Dr Kate Goodhealth Goodhealth Medical Clinic 123 Address Road SUBURBTOWN NSW 2000 Melanie Citizen Referring Doctor Your ref Address Dr John Medico 123 Main Street, SUBURBTOWN NSW 2000 Phone 02 9999 9999 Requested 17 May 2018 Collected 18 May 2018 09:00 am Received 18 May 2018 12:27 pm Reported 25 May 2018 15:46 pm Contextual Genomics Report Full FIND IT Panel Interpretation summary - Potentially responsive to PI3K/mTOR and MEK combination therapy or mtor/mek and PKC combination therapy Test results summary TABLE 1: KEY MUTATIONS PRESENT Gene GNA11 cdna change c.626a>t (NM_002067) Amino Acid Change Q209L (p.gln209leu) Exon Allelic ratio (%) Therapeutic implication 5 48.0 Potentially responsive to PI3K/mTOR and MEK combination therapy or mtor/mek and PKC combination therapy Level of evidence Tier: II.D TABLE 2: KEY MUTATIONS ABSENT Gene Therapeutic implication Level of evidence None found. Interpretation PRESENCE of a GNA11 mutation An activating mutation in the GNA11 gene, as described in the table above, was detected in the tumour DNA of this patient. This mutation is located in the GTPase domain of the GNA11 protein and causes constitutive GNA11 and downstream pathways Page 1 of 5
activity (Van Raamsdonk et.al, 2008). GNA11 mutations are rare in melanoma (1%-2%) except in primary ocular melanoma where they are found in approximately 40% of patients (Raamsdonk, 2008 & 2010). Together with GNAQ, GNA11 mutations are detected in over 80% of primary ocular melanoma (Raamsdonk, 2008 & 2010). Mutations in the GNA11 genes are mutually exclusive with GNAQ genes and often exclusive with those in NRAS and BRAF (Van Raamsdonk et.al, 2008 and Lovly et.al. 2012). Most melanoma associated mutations in the GNA11 gene are detected in the Q209 codon (Raamsdonk et.al, 2008). There are currently no approved targeted therapies for mutated GNA11 or GNAQ melanoma. Because targeting GNA11 and GNAQ has proven challenging, inhibition of the downstream effectors MEK, PKC and PI3K/mTOR has been proposed as a possible strategy. Clinical trials of MEK inhibitors in monotherapy or with dacarbazine have demonstrated limited clinical utility in uveal melanoma with GNAQ or GNA11 mutations (Falchook et al., 2012; Carvajal et al., 2015). However, preliminary data from an open-label phase 1 trial revealed that sotrastaurin, a protein kinase C (PKC) inhibitor, has clinical activity at multiple dose levels in this patient population (Piperno-Neumann et.al. 2014). In vitro and in vivo preclinical studies using GNAQ and GNA11 mutations cell lines and xenograft have demonstrated increased and synergistic activity to combined PI3K or mtor and MEK inhibitors (Khalili et al, 2012), or combined MEK or mtor and PKC inhibition (Chen et.al, 2014 and Carita et.al, 2016). Clinical trials are ongoing to determine the best targeted treatment and best combination in this patient population. Clinical trials Australia Relevant Trials A Phase I, Multi-center, Open-label, Study of LXS196, an Oral Protein Kinase C Inhibitor, in Patients With Metastatic Uveal Melanoma A Phase II Randomized, Open-label, Multi-center Study of the Safety and Efficacy of IMCgp100 Compared With Investigator Choice in HLA-A*0201 Positive Patients With Previously Untreated Advanced Uveal Melanoma TABLE3: Clinical Studies. Study NCT02601378 NCT03070392 The clinical trials included in the report are sourced from Australian trials listed on clinicaltrials.gov and anzctr.org.au. We select trials based on tumour histotype and mutation status, with a specific focus on trials of targeted therapy. The inclusion of a trial in our report does not necessarily mean that the patient would be eligible. Patients eligibility for a trial, and the benefit that they may derive from it, will depend on additional factors that must be assessed by the oncologist. Conversely, the list of potentially relevant trials in our report may not be complete. We may have overlooked relevant trials on these websites, or there may be relevant trials listed elsewhere. Please let us know if you identify a trial of targeted therapy that could have been included in a patient s report. Specimen Information Specimen Collection Date: 2018-Mar-01 Specimen Source: Excision Tumour Cellularity: 90% Histologic Type: Spindle cell melanoma Report ID: 123 Block ID #: 12345 Specimen Type: FFPE Block Primary Site of Tumour: Eye Sample Name: 12345 Patient ID: 12345 Page 21 of 5
Methodology This test includes targeted sequence analysis of hotspot mutations/coding exons of the requested genes and transcripts (listed below). FFPE slides and/or tissue blocks undergo pathology evaluation, and tumour cell enrichment through macro-dissection is performed, if appropriate. Genomic DNA is extracted and targets of interest amplified using a highly multiplexed in-house designed PCR assay. The targeted regions are sequenced using Illumina technology with 151bp paired-end reads. Sequence reads that pass defined quality threshold metrics are aligned to the reference sequence (Genome Build hg19) and variants are identified and annotated using a validated, custom-built bioinformatics pipeline. Standard acceptance criteria for reporting of analytical runs are a minimum read depth of 500, a variant allele fraction of 5%, a base quality score of 30, and a probability score of 0.90 for single nucleotide changes or a quality score of 400 for insertion/deletion events. The probability score is the likelihood that a detected mutation is a true positive. The variant allele fraction (VAF) is defined as the proportion of alleles with a mutation to the total number of alleles present in a sample, expressed as a percentage. Hotspot variants are categorised into clinical significance tiers as per Li et al, J Mol Diagn 2017, 19(1):4-23. HOTSPOT PANEL Gene Hotspot Transcript Gene Hotspot Transcript AKT1 E17 NM_0010144 32.1 JAK1 V658, S703 NM_002227.2 ALK AR BRAF T1151, L1152, C1156, F1174, L1196, L1198, G1202, D1203, S1206, G1269, R1275 V716, S741, W742, H875, F877, T878 Q201, G466, F468, G469, Y472, D594, G596, L597, V600, K601 NM_004304.4 KIT T670, D816, D820, N822, Y823, A829, exons: 9, 11, 13 NM_000222.2 NM_000044.3 KRAS G12, G13, A59, Q61, K117, A146 NM_004985.4 NM_004333.4 MAP2K1 Q56, K57, K59, D67, P387 NM_002755.3 CTNNB1 D32, S33, G34, S37, T41, S45 NM_001904.3 MAP2K2 F57, Q60, K61, L119 NM_030662.3 DDR2 L239, I638, S768 NM_0010147 Y1253, exons: 13, 14+25, 14-50, NM_0011275 MET 96.1 18, 14 00.1 EGFR Exons: 18, 19, 20, 21 NM_005228.3 NRAS G12, G13, A59, Q61, K117, A146 NM_002524.4 ERBB2 G309, S310, L755, exons: 20 NM_004448.3 PDGFRA D842, L839_Y849, N659, R560_E571 NM_006206.4 ESR1 K303, S463, V534, P535, L536, Y537, D538 NM_0011227 42.1 PIK3CA R88, E542, E545, Q546, D549, M1043, N1044, A1046, H1047, G1049 NM_006218.2 FGFR1 N546, K656 NM_023110.2 PTCH1 W844, G1093 NM_000264.3 FGFR2 S252, P253, N549, K659 NM_000141.4 PTEN I122_M134, K254_K267, R130, R173, S170_Y188, Y225_F243 NM_000314.4 GNA11 Q209 NM_002067.4 RET C634, V804, M918 NM_020975.4 GNAQ Q209 NM_002072.4 ROS1 L2026, G2032 NM_002944.2 GNAS R201 NM_000516.5 SMO D473, S533, W535 NM_005631.4 HRAS G12, G13, Q61 NM_005343.2 STK11 Q37, P281 NM_000455.4 IDH1 R132 NM_005896.3 TP53 R158, R175, R213, Y220, G245, R248, R273, R282, exons: 4, 5, 6, NM_000546.5 7, 8, 9 IDH2 R140, R172 NM_002168.3 TABLE4: Hotspot Panel: CG001.v3.4_Hotspot_Manifest_Panel3.4.5_20170921.tsv Page 31 of 5
Quality metrics The figure below displays the correlation between the expected variant allelic fraction (VAF) and the observed VAF for the quality control sample. Melanie Citizen The table below summarises the average amplicon coverage for this patient. Status Coverage depth All 109 amplicons > 1000 TABLE5: Amplicon Coverage FIGURE1: Correlation between the expected and observed variant allelic fraction (VAF) for the quality control sample for this run. Limitations Single nucleotide changes, insertions and deletions at the targeted hotspots and exons will be reported. The size limit for the detection of insertion and deletion events is 1 to 24 base pairs for deletions and 1 to 18 base pairs for insertions. Variants with 5% allele ratio will be reported. Rare genetic variation can interfere with this assay. The presence of gene amplification and gene rearrangement events will not be detected by this assay. The ability to detect a particular variant in a given specimen will depend upon the allele proportion of the variant in the extracted DNA combined with the lower limit of detection of the assay. This assay does not differentiate between germline (hereditary) and somatic mutations. This assay does not detect copy number variation (CNV) including amplification of HER2/neu nor does it detect gene fusions such as those leading to ALK gene rearrangements. In addition, the assay does not assess for mutations in BRCA or other genes associated with homologous recombination and DNA damage repair which could be associated with response to PARP inhibitors. Page 41 of 5
References J Clin Oncol 32:5s, 2014 (suppl; abstr 9030). 5049 Khalili JS, Yu X, Wang J, et al. "Combination small molecule MEK and PI3K inhibition enhances uveal melanoma cell death in a mutant GNAQ- and GNA11-dependent manner." 2012 Jun 25. Clin. Cancer Res. - PMID22733540 Falchook GS, Lewis KD, Infante JR, et al. "Activity of the oral MEK inhibitor trametinib in patients with advanced melanoma: a phase 1 dose-escalation trial." 2012 Jul 16. Lancet Oncol. - PMID22805292 Chen X, Wu Q, Tan L, et al. "Combined PKC and MEK inhibition in uveal melanoma with GNAQ and GNA11 mutations." 2013 Oct 21. Oncogene - PMID24141786 Society for Melanoma Research Congress San Francisco, CA, USA. 18 21 November 2015. 5045 Carita G, Frisch-Dit-Leitz E, Dahmani A, et al. "Dual inhibition of protein kinase C and p53-mdm2 or PKC and mtorc1 are novel efficient therapeutic approaches for uveal melanoma.". Oncotarget - PMID27507190 Van Raamsdonk CD, Griewank KG, Crosby MB, et al. "Mutations in GNA11 in uveal melanoma." 2010 Nov 17. N. Engl. J. Med. - PMID21083380 Van Raamsdonk CD, Bezrookove V, Green G, et al. "Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi." 2008 Dec 10. Nature - PMID19078957 Lovly CM, Dahlman KB, Fohn LE, et al. "Routine multiplex mutational profiling of melanomas enables enrollment in genotype-driven therapeutic trials." 2012 Apr 20. PLoS ONE - PMID22536370 Page 51 of 5