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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. Normal 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 1st hit Premalignant Cancer In situ Metastatic Cancer Microarray FISH testing Massively Parallel Sequencing Current Offerings MUSC Test Directory and Specimen Collection Information http://pathology.musc.edu/ or https://www.testmenu.com/musclabservices 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 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 Why is Copy Number So Important? Copy number loss Copy number gain Whole gene Partial gene Contiguous genes Regulatory sequences Targeted resequencing panel based on PCR amplification Current Testing: 26-Gene Solid Tumor Cancer Panel Batched once per week, start date on Monday Turn-around-time of 7-11 days Mixture of hotspot and full exon coverage In process of validating a 50-gene solid tumor panel Will include all of the genes currently covered Future Testing for Solid Tumors ABL1 EGFR GNAQ KRAS PTPN11 AKT1 ERBB2 GNAS MET RB1 ALK ERBB4 HNF1A MLH1 RET APC EZH2 HRAS MPL SMAD4 ATM FBXW7 IDH1 NOTCH1 SMARCB1 BRAF FGFR1 IDH2 NPM1 SMO CDH1 FGFR2 JAK2 NRAS SRC CDKN2A FGFR3 JAK3 PDGFRA STK11 CSF1R FLT3 KDR PIK3CA TP53 CTNNB1 GNA11 KIT PTEN VHL In process of validation of myeloid panel of 49-genes by nextgeneration sequencing Future Testing for Hematological Malignancies ASXL1 BCOR BCOR1 BRAF CALR CBL CBLB CEBPA CSF3R DNMT3A ETV6 EZH2 FLT3 GATA1 GATA2 GNAS HRAS IDH1 IDH2 JAK1 JAK2 JAK3 KDM6A KIT KMT2A/ MLL-PTD KRAS MEK1 MPL MYD88 NOTCH1 NPM1 NRAS PHF6 PML PTEN PTPN11 RAD21 RUNX1 SETBP1 SF3B1 SMC1A SMC3 SRSF2 STAG2 TET2 TP53 U2AF1 WT1 ZRSR2 Solid Tumor Testing A Brief 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 Solid Tumor Testing History Molecular Testing Volumes for Solid Tumors 266 224 209 191 133 118 117 103 56 54 62 42 29 36 0 38 0 2012 2013 Total Cases Lung 44 21 2014 Melanoma Colorectal 2015 Other 2015 2014 2015 TruSight Tumor Solid Cancer Panel Analysis By Tumor Type 2014 TruSight Tumor Solid Cancer Panel Analysis By Tumor Type Unable to Process Unable to Process 15 Colon Colon 43 Melanoma Lung 1 2 Langerhans 1 0 20 19 HCC Trial 14 GISTs 141 Other 3 Thyroid 41 Lung 96 HCC Trial 51 Melanoma 54 Other 35 40 60 80 100 120 9 Thyroid 1 GISTs 1 Langerhans 1 0 20 40 60 80 100 120 140 • During 2015, we started creating final reports for cases with insufficient tissue 160 2015 Insufficient Samples Tissue 11% 43% 46% DNA Quality Control failure • 43% of insufficient specimens were due to insufficient tissue prior to any molecular analysis 2014 Positivity by Tumor Type KIT, 2, 2.4% MET 6 7.1% BRAF, 18, 5.9% APC, 24, 25.3% BRAF 6 3.9% EGFR, 10, 6.5% KRAS, 21, 22.1% KRAS, 35, 22.9% MET 8 5.2% NRAS 10 11.8% PIK3CA 6 6.3% TP53, 21, 24.7% TP53, 30, 31.6% TP53, 62, 40.5% 2015 Positivity by Tumor Type BRAF 16 18.8 % AKT1 2 3.6% KIT 5 5.9% NRAS 5 5.9% TP53 9 10.6% BRAF, 4, 4.2% KRAS 27 28.4 % APC 41 43.2% PIK3C A 9 9.5% TP53, 41, 43.2% BRAF, 5, 3.3% EGFR 28 18.3 % KRAS 46 30.1% SKT11 16 10.5% TP53, 86, 56.2% 2014 Frequency of Variant Classification 2015 113 127 18 60 240 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 276 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 Original Intent of the Tumor Board 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. Global review of Molecular Testing from the previous month by Molecular laboratory Cases identified by clinicians for Tumor Board Discussion Suggested Format of the Tumor Board 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 PROPOSAL FOR CLINICAL PRACTICE STANDARDS DIRECTIVE PROTOCOL Standards Directive for Mutation Testing 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 Diagnostic studies Type Renal Tumor % Chromosome Abnormality Clear Cell Renal Cell ~70 Carcinoma Loss of 3p Papillary Renal Cell Carcinoma 10-20 Extra copies of 7 and 17 Chromophobe Renal Cell Carcinoma 5 Loss of chromosomes 1, 2, 6, 10, 13, 17, 21 Oncocytoma <5 Normal or loss of 1p Microarray Result Prognostic Significance: Clear Cell Renal Cell Carcinoma Clear Cell RCC with 3p- Better prognosis Clear Cell RCC with 3pPlus other genetic abnormalities Worse prognosis Aberrations Associated with Adverse Prognosis Clear Cell Renal Cell Carcinoma ** ** * * ** * * * * * * * * Percent with abnormality * * P <0.5 ** P <0.01 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 Karyotype here 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 LOH Associated Gene 1p NRAS 4q TET2 7q EZH2 9p JAK2, CDKN2A, PAX5 11p WT1, PAX6 11q CBL 13q FLT3 17p TP53 19q CEBPA 21q RUNX1 cnLOH control Percent survival HR 1.87 Percent relapse Importance of LOH in Prognosis in Acute Myeloid Leukemia and Myelodysplastic Syndromes 13q cnLOH No 13q cnLOH P=0.006 Relapse HR 1.82 cnLOH P=0.03 Percent survival HR 6.64 Percent relapse P=0.04 control No 13q cnLOH 13q cnLOH P=0.02 Survival Min Fang, personal communication; Cancer. 2015 Sep 1;121(17):2900-8. HR 3.45 Among FLT3-ITD patients, 13qLOH associated with poor prognosis P=0.01 P=0.01 n.s. Min Fang, personal communication; Cancer. 2015 Sep 1;121(17):2900-8. Diagnosis/Prognosis for Glioma Loss 1p/19q: Oligodendroglioma Survival >10 years* MA better than FISH *Need molecular assessment of IDH1/2 gene Gain7/Loss 10, amp 4q (PDGFRA,KIT): High grade glioblastoma; proneural Survival <1 year 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 Activates a tryrosine kinase that can be targeted by imatinib EBF1 -PDGFRB
