Patricia Aoun MD, MPH Professor and Vice-Chair for Clinical Affairs Medical Director, Clinical Laboratories Department of Pathology City of Hope

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1 Patricia Aoun MD, MPH Professor and Vice-Chair for Clinical Affairs Medical Director, Clinical Laboratories Department of Pathology City of Hope National Medical Center

2 Disclosures I have no disclosures and no conflicts of interest.

3 Diagnosis of Lymphoma in the Era of Precision Medicine Morphological Assessment (Pattern Recognition) Atypical Benign Immunophenotyping, including for therapeutic targets (CD20, CD30, CD38,CD138, ALK, PD-1, PDL-1, etc.) Malignant Histological Subtyping Molecular Genetic Testing Clonality Classification Therapeutic targets Disease monitoring

4 The Landscape of Genetic Alterations in Lymphoma Immunoglobulin heavy and light chain gene rearrangements T-cell receptor gamma and beta chain gene rearrangements by PCR with BIOMED-2 primers Gene fusions (many) Gene mutations: single or multiple nucleotide variations, insertions, deletions, amplifications, copy number alterations (thousands )

5 A Few Examples Non-Hodgkin Lymphoma Subtype Diffuse Large B-cell Lymphoma Chronic Lymphocytic Leukemia Burkitt Lymphoma PTCL Genetic Alterations Translocations: BCL2, BCL6, CMYC Mutations: ARID1A, ARID1B, BCL6, CARD11, CD79B, CREBPP/EP300, EZH2, KDM2, KDM6A, KDM6B, MLL2, MLL3, MYD88, SETD2, SGKI, SMARCA, SMARCB, SMARC, TP53, and many more. Aberrations: del 17p, del 11q, +12, del 13q Mutations: ATM, DDX3X, IDH1, NOTCH1, SF3B1, TP53, and many more. Translocations: CMYC Mutations: DDX3X,GNA13, ID3,MKI67, PDCD11, SMARCA4, TCF3, TP53, and many more. Mutations: DNMT3A, ETV6, IDH2, JAK2, JAK3, NOTCH1, RHOA, STAT3, STAT5, TET2, and many more.

6 Making Sense of the Landscape of Genetic Alterations in Lymphoma Cancer (lymphoma) is the result of Gain of function mutations in oncogenes, and Loss of function mutations in tumor suppressor genes. But, not all mutations matter. Driver mutations, passenger mutations, backseat driver mutations And, the cancer (lymphoma) genome is always changing. Primary and metastatic tumors have different mutations. Within the same tumor, there is genomic heterogeneity.

7 Making Sense of the Landscape of Genetic Alterations in Lymphoma Clinically relevant mutations are actionable: Diagnostic Prognostic Predictive On the other hand, our understanding of specific mutations is constantly changing.

8 2011: Case 1: 55 year-old man with acute onset of hip pain Imaging XRAY: Bony abnormality at obturator foramen CT: Incomplete fracture of femoral neck, c/w pathological fracture Normal CBC and peripheral blood smear Normal LDH, BUN/Creatinine Elevated Alkaline Phosphatase Normal total protein with low albumin Quantitative immunoglobulins: Normal Serum protein electrophoresis: Acute phase reaction Immunofixation electrophoresis: Negative

9 Case 1: Femoral head fragments

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11 Case 1: Bone Marrow- No morphological, immunophenotypic or cytogenetic abnormality.

12 Case 1: Femoral head fragments TRAP and DBA44 expression: Characteristic of Hairy cell leukemia (HCL) but not 100% specific Cyclin D1: Hairy cell leukemia, mantle cell lymphoma, myeloma CD10 expression (Jasionowski et al, AJCP 2003): Described in about 10% of HCL cases, all BCL-6 negative BCL-6 expression: Usually negative in HCL and therefore significance unclear. Next steps??

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14 Tiacci et al, N Engl J Med 2011; 364: Whole exome sequencing of enriched, purified leukemic and matched normal peripheral blood cells from index patient with HCL Sanger sequencing of 47 additional patients with HCL Identified 5 missense clonal somatic mutations, including a heterozygous mutation=> BRAF V600E variant protein BRAF V600E not present in 195 patients with other peripheral blood lymphomas/leukemias

15 Case 1: BRAF mutation analysis by pyrosequencing

16 2015 Case 2: 3-year old boy with skin nodules

17 Case 2: Bone Marrow

18 Case 2: Clinical course Failed multiple conventional therapies. Underwent hematopoietic stem cell transplantation. Relapsed with high burden of disease. Further therapeutic options?

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20 COH Clinical Molecular Diagnostics Laboratory Comprehensive tumor testing by Next Generation Sequencing Onco Gene Mutation Panel Targets >2800 mutations in 49 key cancer genes ABL1 AKT1 ALK APC ATM BRAF CDH1 CDKN2A CSF1R CTNNB1 EGFR ERBB2 ERBB4 EZH2 FBXW7 FGFR1 FGFR2 FGFR3 GNA11 GNAQ GNAS HNF1A HRAS IDH1 IDH2 JAK2 JAK3 KDR KIT KRAS MET MLH1 MPL NOTCH1 NPM1 NRAS PDGFRA PIK3CA PTEN PTPN11 RB1 RET SMAD4 SSMARCB1 SMO SRC STK11 TP53 VHL

21 Case 2: BRAF mutation testing of skin biopsy BRAF gene analysis by Next Generation Sequencing (NGS) 77 mutations within exons 11 and 15 Genomic alteration identified in skin biopsy: c1799t>a; p. V600E

22 EGFR Signaling pathway: KRAS, NRAS, BRAF, PIK3CA, PTEN

23 BRAF mutations are driver mutations

24 Vemurafenib

25 Molecular diagnostics enable precision medicine Clayton Christensen, Harvard Business School 2008

26 2011National Research Council Task Force: Toward Precision Medicine Is it possible to develop a new taxonomy of human disease by classifying diseases based on molecular information instead of by groups of symptoms or organ sites?

27 Vemurafenib in Multiple Nonmelanoma Cancers with BRAF V600 Mutations Hyman et al, N Engl J Med 2015;373: Histology-independent early phase 2 basket trial 122 patients from 23 centers in specific cancer cohorts Langerhans Cell Histiocytosis/Erdman-Chester Disease Non-small Cell Lung Carcinoma Colorectal Carcinoma Vemurafenib monotherapy Vemurafenib + Cetuximab (anti-egfr agent) Cholangiocarcinoma Anaplastic Thyroid Carcinoma Others: Primary Brain Tumors, Multiple Myeloma, Ovarian, Breast, Salivary Duct, Pancreatic, Clear Cell Sarcoma, Unknown Primary Excluded: Melanoma, Papillary Thyroid Carcinoma, Leukemia, Lymphoma Primary Endpoint: Response at week 8

28 Vemurafenib in Multiple Nonmelanoma Cancers with BRAF V600 Mutations Hyman et al, N Engl J Med 2015; 373: Response LCH/ECD (N=14) NSCLC (N=19) CRC MonoRx (N=10) CRC ComboRx (N=26) Cholangio CA (N=8) Anaplastic Thyroid (N=7) Complete Response Partial Response Stable Disease Progressive Disease 7% 36% 57% 0% 0% 42% 42% 11% 0% 0% 50% 50% 0% 4% 69% 27% 0% 12% 50% 38% 14% 14% 0% 57%

29 Precision Medicine: Towards a New Molecular Taxonomy of Cancer? Not all BRAF-mutated tumor types respond to BRAF-targeted therapy. Histology still matters. In 2016, histology with molecular subtypes matters most. But, the medical community has no practice standards for which genes to test in order to assign molecular subtypes.

30 What Test Do I Order????? Specimens Molecular Diagnostics Lab Results

31 Developing Practice Standards for Genomic Testing

32 Number of genes/panel: Total number of genes across all panels: 611 Number of genes present in EVERY panel: 0 54 genes were listed in 3 panels 43 genes were listed in 4 panels 393 genes were listed in only 1 or 2 panels

33 Foundation Heme Entire coding sequence of 405 genes Base substitutions, indels, copy number alterations Selected introns of 31 genes involved in rearrangements (gene fusions) RNA sequencing of 265 genes involved in hematological malignancies, sarcoma, and pediatric cancers List Price = $7200

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35 2016 Development Pipeline: Hematological Malignancies NGS 74 gene mutation panel ASXL1 CCND1 EP300 IDH1 KMT2D NRAS SF3B1 WHSC1L1 ATM CCND3 ETV6 IDH2 KRAS PAX5 SPI1 WT1 BCOR CD79A EZH2 IL7R MEF2B PDGFRA SRSF2 ZRSR2 BIRC3 CD79B FBXW7 JAK1 MIR142 PHF6 STAG2 BRAF CDKN2A FLT3 JAK2 MPL PIK3CA SUZ12 BTK CEBPA GATA1 JAK3 MYC PTEN TCF3 CALR CHEK2 GATA2 KDM6A MYD88 PTPN1 TET2 CARD11 CREBBP HRAS KIT NOTCH1 PTPN11 TP53 CBL CSF3R ID3 KMT2A NOTCH2 RUNX1 U2AF1 CBLB DNMT3A IKZF1 KMT2C NPM1 SETBP1 WHSC1

36 2016 Development Pipeline: Hematological Malignancies Fusion Panel by NGS ABL1 ABL2 ALK BCR CBFB CRLF2 CSFIR FGFR1 JAK2 KMT2A MECOM MKL1 NOTCH1 NUP214 PDGFRA PDGFRB PICALM RARA RBM15 RUNX1 RUNX1T1 TAL1 TCF3

37 Developing Practice Standards for Genomic Testing Remember, clinically relevant mutations are actionable: Diagnostic, Prognostic, Predictive Single gene tests: Still useful for diagnosis, prognosis, or prediction of therapeutic response when applied in the context of morphological findings. Targeted gene panels: Focus on clinically relevant mutations. Allow for greater depth of sequencing to detect mutations found at low frequencies. Large gene panels: Rare tumors for which the driver mutations are unknown. Common tumors that have been shown not to have the usual mutations. Relapsed tumors not responsive to standard of care therapies

38 Leveraging Tumor Mutation Profiles to Monitor Patients During/After Therapy: Liquid Biopsies Peripheral blood: Quantitative allele specific, real-time polymerase chain reaction test for the BRAF V600E mutation Lower limit of detection 0.02% in whole blood Urine Qualitative droplet digital PCR assay for cell free DNA Lower limit of detection 0.03%

39 Leveraging Tumor Mutation Profiles to Monitor Patients During and After Therapy Next Generation Sequencing of B and T receptor genes in lymphoma and leukemia Identification of multiple clones when present Subsequent testing of peripheral blood for circulating tumor DNA to detect minimal residual disease or early relapse Lower limit of detection % (1 in a million) Studied in Diffuse large B-cell lymphoma Mantle cell lymphoma Chronic lymphocytic leukemia Acute lymphoblastic leukemia Multiple myeloma Cutaneous T-cell lymphoma Classical Hodgkin Lymphoma Commercially available as clonoseq ID and clonoseq MRD assays for circulating tumor DNA

40 Leveraging Tumor Mutation Profiles to Monitor Patients During and After Therapy The medical community has few practice standards for monitoring patients with liquid biopsies. Which test and how often? Outside of a Clinical Trial? How much does it cost? Peripheral blood BRAF quantitative PCR assay: $890 Urine cf DNA BRAF assay: $1495 Initial tumor profiling, clonoseq ID $1200 Peripheral blood monitoring, clonoseq MRD $1300

41 Molecular Testing in the Era of Precision Medicine Lymphoma and Other Malignancies Understand and acknowledge the goal of performing each test for each patient each time. Clinical utility (drive patient care) Discovery (research) Both Decide up front when, why, and how often testing will be performed. Consider the cost and the return on the investment. Select tests and reference labs thoughtfully. The rate of genomic discovery and test design is much faster than the rate of clinical utility. Molecular testing for hematological malignancies (and all cancer) is about balancing discovery with clinical utility and cost.

42 COH Clinical Molecular Diagnostics Laboratory