a drug does to you

Pharmacogenomic Targets, Molecular Tests, & Therapies BREAST Gene Expression Tested: Tests Used: Treatments Indicated by Positive Results: Examples: Her2neu Immunohisto-chemistry (IHC) & FISH Her2neu antagonists Herceptin & Tykerb ER/PR (IHC) Endocrine Therapy Tamoxifen, aromatase inhibitors HER2 – breast & lung, possibly gastric cancer; over-expression seen in colon, bladder, ovarian, endometrial, uterine, head & neck, esophageal. Targeted RX: Herceptin, Tykerb TKI – CML BCR/ABL Ph+ causes aberrant TKI activity. Target RX: imatinib (Gleevec). GIST over-expression of c-kit, also Target RX: Gleevec and Dasatinib. Dasatinib for resistant CML and ALL Ph+. Nilotinib. Now Posatinib in 513i mutation CML. CD20 – located on B-lymphocytes, >90% NHL, also in CLL; Targeted RX: Rituximab. Refractory CLL: Targeted RX: Ofatumumab. EGFR – Targeted RX: Tarceva (only 10% NSCLC responded to Iressa) KRAS – EGFR mutations are more commonly found in tumors from patients who never smoked cigarettes (1), while KRAS mutations are present in those with significant tobacco exposure (KRAS mutations as a negative prognostic factor with a hazard ratio (HR) for death of 1.40 (95% confidence interval [CI], 1.18–1.65). Among adenocarcinomas—the histology most likely to have KRAS mutations—the HR was 1.50 (95% CI, 1.26–1.80). Unfortunately, since all prognostic factors were not available for all studies, KRAS mutations have been investigated as negative predictors of benefit from erlotinib or gefitinib treatment. Since KRAS is a downstream effector of EGFR, the target of erlotinib and gefitinib, we hypothesized that inhibition of EGFR would be ineffective in controlling tumors with KRAS mutations. Eval in colon cancer.; RAF family of kinases VEGF – Renal Ca, anti-angiogenesis Rx. Avastin for Brst Ca, etc. Brentuximab vedotin is a novel antibody-drug conjugate directed against the CD30 receptor present primarily in Hodgkin and Reed Sternberg cells and in T-cell anaplastic large cell lymphoma. chimeric monoclonal antibody brentuximab (which targets the cell-membrane protein CD30) linked to three to five units of the antimitotic agent monomethyl auristatin E (MMAE, reflected by the 'vedotin' in the drug's name). The antibody portion of the drug attaches to CD30 on the surface of malignant cells, delivering MMAE which is responsible for the anti-tumour activity.[3][4] Hence it is an antibody-drug conjugate.

Breast Cancer – Estrogen Receptor Immunohistochemistry (IHC) 1+ IHC Breast Cancer – Estrogen Receptor 2+ IHC DEF: Protein detection by antigen/antibody recognition Visual stain to detect presence/absence of the protein Advantages: Need only small amount of tissue to test Use on fixed or fresh tissue & can be stored indefinitely for future reference Relatively inexpensive Disadvantages: Results are qualitative (i.e. positive, negative, indeterminate) Inconsistent results depending on quality/degree of staining Interpretation variability Presence of protein only, cannot discern its function 3+ IHC Rohit Bhargava, William L Gerald, Allan R Li, Qiulu Pan, Priti Lal, Marc Ladanyi and Beiyun Chen

Pharmacogenomic Targets, Molecular Tests, & Therapies Chronic Myelogenous Leukemia (CML) Gene Expression Tested: Tests Used: Treatments Indicated by Positive Results: Examples: Tyrosine Kinase Cytogenetics & Fluorescent In Situ Hybridization (FISH) Tyrosine Kinase Inhibitors (TKI) Imatinib (Gleevec), Dasatinib, Nilotinib Tyrosine Kinase with T315i mutation FISH Posatinib HER2 – breast & lung, possibly gastric cancer; over-expression seen in colon, bladder, ovarian, endometrial, uterine, head & neck, esophageal. Targeted RX: Herceptin, Tykerb TKI – CML BCR/ABL Ph+ causes aberrant TKI activity. Target RX: imatinib (Gleevec). GIST over-expression of c-kit, also Target RX: Gleevec and Dasatinib. Dasatinib for resistant CML and ALL Ph+. Nilotinib. Now Posatinib in 513i mutation CML. CD20 – located on B-lymphocytes, >90% NHL, also in CLL; Targeted RX: Rituximab. Refractory CLL: Targeted RX: Ofatumumab. EGFR – Targeted RX: Tarceva (only 10% NSCLC responded to Iressa) KRAS – EGFR mutations are more commonly found in tumors from patients who never smoked cigarettes (1), while KRAS mutations are present in those with significant tobacco exposure (KRAS mutations as a negative prognostic factor with a hazard ratio (HR) for death of 1.40 (95% confidence interval [CI], 1.18–1.65). Among adenocarcinomas—the histology most likely to have KRAS mutations—the HR was 1.50 (95% CI, 1.26–1.80). Unfortunately, since all prognostic factors were not available for all studies, KRAS mutations have been investigated as negative predictors of benefit from erlotinib or gefitinib treatment. Since KRAS is a downstream effector of EGFR, the target of erlotinib and gefitinib, we hypothesized that inhibition of EGFR would be ineffective in controlling tumors with KRAS mutations. Eval in colon cancer.; RAF family of kinases VEGF – Renal Ca, anti-angiogenesis Rx. Avastin for Brst Ca, etc. Brentuximab vedotin is a novel antibody-drug conjugate directed against the CD30 receptor present primarily in Hodgkin and Reed Sternberg cells and in T-cell anaplastic large cell lymphoma. chimeric monoclonal antibody brentuximab (which targets the cell-membrane protein CD30) linked to three to five units of the antimitotic agent monomethyl auristatin E (MMAE, reflected by the 'vedotin' in the drug's name). The antibody portion of the drug attaches to CD30 on the surface of malignant cells, delivering MMAE which is responsible for the anti-tumour activity.[3][4] Hence it is an antibody-drug conjugate.

Example: Philadelphia Chromosome in CML Cytogenetics – Chromosome analysis Example: Philadelphia Chromosome in CML Erroneous sharing of chromosomal material btw chromosomes 9 & 22; Creating the fusion of the BCR region and ABL region = Philadelphia chromosome http://en.wikipedia.org/wiki/File:Philadelphia_Chromosom.svg

CML – Philadelphia Chromosome t 9:22 Fluorescent in situ Hybridization (FISH) CML – Philadelphia Chromosome t 9:22 Translocation FISH probe shows: Signals of BCR gene (green signal) ABL1 gene (red signal) Arrows denote fusion signal on chromosome 9 = BCR/ABL gene that is diagnostic of CML.

Current applications of Genomic Science Gene Testing Colon Cancer MSH gene test – at time of surgical resection to stratify risk UGT1A1 – Screening, positivity predicts toxicity to Irinotecan Acute Myelogenous Leukemia FLT3-ITD – worse outcomes, implement more aggressive therapy, and/or stem cell transplant NPM1 – favorable outcomes (only in the absence of FLT3+), may be cured with chemo alone Non-Small Cell Lung Cancer (NSCLC) EGFR & EML4 – increased malignant behavior, implement more aggressive therapy ALK – worse prognosis, use ALK inhibitors

Current applications of Genomic Science Genomic Testing Expression Profiles Breast Cancer To Direct the Use of Pre-operative Chemotherapy for Early Stage Breast Cancer Goal – Predict Chemosensitivity

Summary Consider how genetics/genomics in oncology impact the personalized healthcare you provide Become more familiar with the ‘red flags’ on a hereditary cancer risk assessment Understand the difference between: Genetic testing Genomic testing Identify molecular tests used to evaluate cancer genetic/genomic expression: IHC FISH Cytogenetics i.e. BRCA 1 and 2 - gene i.e. Oncotype DX - genomic

The Challenge No single technology at present that will detect all the types of abnormality--deletions, rearrangements, point mutations, frameshift insertions, amplifications, imprinting, and epigenetic changes--implicated in cancer. Microarrays and gene chip analysis, however, are beginning to unveil some key genomic drivers. (Please see Molecular Diagnostics for more information.) Many clinical trials now include genomic profiles of cancer patients as prognostic and diagnostic indicators. Genomic profiles are even used to monitor where and how the cancer genome has been hit during molecularly targeted therapies. Mining and sharing all this data should eventually help oncologists to better integrate the genotypic and phenotypic changes that occur in a biosystem during cancer's progression. This knowledge will be used to bring earlier and better interventions to cancer patients.

Resources American Association for Cancer Research http://clincancerres.aacrjournals.org/content/9/10/3645/F2.expansion?ck=nck Bharhava, R., et al (2011). Modern Pathology. Retrieved 9/18/11, fromhttp://www.nature.com/modpathol/journal/v18/n8/fig_tab/3800438f1.html Eastman, P. (2007, April 25). IOM Report: Much Stronger Leadership Needed to Fuel Biomarker R&D. Oncology Times, XXIX No. 8, p. 11. Genomic Health: Oncotype DX Nursing Education Materials Goetsch, C.M. (2011). Genetic Tumor Profiling and Genetically Targeted Cancer Therapy. Seminars in Oncology Nursing, 27, 34-44. http://www.nature.com/bjc/journal/v100/n1/fig_tab/6604781f1.html#figure-title http://en.wikipedia.org/wiki/File:Philadelphia_Chromosom.svg International Society of Nurses in Genetics www.isong.org/ Lab Med Online (2011). A Case of Concomitant Inv(3)(q21q26) and Cryptic BCR/ABL1 Rearrangement in the Blast Crisis of Chronic Myeloid Leukemia . Retrieved 9/19/11, from http://dx.doi.org/10.3343/lmo.2011.1.3.7 Lee, H., et al., (2011). Lab Med Online. 2011 Jul;1(3):163-167. Published online 2011 July 05.  http://dx.doi.org/10.3343/lmo.2011.1.3.7, Laboratory Medicine Online Nature Reviews Drug Discovery 3, 739-748 (September 2004) National Human Genetics Research Institute: www.nhgri.gov N EnglandJ Med 2009;360:753-764. Oncology Nursing Society: www.ons.org Stenger, E. (2011). Genetic Profiling in Non-Small Lung Cancer. ASCO Post, 5, 6-8. Abstract retrieved 9/09/11, from www.ASCOPost.com. Susman, E. (2007, April 25). Increasing Interest in Stopping Cancer Stem Cells as New Treatment Method. Oncology Times, XXIX No. 8, p. 24. www.cancer.gov/cancertopics/understandingcancer/ www.cancer.gov/cancertopics/understandingcancer/genetesting/AllPages www.cellulargenetix.com/pharmacogenomics.asp www.genomics.gov www.insidecancer.org/ www.medscape.com/viewarticle/487451_4 www.nature.com/cancer/index.html www.nature.com/nrd/journal/v3/n9/images/nrd1497-i1.jpg www.ncbi.nlm.nih.gov/About/primer/microarrays.html www.nci.gov