Variant interpretation exercise. ACGS Somatic Variant Interpretation Workshop Joanne Mason 21/09/18

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1 Variant interpretation exercise ACGS Somatic Variant Interpretation Workshop Joanne Mason 21/09/18

2 Format of exercise Compile a list of tricky variants across solid cancer and haematological malignancy. Also include a couple of more straightforward variants. Include referral reason/tumour site, gene, HGVS for variant and VAF (all >20%). Limit cells for ease of compilation. - Classification (clinically significant, potentially clinically significant, VUS, likely benign, benign) - Would the variant would be reported externally? (yes/no/maybe) - Ask for a brief summary for the reasoning behind the responses Distribute to everyone on mailing list/slack 5-14 responses per variant

3 Solid tumours

4 Colorectal Dukes C1 adenocarcinoma PIK3CA frameshift Potentially clinically significant: - Nonsense variant, COSMIC similar variants at penultimate aa in solids - We think this is a frameshift/ premature stop in the last codon of the exon, not a recognised driver mutation therefore would not report out - There are clinvar entries and we report fs routinely, regardless of where they are located in the gene. - Variant has been previously reported in adenocarcinoma and functional studies suggest that it is an activating mutation (Ng et al. Cancer Cell. 2018; 33:450)

5 Colorectal Dukes C1 adenocarcinoma PIK3CA frameshift VUS: - Not included in COSMIC or My Cancer Genome and clinical significance is unclear. Besides this mutation is located in the codon before the STOP codon, resulting in an elongated protein with only extra 4 amino acids. - Some evidence activating- frameshift causing protein extention (PMID: ). No evidence clinically actionable Likely benign - Truncating mutations in PIK3CA show little recurrence outside COSMIC and are not known to be pathogenic. Mutation is very close to C-terminus.

6 Colorectal Dukes C1 adenocarcinoma PIK3CA frameshift Potentially CS: 2/5 labs Yes, 1/5 Maybe, 2/5 No ( we don t currently report variants in PIK3CA outside of specific codons ). VUS: 3/4 No, 1/4 Maybe ( No evidence clinically actionable ) Likely benign: No

7 Colorectal Dukes C1 adenocarcinoma PIK3CA c.3201_3202insa p.asn1068lysfs*5 Summary points Lack of consensus on likely effect of insertion. Some labs think this frameshift creates a premature stop in the last codon of the exon, one lab called it a nonsense variant and other labs consider this results in an elongated protein with 4 extra amino acids. Many labs commented on it s position in the last codon and next to the C-terminus Some labs would not report on basis that this is not a recognised driver mutation, despite assessing it as pathogenic (i.e. not clinically actionable)

8 Lung tumour BRAF missense Clinically significant/potentially CS: - Cannonical driver mutation - Clinvar and multiple COSMIC entries - Located in highly conserved motif in KD domain, other variants reported- ClinVar - Described as oncogenic in OncoKB, associated with gain of function - Reported in multiple papers in NSCLC but insufficient evidence regarding actionability

9 Lung tumour BRAF missense VUS: - This is not an activating mutation in codon 600/exon 15, the standard of care test for NICE-approved BRAF/MEK inhibitor therapy but may be one of the less common BRAF inactivating mutations. Not clinically significant based on current knowledge. - Well documented activating mutation in multiple tumour types, but prognostic significance/actionability of this variant in lung cancer is unclear.

10 Lung tumour BRAF missense CS: 2/2 Yes Potentially CS: 5/6 Yes, 1/6 Maybe When assessing this type of somatic variant in solid tumours an additional level of assessment is required. The variant may be established as likely / pathogenic but the effect on the kinase domain may be different depending on the amino acid substitution and thus the response to targeted treatments may differ. It is important to establish the effect on kinase activity and then source information on response to targeted drugs. VUS: 1/3 Yes, 2/3 No

11 Lung tumour BRAF c.1406g>t p.(gly469val) Summary - All agreed this was a pathogenic activating mutation, but some labs annotated as VUS/would not report because of questionable actionability in lung tumours.

12 Lung tumour CCND1 del ins Benign / likely benign - Apparent indels can be the result of sequencing artefacts - Not included in COSMIC, gnomad/exac or My cancer genome or found in literature search. No evidence found to support clinical reporting or significance. - Not recurrent

13 Lung tumour CCND1 del ins VUS - Not much evidence - The only indicator to report is the VAF, so it might be reportable as a simple clonal marker. - Variant affects a well conserved residue within the CCND1 protein - 2/8 might report externally

14 Lung tumour CCND1 c.364_366delinsaaa p.glu122lys Summary points - Suggestion this could be an artefact what are labs doing to reassure themselves they are excluding NGS artefacts? - Is non-recurrence / absence from databases sufficient to annotate as benign/likely benign? - One lab may report as evidence of clonality on VAF alone

15 Ovarian cancer TP53 (dup) nonsense Clinically significant/potentially - Nonsense - Frameshift in tumour suppressor, predict likely pathogenic. - Classic disrupting mutation - Presumed LOF. Always report fs/stops. - R175 is a hotspot for mutations - may be eligible for NICE-approved PARP inhibitor therapy

16 Ovarian cancer TP53 c.518_522duptgagg p.(arg175*) Summary points - Good consensus on clinical significance LOF mutation in TS gene - Lack of consensus on nomenclature regarding this duplication: frameshift or nonsense or either? - One lab stated in IARC, one lab could only find point mutation of this codon in IARC, not exact variant, a third lab stated not in mutation database/s

17 Haem Malignancy

18 Monocytosis MPL missense Clinically significant/potentially - Known activating mutation with supporting functional evidence (Ding J, et al. (2004) - Known clonal marker for MPN (entries on COSMIC, literature). Recurrent mutation found in a range of myeloid malignancies including myelofibrosis - Clinvar shows pathogenic/likely pathogenic (can be germline) - absent in GnomAD

19 Monocytosis MPL c.1514g>a p.(ser505asn) 12/12 would report Summary points - Good consensus lots of evidence available - This is one of a rare class of mutations than can be seen as either a germline or a somatic event (see Beer et al 2008 PMID: ). It is a pathogenic mutation in both circumstances, associated with inherited thrombocytosis or ET respectively. Given the VAF, germline testing should be recommended

20 ?transformation to AML TET2 missense Potentially clinically significant - Confirmed somatic variant in haematopoietic tissue (COSMIC, gnomad/exac) - Not common variant, highly conserved amino acid, in silico not tolerated. TET2 mutations common in AML. Likely benign - Likely passenger event: no support outside of COSMIC.

21 ?transformation to AML TET2 missense VUS - Not seen in Clinvar; COSMIC: 5 calls at this position; large physicochemical difference; moderately conserved nucleotide; highly conserved amino acid. - No good evidence of pathogenicity, although predictive scores are high. - not a confirmed SNP (GnomAD/dbSNP) however note VAF - VAF would suggest a heterozygous variant, not clonal. - Insufficient evidence that the variant is actionable. - Does not fit MoP for gene (LOF).

22 ?transformation to AML TET2 missense Reflecting the fact that most labs think VUS

23 ?transformation to AML TET2 c.5609c>t p.(ser1870leu) Summary points - Labs are using same evidence to come to different conclusions - Some labs are anti-vaf ( reluctant to place any reliance on the VAF), others are using VAFs as evidence of potential germline origin (either for SNP or for cancer associated syndrome) - Caution with VAFs if using amplicon NGS - Very few defined actionable variants (in terms of treatment) in haem malignancy (examples include FLT3-ITDs and TKD mutations and midostaurin, IDH1(R132) and IDH2(R140) and IDH inhibitors). Clinical significance often at the gene level once pathogenicity established >diagnosis and prognosis, only occasionally treatment.

24 ?MDS TET2 missense Potentially clinically significant /clinically significant - COSMIC: 44 calls at this position, 21 calls corresponding to this variant. - Recurrent across multiple databases with multiple different alternative amino acids, both of which are strong evidence for pathogenicity - strong predictive scores - Hu et al describe this as a 'critical residue' in Loop L1 of the DNA binding domain - VAF is not heterozygous/homozygous

25 ?MDS TET2 missense VUS - COSM87130; Not found in dbsnp. ExAC ~0.005% for p.arg1261his - Metzeler et al Somatically acquired mutation in 2 patients (3rd patient had no matched normal tissue) - however all three patients also had truncating mutations in TET2 - therefore its significance is uncertain

26 ?MDS TET2 missense 2 labs would not report externally 1 VUS and 1 Potentially CS

27 ?MDS TET2 c.3781c>t p.(arg1261cys) Summary points - Majority of labs agreed this is (potentially) clinically significant. Consistent with a diagnosis of MDS. Not enough to diagnose MDS in absence of other findings. - No-one mentioned possibility of CHIP. How many labs mention this in their reports, and when? - Some labs again mentioned the relevance of the VAF (21%) in helping them decide this was probably a clonal marker, probably/possibly a driver

28 Thrombocytosis DNMT3A nonsense CS/potentially CS - Nonsense variant presumed pathogenic. Fits MoP for DNMT3A. - Not found in COSMIC, dbsnp, HMGD, ExAC; but likely oncogenic due to lose of function. - MoP can be dominant negative (e.g. R882H common missense) or haploinsufficiency, the latter acting over a more extended period of time (Cole et al)

29 Thrombocytosis DNMT3A nonsense VUS - Similar evidence quoted as labs assigning clinical significance or potential clinical significance

30 Thrombocytosis DNMT3A c.1443c>g p.(tyr481*) 1 lab would not report (VUS) Summary points - Good consensus - some labs seem to have a higher threshold for assigning clinical significance or potential clinical significance

31 ?MDS DNMT3A missense Potentially CS Not common variant, absent in GnomAD (PM2). Highly conserved amino acid, in silico not tolerated. Ser663 in MTase domain of protein (?PM1), catalytic activity, aa 664 binding site. Benign - Not recurrent

32 ?MDS DNMT3A missense VUS Not seen in population databases, seen once in COSMIC Highly conserved amino acid. Missense variants S669F, A662G reported as oncogenic while Y660C reported as possibly oncogenic in AML. Variant is in the methyltransferase domain and in silico tools suggest it could be pathogenic. MoP is dominant negative with missense variants being common in methyltransferase domain

33 ?MDS DNMT3A c.1988c>g p.(ser663trp) 11/13 labs assigned VUS > Yes, No and Maybe! Summary points Most labs agree not enough evidence to call either way One lab thought enough evidence (of lack of recurrence) to call benign One lab mentioned possibility of CHIP

34 ?biphenotypic leukaemia TP53 missense CS/ Potentially CS Multiple Clinvar and COSMIC entries IARC: pathogenic/non-functional Canonical driver mutation 12/12 would report externally

35 ?AML RUNX1 frameshift CS/ Potentially CS Frameshift mutation at the 3' end of RUNX1 predicted to disrupt the transactivation domain resulting in LOF: MoP in RUNX1 This particular variant not listed on COSMIC but a cluster of other frame shifting indels in the region This would be reported as 'likely pathogenic', given the diagnosis, the allele fraction and that it is a truncating mutation. This is a difficult one as the mutation is very close to the C-terminus. Needs to be considered carefully in the clinical context. WHO provisional entity AML with mutated RUNX1, as a reflection of the evidence base.

36 ?AML RUNX1 frameshift VUS Frameshift mutation located in the Runx inhibition domain. Majority of the pathogenic mutations described are missense. This mutation is localized in a CG-rich region, which is prone to originate sequencing artefacts. Not reported in COSMIC, mycancer genome or gnomad/exac Insufficient evidence that the variant is actionable.

37 ?AML RUNX1 c.1214_1218dup p.(tyr407valfs*194) 10/10 labs CS / Potentially CS would report externally 2/2 labs VUS would not report

38 ?AML RUNX1 c.1214_1218dup p.(tyr407valfs*194) Summary points Most labs agree that frameshift mutations, even at the C- terminus of RUNX1, are likely to disrupt the TAD >pathogenic. One lab stated this is a truncating mutation (?unfamiliar with gene). Actually predicted to result in a longer last exon with removal of VWRPY motif in TAD (fitting LOF model). In AML, RUNX1 (pathogenic) variants are highly relevant: WHO provisional entity for classification, associated with poor prognosis (ELN Guidelines, Döhner et al 2017).

39 Summary There is some consensus already. There are differences in emphasis between solids and haem. Emphasis on actionability in terms of targeted therapies much more evident for solid tumours. Haem malignancy once pathogenicity established, variants in relevant genes most often used to corroborate diagnosis or for risk stratification. There is plenty of evidence that labs are approaching SVI with much caution. When a variant was assigned to have clinical significance by any lab, this was associated with a general consensus for clinical significance or potential clinical significance i.e. only when the evidence was strong. There were also high levels of consensus for the use of VUS i.e. when there was lack of sufficient evidence.

40 Summary Many labs are using the same evidence commonly used in germline variant annotation to support assignment of pathogenicity (or not) to somatic variants (nucleotide/aa conservation; functional domain?; in silico prediction tools etc) Evidence that some labs are using VAFs to aid interpretation (or at least report clonality) On occasion, labs are highlighting the potential for a germline variant rather than somatic (can we establish when?) Are haem labs highlighting potential for CHIP?

41 Summary There is inconsistency in reporting out perhaps due to pathogenicity v actionability Potentially clinically significant variants may not always be reported VUS sometimes reported, sometimes not. (Concept of hot 3s?)

42 Thanks Many thanks to everyone that participated