Collaboration between CEQAS and UK NEQAS for Molecular Genetics Solid Tumour EQAs and variant interpretation

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1 Collaboration between CEQAS and UK NEQAS for Molecular Genetics Solid Tumour EQAs and variant interpretation Dr Jenni Fairley Deputy Scheme Director (Molecular Pathology), GenQA, Edinburgh, UK

2 From 1 st January 2018 Hosted by NHS Lothian within Laboratory Medicine, Royal Infirmary, Edinburgh 78 Hosted by OUH NHS Foundation Trust in Women s Centre, John Radcliffe Hospital, Oxford

3 Haematology Newborn screening Molecular Genetics core diseases Delivering 78 EQAs across 14 specialities Constitutional - postnatal Constitutional - prenatal Molecular Pathology Preimplantation Genetic Testing Next Generation Sequencing EQAs Noninvasive prenatal testing DNA extraction and quality measurement Tumour Assessment Training/ Competency Clinical Genetics Educational Molecular Rapid Aneuploidy testing

4 Molecular Pathology EQAs Colorectal cancer (KRAS, NRAS, BRAF) Lung cancer (EGFR, ALK, KRAS, NRAS) Melanoma (BRAF, NRAS, KIT) Gastrointestinal stromal tumours (KIT, PDGFRA) BRCA testing in ovarian cancer (germline and somatic BRCA1 and BRCA2)* CNS tumours (1p19q, MGMT methylation, IDH1/2) Sarcoma Neuroblastoma Mismatch repair (BRAF, MLH1 methylation) MSI (microsatellite instability, BRAF, MLH1 methylation) Molecular Tissue Idenitification Additional lung biomarkers (ROS1, RET, MET) cfdna (EGFR) * Joint with EMQN

5 Molecular Pathology EQAs Colorectal cancer (KRAS, NRAS, BRAF) Lung cancer (EGFR, ALK, KRAS, NRAS) Melanoma (BRAF, NRAS, KIT) Gastrointestinal stromal tumours (KIT, PDGFRA) BRCA testing in ovarian cancer (germline and somatic BRCA1 and BRCA2) Next Generation Sequencing most common method for testing * Joint with EMQN

6 Reporting of Variants Laboratories only requested to report variants in genes stated on referral Some laboratories reporting variants in other genes Validation performed by NGS on all cases identification of novel variants in specified genes

7 Issues in Variant Reporting (1) BRCA testing in ovarian cancer Germline and somatic versions of EQA Fully interpretative EQAs Response to PARP inhibitors

8 Issues in Variant Reporting (1) Case Patient name Sex Date of birth (dob) Validated result BRCA1 (LRG_292t1) BRCA2 (LRG293_t1) 3 Bernadette YAFFE Female 31/10/1964 Heterozygous for BRCA1 duplication of exon 12 (using systematic / sequential exon numbering) OR Heterozygous for BRCA1 (NM_ ) duplication of exon 13 (using legacy / custom exon numbering) No pathogenic variant detected

9 Issues in Variant Reporting (1)? Pathogenicity of variant?

10 Issues in Variant Reporting (1)? Pathogenicity of variant? Classified as class 5 according to vast majority of laboratories and the expert advisors to the EQA In many populations this is a founder mutation However, a few laboratories classified this as a class 3 or 4 as there is a (probably very small) risk that the duplication is not in tandem (method dependent -MLPA)

11 Issues in Variant Reporting (2) Molecular Testing in melanoma (16-17 Run 2) Validated Results Clinical Case Number Block Numb er Patient Name BRAF NRAS KIT Dana BRANSON c.1801a>g p.(lys601glu) No mutations detected No mutations detected

12 Issues in Variant Reporting (2)? Interpretation of variant?

13 Issues in Variant Reporting (2)? Interpretation of variant? Evidence that c.1801a>g p.(lys601glu) is an activating mutation of BRAF Little information in literature regarding clinical utility for BRAF/MEK therapies Strong evidence that BRAF V600 mutations respond to BRAF and MEK inhibitors

14 Issues in Variant Reporting (2)? Interpretation of variant? The patient may respond to BRAF- or MEK-targeted therapy. The prognostic and/or predictive value of this mutation is currently unclear. Recent studies, however, have shown that patients with a BRAF mutation in codon 600 may benefit from treatment with a BRAF inhibitor These results are consistent with a BRAF V600 mutant melanoma, therefore, this patient may benefit from BRAF inhibitors alone or in combination with MEK inhibitors The analysis revealed that Dana Branson HAS a mutation (p.lys601glu) in the BRAF gene and therefore is eligible for targeted treatment

15 Issues in Variant Reporting (2)? Interpretation of variant? The patient may respond to BRAF- or MEK-targeted therapy. The prognostic and/or predictive value of this mutation is currently unclear. Recent studies, however, have shown that patients with a BRAF mutation in codon 600 may benefit from treatment with a BRAF inhibitor These results are consistent with a BRAF V600 mutant melanoma, therefore, this patient may benefit from BRAF inhibitors alone or in combination with MEK inhibitors The analysis revealed that Dana Branson HAS a mutation (p.lys601glu) in the BRAF gene and therefore is eligible for targeted treatment

16 Issues in Variant Reporting (2)? Interpretation of variant? In the absence of extensive clinical trials, it is unknown at this time how this patient with c.1801a>g, p.(lys601glu)(or K601E) mutation at codon 601 in exon 15 of the BRAF gene may respond to BRAF-inhibitor therapies. Preclinical studies suggest that downstream signalling induced by the K601E mutant may be blocked by the BRAF inhibitor, vemurafenib (Dahlman et al. 2012) Whilst data on therapeutic implications of this mutation is limited, there is evidence to suggest that patients are unlikely to respond to BRAF kinase inhibitors (Falchook et al 2012 Lancet 379: ; Sullivan and Flaherty 2011 J Skin Cancer ) but may respond to MEK inhibitors (Bowyer et al 2014 Melanoma Research 24: ; Kim et al 2012 J Clin Oncol 31: )

17 Issues in Variant Reporting (3) Molecular Testing in colorectal cancer (17-18 Run 1) Clinical Case Number Block Number Patient Name Validated Results KRAS NRAS BRAF PIK3CA Sylvia REED No mutation detected* No mutation detected No mutation detected** c.1633g>a p.(glu545lys)

18 Issues in Variant Reporting (3) Molecular Testing in colorectal cancer (17-18 Run 1) Clinical Case Number Block Number Patient Name Validated Results KRAS NRAS BRAF PIK3CA Sylvia REED No mutation detected* No mutation detected No mutation detected** c.1633g>a p.(glu545lys) * KRAS c.99t>g p.(asp33glu) variant detected (outwith codons stated for validation) ** BRAF c. 1781A>G p.(asp594gly) variant detected (outwith codons stated for validation)

19 Issues in Variant Reporting (3)? Interpretation of variant?

20 Issues in Variant Reporting (3)? Interpretation of variant? KRAS c.99t>g p.(asp33glu) (exon 2 of KRAS) reported by 8 laboratories in EQA In vitro data suggests it is an activating mutations. reported previously in colorectal cancer Prescribing guidelines for EGFR monoclonal antibodies state they should not be used in the treatment of colorectal cancer patients whose tumours have RAS mutations or for whom RAS tumour status is unknown. BRAF c. 1781A>G p.(asp594gly) Reported by 15 laboratories in the EQA Likely to be a BRAF activating mutation Previously identified in colorectal cancer No current evidence regarding clinical utility

21 Acknowledgements Peer assessors UK NEQAS for Molecular Genetics/GenQA team UK NEQAS for Molecular Pathology EQA Specialist Advisory Group The participants Contact us on