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802 Next generation sequencing for diagnosis of patients with hereditary breast and ovarian cancer
S134
Abstracts
mutually exclusive of ERBB2 amplification. The majority of ERBB2 ECD mutations are S310F and S310Y base substitutions and tumors with these genomic alterations have recently been associated with dramatic responses to anti-HER2 targeted therapy. Material and Methods: Comprehensive genomic profiling (CGP) of 37,772 clinical FFPE cancer specimens using hybridization capture of exonic regions from 315 cancer-related genes and select introns from 19 genes commonly rearranged in cancer was applied to 50ng of DNA extracted and sequenced to high, uniform median coverage (623X) and assessed for base substitutions, short INDELs, copy number alterations and gene fusions/rearrangements. Clinically relevant genomic alterations (CRGA) were defined as those for which anti-cancer drugs on the market or in registered clinical trials could be identified. Results: Of 37,772 sequenced clinical samples, 177 (0.5%) featured either an S310F or S310Y ERBB2 ECD mut. In contrast, only 34 (.01%) non-ECD ERBB2 point mutations were identified in this series. The 7 tumor types listed in the table accounted for 76% of the total ERBB2 ECD mut. Tumor type
Tumors with ERBB ECD Mut Total % of total % in this cases ERBB2 ECD Mut tumor type
Bladder/kidney urothelial carcinoma (UC) 32 Unknown primary carcinoma (CUP) 32 Lung carcinoma 22 Breast carcinoma 14 Biliary tract carcinoma (BTC) 13 Gastroesophageal carcinoma 12 Colorectal carcinoma 10
18% 18% 12% 8% 7% 7% 6%
3.20% 0.96% 0.35% 0.26% 1.10% 0.71% 0.19%
Comparison of this tumor type to all tumor types P < 0.0001 P=0.0004 NS NS P=0.0045 NS NS
A wide variety of greater than 30 other tumor types accounted for the remaining 24% of the ERBB2 ECD mut. Of the ERBB2 ECD mutated cancers, 154 (87%) of had normal ERBB2 copy number and would not be identified as ERBB2 “positive” by FISH or IHC testing. ERBB2 ECD mut were significantly enriched in UC (p < 0.0001), BTC (P = 0.0045) and CUP (P = 0.0004) versus all other tumor types. ERBB2 ECD mut was also highly associated with the micropapillary variant of UC vs conventional UC (p < 0.0001). Multiple cases of ERBB2 ECD mut positive tumors responding to anti-HER2 targeted therapies including both currently available and experimental kinase inhibitors will be presented. Conclusion: ERBB2 ECD mutations are rare but important targetable genomic alterations across a wide range of tumor types. These ERBB2 sequence alterations are not detectable by IHC or FISH and represent a promising new opportunity to utilize comprehensive genomic profiling to personalize treatment for patients with advanced and refractory malignancies. Conflict of interest: Ownership: JS Ross, ZR Chalmers, K Wang, R Yelensky, D Lipson, JA Elvin, J Vergilio, J Chmielecki, SM Ali, VA Miller, PJ Stephens have ownership in Foundation Medicine, Inc. Corporatesponsored Research: JS Ross has corporate sponsored research from Foundation medicine, Inc. 802 POSTER Next generation sequencing for diagnosis of patients with hereditary breast and ovarian cancer P. Bhai1 , D. Gupta1 , R. Saxena1 , I.C. Verma1 . 1 Sir Ganga Ram Hospital, Center of Medical Genetics, Delhi, India Background: BRCA1 and BRCA2 genes are the two most commonly mutated genes in families with Hereditary Breast and Ovarian Cancer (HBOC). However several additional breast cancer predisposition genes are now known to be associated with HBOC. Parallel sequencing of these multiple genes is possible with customized next generation sequencing panels. In the present study we evaluated the sensitivity and efficacy of NGS panel testing for clinical diagnosis of HBOC families. Material and Methods: Fifteen breast cancer patients with positive family history of breast and ovarian cancer were subjected to high throughput next generation sequencing panel testing. Genes analyzed in the panel are ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1,MRE11A,MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, STK11,and TP53. Mutations identified by NGS were validated using sanger sequencing. Software analyses by Polyphen-2, SIFT, Mutation Taster and Align-GVGD was done to predict damaging effect of novel variants. Predictive genetic testing and counselling was done for the first degree relatives. Results: Pathogenic mutations in 6 out of 15 patients (40%) were identified. In rest of the seven patients we found several variants of unknown significance (VUS). Four patients had mutations in BRCA1 gene (c.5074+1G>A, c.4484+1G>A, c.4552C>C/T p.Q1518Ter, c.7480C>T; R2494X) and one patient had mutation in BRCA2 gene (c.9215T>A p.Val3072Glu). One of the patients had a novel nonsense mutation in
MRE11 gene (c.1090C>T: p.Arg364Ter). Another patient was a double heterozygote for mutations in MSH6 and BARD1 gene. In-silico analysis, segregation study in the family and 100 normal controls were studied to confirm the pathogenicity of novel mutations. Conclusion: Multi gene parallel sequencing allowed more effective and accurate diagnosis of HBOC families and supports incorporation of panel testing into clinical practice. Clinical genetic counselling for patients with novel variants and VUS in intermediate penetrance gene is complex and challenging and further studies are needed to clarify the precise management of these patients No conflict of interest. 803 POSTER Highly sensitive and multiplexed next-generation sequencing MiSeqDx Extended RAS Panel for FFPE colorectal samples N. Udar1 , M. Porter1 , R. Haigis1 , J. Fabian1 , T. Dunn1 , D. Lee1 , D. Lee1 , T. Gros1 , F. Hasnat1 , C. Lofton-Day2 , S. Jung3 , A. Iyer1 . 1 Illumina, Oncology, San Diego, USA; 2 Amgen, Medical Sciences, Thousand Oaks, USA; 3 Amgen, Global Development Hematology and Oncology, Thousand Oaks, USA Background: Next generation sequencing (NGS) is a highly sensitive method for the detection of somatic mutations (Wagle et al). Some mutations in the RAS oncogenes allow constitutive activation of downstream signaling resulting in increased survival and proliferation of tumor cells. Overall, mutations in NRAS and KRAS exon 2, 3 and 4 affect approximately 50% of patients with mCRC. Recent data indicate that mutations in RAS are predictive of a lack of response to panitumumab treatment (Douillard et al). An Extended RAS panel* was designed to encompass a total of 56 mutations within exons 2, 3 and 4 of the KRAS and NRAS genes as a single multiplex assay. A dual strand approach was used to clearly distinguish true mutations from artifacts commonly found in DNA from Formalin Fixed Paraffin Embedded (FFPE) tissue. Performance characteristics of the controls as well as FFPE samples was determined for analytical sensitivity and specificity. Material and Methods: Pathological assessment of the FFPE tissue was conducted for neoplastic content and area. DNA was extracted from FFPE tissue, cell lines and plasmids. The DNA quality and quantity was simultaneously determined through a quantitative real-time PCR measurement of amplifiability (dCq). TruSeq Custom Amplicon technology (TSCA) targeting each strand of DNA individually was used to construct the NGS libraries. Sequencing was performed on the MiSeqDx platform using a minimum depth of 2400 reads per target. The assay is in early development. Results: The dual-strand amplicon approach results in high accuracy by eliminating false positive calls observed on only one strand of DNA typical of an FFPE artifact. The number of sections and FFPE tissue area influence the performance of this NGS test. FFPE specimen encompassing a range of tissue area were tested using 1, 3, 5, and 8 sections per sample. 80mm2 minimum (240mm2 recommended) cumulative tissue area using 5uM slices was optimal for assay performance and accuracy. A sensitivity of 5% minor allele frequency (MAF) for 143/144 (99%) nucleotide changes was achieved in the target codons, as well as specificity of 100% in 7920/7920 non mutant nucleotide positions. We also demonstrated that DNA quantity and quality heavily influence test accuracy and sensitivity. A study with 40 FFPE specimens assayed over multiple operators and sequencing instruments produced an average coverage of 32,000 reads per target per sample, with over 93% alignable reads. Reproducibility of mutations detected over multiple operators and instruments was 100%, and results were 100% concordant with Sanger sequencing. We also demonstrated the detection of all 56 distinct mutations in a single run. Conclusion: This multiplex assay can achieve high sensitivity and specificity for the detection of somatic mutations from DNA extracted from FFPE mCRC tissue samples. *In development Conflict of interest: Ownership: Authors are either employees of Illumina or Amgen. 804 POSTER Experiencing online cancer communities − a qualitative study L. Harkin1 , K. Beaver1 , P. Dey2 , K. Choong3 . 1 University of Central Lancashire, School of Health, Preston, United Kingdom; 2 University of Central Lancashire, School of Medicine and Dentistry, Preston, United Kingdom; 3 University of Central Lancashire, Lancashire Law School, Preston, United Kingdom Introduction: Europe’s 600 million internet users are spending a rapidly increasing proportion of time communicating in social media.
Abstracts
mutually exclusive of ERBB2 amplification. The majority of ERBB2 ECD mutations are S310F and S310Y base substitutions and tumors with these genomic alterations have recently been associated with dramatic responses to anti-HER2 targeted therapy. Material and Methods: Comprehensive genomic profiling (CGP) of 37,772 clinical FFPE cancer specimens using hybridization capture of exonic regions from 315 cancer-related genes and select introns from 19 genes commonly rearranged in cancer was applied to 50ng of DNA extracted and sequenced to high, uniform median coverage (623X) and assessed for base substitutions, short INDELs, copy number alterations and gene fusions/rearrangements. Clinically relevant genomic alterations (CRGA) were defined as those for which anti-cancer drugs on the market or in registered clinical trials could be identified. Results: Of 37,772 sequenced clinical samples, 177 (0.5%) featured either an S310F or S310Y ERBB2 ECD mut. In contrast, only 34 (.01%) non-ECD ERBB2 point mutations were identified in this series. The 7 tumor types listed in the table accounted for 76% of the total ERBB2 ECD mut. Tumor type
Tumors with ERBB ECD Mut Total % of total % in this cases ERBB2 ECD Mut tumor type
Bladder/kidney urothelial carcinoma (UC) 32 Unknown primary carcinoma (CUP) 32 Lung carcinoma 22 Breast carcinoma 14 Biliary tract carcinoma (BTC) 13 Gastroesophageal carcinoma 12 Colorectal carcinoma 10
18% 18% 12% 8% 7% 7% 6%
3.20% 0.96% 0.35% 0.26% 1.10% 0.71% 0.19%
Comparison of this tumor type to all tumor types P < 0.0001 P=0.0004 NS NS P=0.0045 NS NS
A wide variety of greater than 30 other tumor types accounted for the remaining 24% of the ERBB2 ECD mut. Of the ERBB2 ECD mutated cancers, 154 (87%) of had normal ERBB2 copy number and would not be identified as ERBB2 “positive” by FISH or IHC testing. ERBB2 ECD mut were significantly enriched in UC (p < 0.0001), BTC (P = 0.0045) and CUP (P = 0.0004) versus all other tumor types. ERBB2 ECD mut was also highly associated with the micropapillary variant of UC vs conventional UC (p < 0.0001). Multiple cases of ERBB2 ECD mut positive tumors responding to anti-HER2 targeted therapies including both currently available and experimental kinase inhibitors will be presented. Conclusion: ERBB2 ECD mutations are rare but important targetable genomic alterations across a wide range of tumor types. These ERBB2 sequence alterations are not detectable by IHC or FISH and represent a promising new opportunity to utilize comprehensive genomic profiling to personalize treatment for patients with advanced and refractory malignancies. Conflict of interest: Ownership: JS Ross, ZR Chalmers, K Wang, R Yelensky, D Lipson, JA Elvin, J Vergilio, J Chmielecki, SM Ali, VA Miller, PJ Stephens have ownership in Foundation Medicine, Inc. Corporatesponsored Research: JS Ross has corporate sponsored research from Foundation medicine, Inc. 802 POSTER Next generation sequencing for diagnosis of patients with hereditary breast and ovarian cancer P. Bhai1 , D. Gupta1 , R. Saxena1 , I.C. Verma1 . 1 Sir Ganga Ram Hospital, Center of Medical Genetics, Delhi, India Background: BRCA1 and BRCA2 genes are the two most commonly mutated genes in families with Hereditary Breast and Ovarian Cancer (HBOC). However several additional breast cancer predisposition genes are now known to be associated with HBOC. Parallel sequencing of these multiple genes is possible with customized next generation sequencing panels. In the present study we evaluated the sensitivity and efficacy of NGS panel testing for clinical diagnosis of HBOC families. Material and Methods: Fifteen breast cancer patients with positive family history of breast and ovarian cancer were subjected to high throughput next generation sequencing panel testing. Genes analyzed in the panel are ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1,MRE11A,MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, STK11,and TP53. Mutations identified by NGS were validated using sanger sequencing. Software analyses by Polyphen-2, SIFT, Mutation Taster and Align-GVGD was done to predict damaging effect of novel variants. Predictive genetic testing and counselling was done for the first degree relatives. Results: Pathogenic mutations in 6 out of 15 patients (40%) were identified. In rest of the seven patients we found several variants of unknown significance (VUS). Four patients had mutations in BRCA1 gene (c.5074+1G>A, c.4484+1G>A, c.4552C>C/T p.Q1518Ter, c.7480C>T; R2494X) and one patient had mutation in BRCA2 gene (c.9215T>A p.Val3072Glu). One of the patients had a novel nonsense mutation in
MRE11 gene (c.1090C>T: p.Arg364Ter). Another patient was a double heterozygote for mutations in MSH6 and BARD1 gene. In-silico analysis, segregation study in the family and 100 normal controls were studied to confirm the pathogenicity of novel mutations. Conclusion: Multi gene parallel sequencing allowed more effective and accurate diagnosis of HBOC families and supports incorporation of panel testing into clinical practice. Clinical genetic counselling for patients with novel variants and VUS in intermediate penetrance gene is complex and challenging and further studies are needed to clarify the precise management of these patients No conflict of interest. 803 POSTER Highly sensitive and multiplexed next-generation sequencing MiSeqDx Extended RAS Panel for FFPE colorectal samples N. Udar1 , M. Porter1 , R. Haigis1 , J. Fabian1 , T. Dunn1 , D. Lee1 , D. Lee1 , T. Gros1 , F. Hasnat1 , C. Lofton-Day2 , S. Jung3 , A. Iyer1 . 1 Illumina, Oncology, San Diego, USA; 2 Amgen, Medical Sciences, Thousand Oaks, USA; 3 Amgen, Global Development Hematology and Oncology, Thousand Oaks, USA Background: Next generation sequencing (NGS) is a highly sensitive method for the detection of somatic mutations (Wagle et al). Some mutations in the RAS oncogenes allow constitutive activation of downstream signaling resulting in increased survival and proliferation of tumor cells. Overall, mutations in NRAS and KRAS exon 2, 3 and 4 affect approximately 50% of patients with mCRC. Recent data indicate that mutations in RAS are predictive of a lack of response to panitumumab treatment (Douillard et al). An Extended RAS panel* was designed to encompass a total of 56 mutations within exons 2, 3 and 4 of the KRAS and NRAS genes as a single multiplex assay. A dual strand approach was used to clearly distinguish true mutations from artifacts commonly found in DNA from Formalin Fixed Paraffin Embedded (FFPE) tissue. Performance characteristics of the controls as well as FFPE samples was determined for analytical sensitivity and specificity. Material and Methods: Pathological assessment of the FFPE tissue was conducted for neoplastic content and area. DNA was extracted from FFPE tissue, cell lines and plasmids. The DNA quality and quantity was simultaneously determined through a quantitative real-time PCR measurement of amplifiability (dCq). TruSeq Custom Amplicon technology (TSCA) targeting each strand of DNA individually was used to construct the NGS libraries. Sequencing was performed on the MiSeqDx platform using a minimum depth of 2400 reads per target. The assay is in early development. Results: The dual-strand amplicon approach results in high accuracy by eliminating false positive calls observed on only one strand of DNA typical of an FFPE artifact. The number of sections and FFPE tissue area influence the performance of this NGS test. FFPE specimen encompassing a range of tissue area were tested using 1, 3, 5, and 8 sections per sample. 80mm2 minimum (240mm2 recommended) cumulative tissue area using 5uM slices was optimal for assay performance and accuracy. A sensitivity of 5% minor allele frequency (MAF) for 143/144 (99%) nucleotide changes was achieved in the target codons, as well as specificity of 100% in 7920/7920 non mutant nucleotide positions. We also demonstrated that DNA quantity and quality heavily influence test accuracy and sensitivity. A study with 40 FFPE specimens assayed over multiple operators and sequencing instruments produced an average coverage of 32,000 reads per target per sample, with over 93% alignable reads. Reproducibility of mutations detected over multiple operators and instruments was 100%, and results were 100% concordant with Sanger sequencing. We also demonstrated the detection of all 56 distinct mutations in a single run. Conclusion: This multiplex assay can achieve high sensitivity and specificity for the detection of somatic mutations from DNA extracted from FFPE mCRC tissue samples. *In development Conflict of interest: Ownership: Authors are either employees of Illumina or Amgen. 804 POSTER Experiencing online cancer communities − a qualitative study L. Harkin1 , K. Beaver1 , P. Dey2 , K. Choong3 . 1 University of Central Lancashire, School of Health, Preston, United Kingdom; 2 University of Central Lancashire, School of Medicine and Dentistry, Preston, United Kingdom; 3 University of Central Lancashire, Lancashire Law School, Preston, United Kingdom Introduction: Europe’s 600 million internet users are spending a rapidly increasing proportion of time communicating in social media.