Cancer validation meeting

RMH Case Clinical History (47year old female) Diagnosis Disease cohort: Renal Subtype: Unknown Diagnosed July 2016 G3 pT2b; pN0; pMx (TNM 7th edition) 7th Edition) Previous molecular testing Germline: Known p53 germ line carrier Li Fraumeni syndrome (C>T codon 196, premature stop) No routine somatic testing performed in renal cases Young son; tested for p53 germ line mutation-confirmed mutation (under care of GOS) History Complex clinical history; prophylactic double mastectomy (2000), right oopherectomy (2007), several benign moles and cervical biopsy (benign) Participant in SIGNIFY study-use of whole body MRI to screen patients with rare p53 germline mutations 11cm renal mass was identified plus a uterine fibroid Treatment Nephrectomy/hysterectomy/unilateral salpingoophorectomy/utererolysis Follow-up MRI imaging-admitted with left sided weakness and vertigo Dec 2016 Brain lesion observed; histology confirmed high grade glioma-new primary and not metastasis from RCC Referred for phase 1 trials; debulking of posterior fossa glioblastoma multiforme-concomitant chemo-radiotherapy, completed 6 cycles of temozolomide; recurrent disease-commenced second line PVC chemotherapy No further treatment options at time of tumour board review due to declining PS; sadly she has since passed away Most Diagnosis based on immunophenotyping, morphology aspirate/trephine and histo/IHC; Subtypes/sub-classifications based on molecular results and also prognostic information which will be reflected in therapy choices Minimal residual disease monitoring (MRD)

Results Technical aspects of sequencing Tumour content % : 50 Pathology CPD club 10-12-15 Results Technical aspects of sequencing Tumour content % : 50 Genome wide coverage mean 92X Total somatic SNVs 16645, total somatic indels 21680, total SVs Domain 1 None Domain 2 SMAD2 SMAD Family Member 2 c.1109A>G p.(Gln370Arg) 55% VAF SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways. This protein mediates the signal of the transforming growth factor (TGF)-beta, and thus regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation.

Results RAD21 RAD21 Cohesin Complex Component Pathology CPD club 10-12-15 Results RAD21 RAD21 Cohesin Complex Component c.815-5delT 25% VAF (12/48)-quite a noisy region and adjacent to regions of homopolymers • This protein is a nuclear phospho-protein, which becomes hyperphosphorylated in cell cycle M phase. The highly regulated association of this protein with mitotic chromatin specifically at the centromere region suggests its role in sister chromatid cohesion in mitotic cells. NUTM2B NUT Family Member 2B c.925A>G p.(Ser309Gly) 34% VAF (13/38) NUTM2B (NUT Family Member 2B) is a Protein Coding gene. Diseases associated with NUTM2B include Kidney Clear Cell Sarcoma and Endometrial Stromal Sarcoma. LYL1, Basic Helix-Loop-Helix Family Member c.718G>A p.(Gly240Arg) 39% VAF (20/51) Represents a basic helix-loop-helix transcription factor. The encoded protein may play roles in blood vessel maturation and hematopoeisis. A translocation between this locus and the T cell receptor beta locus (GeneID 6957) on chromosome 7 has been associated with acute lymphoblastic leukemia.

Results and summary Domain 3 Pathology CPD club 10-12-15 Results and summary Domain 3 Available in supplementary report (not reviewed) Pertinent Germline findings No reported in line with disease setting https://www.genomicsengland.co.uk/information-for-gmc-staff/cancer-programme/cancer-genome-analysis/  Discussed with GEL the know p53 germ line status confirmed a TP53 variant was detected in this patient’s germline vcf. https://www.ncbi.nlm.nih.gov/clinvar/variation/43589/ NM_000546.5(TP53):c.586C>T (p.Arg196Ter) No actionable variants detected (no domain 1 variants) Her son is being monitored as confirmed p53 carrier

Pertinent germ line findings Pathology CPD club 10-12-15 Pertinent germ line findings Tumour Type Genes analysed Breast cancer BRCA1, BRCA2, PALB2, PTEN, TP53 Colorectal cancer MLH1, MSH2, MSH6, MUTYH (bi), PMS2, POLD1, POLE, PTEN, SMAD4, STK11 Ovarian cancer BRCA1, BRCA2, MLH1, MSH2, MSH6, BRIP1, RAD51C, RAD51D Prostate cancer BRCA2 Renal Cancer FH, FLCN, PTEN, SDHB, VHL, MET Sarcoma TP53 Melanoma BAP1, (CDK4), CDKN2A Endometrial cancer FH, MLH1, MSH2, MSH6, PMS2, PTEN Adult Glioma APC, ATM (bi), MLH1, MSH2, MSH6, PMS2, TP53 Upper GI MLH1, MSH2, MSH6, PMS2 Head and Neck BRIP1 FANCA FANCB FANCC FANCD2 FANCE FANCF FANCG FANCI FANCL FANCM Neuroendocrine CDKN1B, MAX, MEN1, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, TMEM127, FH, VHL

RMH Case Clinical History (58year old female) Diagnosis Clear cell adenocarcinoma of the ovary. Stage3A1. Involved pelvic lymph node (1/4). CA125 73 No pathogenic BRCA1/2 mutations identified 21.9.2016: Underwent laparotomy; radical abdominal hysterectomy, bilateral salpingoophorectomy, bilateral pelvic node dissection, infracolic and infragastric omentectomy No residual disease Oct 2016-Feb 2017: Adjuvant chemo with Carboplatin/Paclitaxel 6 cycles due to rare & aggressive subtype Progression Follow-up scan in June 2017 showed disease progression to liver; platinum resistance   Entered onto NICCC trial: randomised to Pegylated Liposomal Doxorubicin (caelyx) Progression after two cycles of Caelyx (liver and peritoneal disease) Referral to Drug Development Unit (DDU): Consented for GenambAXL trial (phase 1 trial) Rapid progression on trial - palative Died 07.5.18 Most Diagnosis based on immunophenotyping, morphology aspirate/trephine and histo/IHC; Subtypes/sub-classifications based on molecular results and also prognostic information which will be reflected in therapy choices Minimal residual disease monitoring (MRD)

GEL report Date issued 11.4.17

SNVs All three SNVs confirmed by second NGS panel Variant GEL WGS (VAF%) Mean coverage 96x RMH Ovarian (VAF%) Minimum depth 80x (99.7%) Capture panel ATM c.5644C>T p.(Arg1882*) stop gained 68 (58/85reads) 75 (468/632reads) PIK3CA c.3140A>T p.(His1047Leu) 44 (59/133reads) 40 (245/622reads) ARID1A c.6150G>A p.(Trp2050*) 87 (82/94reads) 81 (459/575reads)

CNV Variant GEL WGS (VAF%) RMH Ovarian (VAF%) % Gene >2.40 = 79% Mean coverage 96x RMH Ovarian (VAF%) Minimum depth 80x (99.7%) Capture panel CDKN2A/CDKN2B amplification chr9:21967753-21975098  low confidence gain. variant was called but did not pass filters % Gene >2.40 = 79% ROI 11/14 SMARCA4 deletion chr19:10961105-11061816 high confidence LOH % Gene <0.5 = 9% ROI 3/35 MAP2K2 Not confirmed % Gene <0.5 = 10% ROI 1/10

CDKN2A/B amplification

MAP2K2 and SMARCA4 deletion