Genomics Tumor Board

Molecular Laboratory Molecular Pathology Director: Frederick Nolte, Ph.D. Cytogenetics and Molecular Genetics Director: Daynna Wolff, Ph.D. Medical Director: Cynthia Schandl, M.D., Ph.D. Associate Director: Julie Woolworth Hirschhorn, Ph.D.

Cancer Genetics  Tumors can be complex  Genetic and genomic information can help with:  Diagnosis  Prognosis  Therapeutic Decisions  Disease Monitoring  Characterization of inherited variation contributing to cancer susceptibility Normal Premalignant Cancer In situ Metastatic 1 st hit

Current Offerings  Cancer Microarray  FISH testing  Massively Parallel Sequencing MUSC Test Directory and Specimen Collection Information or

Current Testing: Cancer Microarray

How can we use microarrays in clinical cancer studies?  Diagnosis  Renal cell carcinoma  Glioblastoma  Prognosis/Disease monitor  Chronic lymphocytic leukemia  Acute myeloid leukemia/MDS  Plasma cell dysplasias  Renal cell carcinoma  Glioblastoma  Therapy  Acute lymphoblastic leukemia  Acute myeloid leukemia Genome Biology 2010, 11:R82

Why is Copy Number So Important?  Copy number variants comprise at least 3X total number SNPs  On average, 2 human differ by 4 – 24 Mb of DNA by CNV; 2.5 Mb due to SNP  Often encompass genes  Important role in human disease and in drug response Copy number lossCopy number gain Whole gene Partial gene Contiguous genes Regulatory sequences

Current Testing: 26-Gene Solid Tumor Cancer Panel  Targeted resequencing panel based on PCR amplification  Batched once per week, start date on Monday  Turn-around-time of 7-11 days  Mixture of hotspot and full exon coverage

Future Testing for Solid Tumors  In process of validating a 50-gene solid tumor panel  Will include all of the genes currently covered ABL1EGFRGNAQKRASPTPN11 AKT1ERBB2GNASMETRB1 ALKERBB4HNF1AMLH1RET APCEZH2HRASMPLSMAD4 ATMFBXW7IDH1NOTCH1SMARCB1 BRAFFGFR1IDH2NPM1SMO CDH1FGFR2JAK2NRASSRC CDKN2AFGFR3JAK3PDGFRASTK11 CSF1RFLT3KDRPIK3CATP53 CTNNB1GNA11KITPTENVHL

Future Testing for Hematological Malignancies  In process of validation of myeloid panel of 49-genes by next- generation sequencing ASXL1BCORBCOR1BRAFCALRCBLCBLB CEBPACSF3RDNMT3AETV6EZH2FLT3GATA1 GATA2GNASHRASIDH1IDH2JAK1JAK2 JAK3KDM6AKIT KMT2A/ MLL-PTD KRASMEK1MPL MYD88NOTCH1NPM1NRASPHF6PMLPTEN PTPN11RAD21RUNX1SETBP1SF3B1SMC1ASMC3 SRSF2STAG2TET2TP53U2AF1WT1ZRSR2

Solid Tumor Testing A Brief History

Solid Tumor Testing History  Molecular Laboratory performed targeted real-time PCR analysis of the EGFR, KRAS, and BRAF genes from late June of 2011 til January of 2014  In Feb of 2014, we began offering a 26-gene solid tumor cancer panel

 2014  2015 During 2015, we started creating final reports for cases with insufficient tissue

43% of insufficient specimens were due to insufficient tissue prior to any molecular analysis

2014 Positivity by Tumor Type BRAF, 18, 5.9% APC, 24, 25.3% KRAS, 35, 22.9% KRAS, 21, 22.1% EGFR, 10, 6.5% NRAS % PIK3CA 6 6.3% TP53, 21, 24.7% TP53, 30, 31.6% TP53, 62, 40.5% BRAF 6 3.9% KIT, 2, 2.4% MET 6 7.1% MET 8 5.2%

2015 Positivity by Tumor Type TP53, 86, 56.2% TP53, 41, 43.2% TP % BRAF % KIT 5 5.9% KRAS % KRAS % APC % EGFR % NRAS 5 5.9% PIK3C A 9 9.5% AKT % BRAF, 4, 4.2% SKT % BRAF, 5, 3.3%

Frequency of Variant Classification Classification 1 – Clinically Actionable / May indicate specific therapeutic intervention Classification 2 – Reported in the literature / Possible clinical relevance Classification 3 – Variant of unknown clinical significance

Original Intent of the Tumor Board  Meeting on the Second Monday of each month at 4 pm in HCC 120  Focus on clinical cases with molecular genetic analyses  Global review of molecular cases analyzed in the previous month  In-depth case discussions (up to 4 cases each month)  For example, cases may be selected because an actionable mutation that was unexpected in a particular tumor type was identified or there was informative referral lab testing information available.  This meeting will also provide a forum to discuss advanced diagnostic tests for cancer and any unmet local needs.

Suggested Format of the Tumor Board  Global review of Molecular Testing from the previous month by Molecular laboratory  Cases identified by clinicians for Tumor Board Discussion  Cases sent for tumor board agenda  Cases should be worked up by fellow(s), with assistance from oncologist and pathologist/laboratory director  Case presented by fellow(s)  Summaries of these cases, including age, diagnosis, tumor site, pathology, molecular pathology test results, and prior therapy will be distributed before the meeting  Additional items included on agenda might be future testing, new testing on the market

Standards Directive for Mutation Testing PROPOSAL FOR CLINICAL PRACTICE STANDARDS DIRECTIVE PROTOCOL  This is a document that will allow mutation analysis to be ordered directly by the diagnosing pathologist if the given criteria are met.  This type of protocol must be accepted by the ordering physicians at the Hollings Cancer Center.  Example:  All metastatic or unresectable melanoma; diagnostic specimen: Mutation status should be assessed to allocate appropriate therapy. Mutation analysis must include BRAF V600E variant testing and KIT activating mutation testing as indicated by NCCN guidelines. Analysis of progressive, newly metastatic (after mutation analysis of the primary site), recurrent disease, and suspected acquired resistance testing will require physician order.  MUSC assay*: Cancer Panel Next Generation Sequencing Analysis  Note: Once diagnosis is made, request for testing may be initiated by the resident / fellow / attending pathologist. The block should accompany the stained slide to molecular pathology for testing. An ideal area for DNA extraction should be circled by the referring pathologist with an associated percent tumor indicated. If insufficient tissue is available on the block, the pathologist should determine whether another specimen (e.g. fine needle aspirate smear, metastatic site biopsy) is available for testing.  Three protocols have been submitted for approval by HCC –colorectal, NSCLC, and melanoma. Myeloid panel may also be submitted once available in-house

Genetic Complexity Simple Chronic Myeloid Leukemia Complex Most solid tumors

Type Renal Tumor%Chromosome Abnormality Microarray Result Clear Cell Renal Cell Carcinoma ~70Loss of 3p Papillary Renal Cell Carcinoma 10-20Extra copies of 7 and 17 Chromophobe Renal Cell Carcinoma 5Loss of chromosomes 1, 2, 6, 10, 13, 17, 21 Oncocytoma<5Normal or loss of 1p Diagnostic studies

Prognostic Significance: Clear Cell Renal Cell Carcinoma Clear Cell RCC with 3p- Plus other genetic abnormalities Better prognosisWorse prognosis

Aberrations Associated with Adverse Prognosis Clear Cell Renal Cell Carcinoma Percent with abnormality ** * * * * * * * * * * * * P <0.5 ** P <0.01

Karyotype here Prognosis/Therapy for Chronic Lymphocytic Leukemia (CLL) 79 yr old male with new dx CLL; Flow 71% WBC; Cyto: deletion 11q21, +12[1/20] 60% del 13q14, 50% +12, 40% del 11q, 60% LOH 17q Clonal Evolution: Patient more likely to need therapy or on therapy

Acute Myeloid Leukemia Prognosis  Standard Cytogenetic Testing  25% Good prognosis: balanced rearrangements [t(8;21),t(15;17),inv(16)]  50% Intermediate prognosis: +8 (10%), NORMAL cytogenetics (40%)  25% Unfavorable prognosis: deletions 5q, 7q, 17p, KMT2A (MLL) rearrangement, complex karyotypes (>4 abn)  Microarray Analysis  Provides exact breakpoints for known cytogenetic aberrations  Reveals cryptic abnormalities  Copy number neutral loss of heterozygosity (10-20% of cases of normal cytogenetics cases; like LOH for 7q)  LOH regions often harbor genes with homozygous mutations

LOH is associated with gene mutations Region of LOHAssociated Gene 1pNRAS 4qTET2 7qEZH2 9pJAK2, CDKN2A, PAX5 11pWT1, PAX6 11qCBL 13qFLT3 17pTP53 19qCEBPA 21qRUNX1

Percent relapse Percent survival cnLOH control No 13q cnLOH 13q cnLOH P=0.02P=0.006 P=0.03P=0.04 HR 1.87 HR 3.45HR 6.64 HR 1.82 RelapseSurvival Importance of LOH in Prognosis in Acute Myeloid Leukemia and Myelodysplastic Syndromes Min Fang, personal communication; Cancer Sep 1;121(17):

Among FLT3-ITD patients, 13qLOH associated with poor prognosis P=0.01 n.s. Min Fang, personal communication; Cancer Sep 1;121(17):

Diagnosis/Prognosis for Glioma Loss 1p/19q: Oligodendroglioma Survival >10 years* MA better than FISH Gain7/Loss 10, amp 4q (PDGFRA,KIT): High grade glioblastoma; proneural Survival <1 year *Need molecular assessment of IDH1/2 gene

Implication for therapy: 23 year old male with Philadelphia-like B-cell Acute Lymphoblastic Leukemia; Cytogenetics and FISH testing negative 8.6 Mb deletion of 5q32q33.3

Molecular Mechanism EBF1 -PDGFRB Activates a tryrosine kinase that can be targeted by imatinib