Illumina TruSeq Amplicon Cancer Panel Research Articles

Four-Year Laboratory Performance of the First College of American Pathologists In Silico Next-Generation Sequencing Bioinformatics Proficiency Testing Surveys.

In 2016, the College of American Pathologists (CAP) launched the first next-generation sequencing (NGS) in silico bioinformatics proficiency testing survey to evaluate the performance of clinical laboratory bioinformatics pipelines for the detection of oncology-associated variants at varying allele fractions. This survey focused on 2 commonly used oncology panels, the Illumina TruSeq Amplicon Cancer Panel and the Thermo Fisher Ion AmpliSeq Cancer Hotspot v2 Panel. To review the analytical performance of laboratories participating in the CAP NGS bioinformatics (NGSB) surveys, comprising NGSB1 for Illumina users and NGSB2 for Thermo Fisher Ion Torrent users, between 2016 and 2019. Responses from 78 laboratories were analyzed for accuracy and associated performance characteristics. The analytical sensitivity was 90.0% (1901 of 2112) for laboratories using the Illumina platform and 94.8% (2153 of 2272) for Thermo Fisher Ion Torrent users. Variant type and variant allele fraction were significantly associated with performance. False-negative results were seen mostly for multi-nucleotide variants and variants engineered at variant allele fractions of less than 25%. Analytical specificity for all participating laboratories was 99.8% (9303 of 9320). There was no statistically significant association between deletion-insertion length and detection rate. These results demonstrated high analytical sensitivity and specificity, supporting the feasibility and utility of using in silico mutagenized NGS data sets as a supplemental challenge to CAP surveys for oncology-associated variants based on physical samples. This program demonstrates the opportunity and challenges that can guide future surveys inclusive of customized in silico programs.

Radiosensitization Through Targeting Molecular Alterations in Adenoid Cystic Carcinoma

Adenoid cystic carcinoma (ACC) is a relatively rare cancer that typically arises in salivary tissues of the head and neck. There are currently no approved systemic agents for ACC and no data supporting the delivery of chemoradiation for ACC patients. The scarcity of validated model systems has hampered research efforts. We report the establishment and propagation of an ACC patient–derived xenograft (PDX), genomic evaluation of cancer-associated mutations, and in vivo response profiles to personalized radiosensitization agents based on next-generation sequencing. An ACC PDX was established and maintained in NOD-SCID gamma (NSG) mice directly from the patient. Common cancer-associated mutations were identified using the Illumina TruSeq Amplicon Cancer panel. PDXs were treated with focal radiation or chemotherapy selected based on the genomic profile of the cancer. Focal radiation was delivered at 5 Gy x 8 fractions twice weekly for 4 weeks. Tumor size was measured over time and comparisons between treatment groups made by repeated measures ANOVA. Target inhibition in vivo was confirmed via western blot of tumor lysates and IHC of FFPE tissue. The histologic and physical characteristics of the primary human tumor are maintained in this ACC PDX. Mutations in the receptor tyrosine kinases (RTKs) cKit and KDR/VEGFR2 were identified. No mutations were identified in EGFR, RAS, or PIK3CA. Several targeted therapies were selected including dovitinib, a multi-RTK inhibitor, BEZ235, a PI3K/mTORC inhibitor, and cetuximab, an EGFR mAB. Target inhibition was confirmed by western blot and IHC. Radiation temporarily halted tumor growth. Treatment with molecularly targeted agents prolonged time to tumor doubling compared to control treatment (p<0.05 for each). Chemoradiation resulted in significant tumor regression which persisted more than 2 months after the end of treatment. PDXs are a powerful model system for investigating potential radiosensitizers based on individual tumor characteristics. Our ACC PDX represents one of only a handful of tools for studying this rare disease. These preliminary data identify the rationale to investigate selected molecular drug/radiation combinations for ACC, particularly when driven by tumor-specific genetic biomarkers. Expansion of these studies may be valuable to advance the design of new treatment strategies for ACC.

Radiosensitization of HRAS Mutated Head and Neck Cancer

To test the hypothesis that head and neck cancer with HRAS activating mutations can be radiosensitized by inhibition of the downstream MEK/ERK or PI3K/MTOR pathways. Mutations in HRAS, KRAS, and NRAS were analyzed using cBioPortal in a head and neck cancer cohort. The Illumina TruSeq Amplicon Cancer panel was used to screen a panel of head and neck cancer cell lines for mutations in RAS-family genes. Radiation survival was assessed by clonogenic survival assay. Immunoblots were used to confirm target activation/knockdown in overexpression and knockdown studies. A tumor control dose 50% (TCD50) assay was employed to investigate radiosensitization of a mutated-HRAS head and neck cancer cell line in a flank xenograft model in nude mice. Activating mutations in one of the three RAS genes are seen in 5-10% of head and neck cancer patients. Mutations in HRAS represent over 50% of these. Screening our panel of head and neck cancer cell lines identified a canonical activating mutation in HRAS (G12V). Consistent with known roles for activated HRASG12V, SCC22B is relatively insensitive to both cetuximab and radiation. Using both in vitro and in vivo studies, cetuximab failed to radiosensitize SCC22B. Cetuximab treatment decreased AKT, but not ERK activation. Direct inhibition of the MEK/ERK or PI3K/MTOR pathway by selumetinib or BEZ235, respectively, decreased target protein activation and resulted in significant growth inhibition compared to control (p<0.05). Treatment with either selumetinib or BEZ235 radiosensitized HRASG12V expressing cells (SER 1.3-1.8) but had no effects on cells expressing wildtype HRAS. siRNA knockdown of HRAS radiosensitized SCC22B, but not SCC1 or SCC6 cells relative to non-targeting control while overexpression of HRASG12Vresulted in less sensitivity to radiation in wildtype cell lines. In vivo assessment of the radiosensitizing effects of these compounds via a TCD50 assay (5 fractions of 0, 0.5, 2, 4, 7, and 10 Gy) is pending. More than 5% of head and neck cancers harbor activating mutations in one of the RAS family of genes. These mutations lead to resistance to cetuximab, either as a single agent, or as a radiosensitizer. Inhibition of downstream targets such as the MEK/ERK and PI3K/MTOR pathways can radiosensitize tumors harboring activating mutations in HRAS. Further investigation of the safety and efficacy of these drugs in combination with chemoradiation is warranted.

Biochemical and genetic predictors of overall survival in patients with metastatic pancreatic cancer treated with capecitabine and nab-paclitaxel

Pancreatic cancer is a dismal disease with a mortality rate almost similar to its incidence rate. To date, there are neither validated predictive nor prognostic biomarkers for this lethal disease. Thus, the aim of the present study was to retrospectively investigate the capability of biochemical parameters and molecular profiles to predict survival of patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) who participated in a phase II clinical trial to test the safety and efficacy of the combination treatment of capecitabine plus nab-paclitaxel. Herein, we investigated the association of 18 biochemical parameters obtained from routine diagnosis and the clinical outcome of the 30 patients enrolled in the clinical trial. Furthermore, we analysed formalin-fixed paraffin-embedded (FFPE) tumour tissue to identify molecular biomarkers via RNA seq and the Illumina TruSeq Amplicon Cancer panel which covers 48 hotspot genes. Our analysis identified SERPINB7 as a novel transcript and a DNA mutation signature that might predict a poor outcome of disease. Moreover, we identified the bilirubin basal level as an independent predictive factor for overall survival in our study cohort.

Abstract 5192: Selection of radiosensitizers based on HRAS mutation in head and neck cancer

Abstract Purpose/Objective(s): The ability of activated HRAS to promote radiation resistance and predict for sensitivity to potential radiosensitizers was tested in a head and neck cancer model system. Materials/Methods: Mutations in HRAS, KRAS, and NRAS were analyzed using cBioPortal in a head and neck cancer cohort. The Illumina TruSeq Amplicon Cancer panel was used to screen a panel of head and neck cancer cell lines for mutations in RAS-family genes. Cell growth and radiation survival was assessed by clonogenic survival assay. Immunoblots were used to confirm target activation/knockdown in overexpression and knockdown studies. A tumor control dose 50% (TCD50) assay was employed to investigate radiosensitization of a mutated-HRAS head and neck cancer cell line in a flank xenograft model in nude mice. Results: Activating mutations in one of the three RAS genes are seen in 5-10% of head and neck cancer patients. Mutations in HRAS represent over 50% of these. Screening our panel of head and neck cancer cell lines identified a canonical activating mutation in HRAS (i.e. G12V) in SCC22B. Consistent with known roles for activated HRASG12V, SCC22B is relatively insensitive to both cetuximab and radiation. Using both in vitro and in vivo studies, cetuximab exhibited no ability to radiosensitize SCC22B. Cetuximab treatment decreased AKT, but not ERK activation. Direct inhibition pathways downstream of HRAS by selumetinib (MEK/ERK) or BEZ235 (PI3K/MTOR) decreased target protein activation and resulted in significant growth inhibition compared to control (p&amp;lt;0.05). Treatment with either selumetinib or BEZ235 radiosensitized HRASG12V expressing cells (SER 1.3-1.8) but had more modest effects on cells with wildtype HRAS. siRNA knockdown of HRAS radiosensitized SCC22B, but not SCC1 or SCC6 cells relative to non-targeting control. Overexpression of HRASG12V conferred relative radioresistance in wildtype cell lines. In vivo assessment of the radiosensitizing effects of these compounds (TCD50) is pending. Conclusion: More than 5% of head and neck cancers harbor activating mutations in one of the RAS family of genes. These mutations lead to resistance to cetuximab, either as a single agent, or as a radiosensitizer. Inhibition of downstream targets such as the MEK/ERK and PI3K/MTOR pathways can radiosensitize tumors harboring activating mutations in HRAS. Identification of additional mutation/drug combinations that result in radiosensitization may be valuable to advance the design of personalized radiation therapy. Citation Format: Michael M. Fisher, Adam D. Swick, Kwangok P. Nickel, Randall J. Kimple. Selection of radiosensitizers based on HRAS mutation in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5192. doi:10.1158/1538-7445.AM2017-5192

Abstract 5187: Potent radiosensitization of adenoid cystic carcinoma in a patient derived xenograft model using the MDM2 inhibitor AMG 232

Abstract Purpose: Adenoid cystic carcinoma (ACC) is a relatively rare cancer that typically arises in major or minor salivary gland tissues of the head and neck. There are currently no approved systemic agents for ACC and no established data supporting the delivery of chemoradiation for ACC patients. The scarcity of validated preclinical model systems has hampered research efforts. We report the successful establishment and propagation of an ACC patient derived xenograft (PDX), genomic evaluation of cancer associated mutations, and in vivo response to MDM2 inhibition combined with radiotherapy. Because TP53 mutations are seen in &amp;lt;5% of ACCs, we investigated the role of a small molecule MDM2 inhibitor, AMG 232. Methods: An ACC PDX was established and assessed for common cancer-associated mutations using the Illumina TruSeq Amplicon Cancer panel. Xenografts were treated with focal radiation with or without AMG 232. Focal radiation was delivered at 0, 2, 5, or 8 Gy x 8 fractions delivered twice weekly, with AMG 232 (50mg/kg) delivered daily by oral gavage. Tumor size was measured by caliper and comparisons between treatment groups made using a repeated measures ANOVA. A TCD50 was calculated by fitting log-transformed data to a dose-response curve and compared using the extra-sum-of-squares f test. Target inhibition and anti-cancer effect was confirmed via immuno-blotting of tumor lysates, and IHC staining or in situ hybridization of relevant targets within FFPE sections of tumors harvested 2 and 48 hrs post treatment. Results: The histologic characteristics of the primary human tumor are maintained in subsequent murine passages of this ACC PDX. Mutational profiling determined that the PDX was wildtype for TP53. AMG 232 alone resulted in modest slowing of tumor growth. Radiation alone produced a tumor growth delay in a dose dependent manner but did not result in sustained local tumor control. Strikingly, the combination of AMG 232 with RT (including low dose radiation of 2 Gy) produced dramatic tumor shrinkage and potent tumor control three months after the end of treatment. Target inhibition of MDM2 was confirmed by western blot of tumor lysates and IHC of FFPE samples taken 2 hrs post treatment. Antiproliferative and apoptotic effects were confirmed by western blot of tumor lysates and IHC of FFPE samples taken at 48 hrs post treatment. Conclusions: This study suggests that MDM2 inhibition may provide potent radiosensitization in TP53 WT ACC. While additional translational models are warranted, the powerful response profile observed suggests that phase I clinical trial evaluation of this combination is worthy for this challenging malignancy. Citation Format: Amal Javaid, Adam D. Swick, Lauryn Werner, Prashanth Prabakaran, Rong Hu, Kwangok P. Nickel, Irene Ong, Emmanuel Sampene, Justine Bruce, Gregory Hartig, Aaron Wieland, Jude Canon, Paul Harari, Randall Kimple. Potent radiosensitization of adenoid cystic carcinoma in a patient derived xenograft model using the MDM2 inhibitor AMG 232 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5187. doi:10.1158/1538-7445.AM2017-5187

Somatic mutations in salivary duct carcinoma and potential therapeutic targets.

BackgroundSalivary duct carcinomas (SDCa) are rare highly aggressive malignancies. Most patients die from distant metastatic disease within three years of diagnosis. There are limited therapeutic options for disseminated disease.Results11 cases showed androgen receptor expression and 6 cases showed HER2 amplification. 6 Somatic mutations with additional available targeted therapies were identified: EGFR (p.G721A: Gefitinib), PDGFRA (p.H845Y: Imatinib and Crenolanib), PIK3CA (p.H1047R: Everolimus), ERBB2 (p.V842I: Lapatinib), HRAS (p.Q61R: Selumetinib) and KIT (p.T670I: Sorafenib). Furthermore, alterations in PTEN, PIK3CA and HRAS that alter response to androgen deprivation therapy and HER2 inhibition were also seen.Materials and MethodsSomatic mutation analysis was performed on DNA extracted from 15 archival cases of SDCa using the targeted Illumina TruSeq Amplicon Cancer Panel. Potential targetable genetic alterations were identified using extensive literature and international somatic mutation database (COSMIC, KEGG) search. Immunohistochemistry for androgen receptor and immunohistochemistry and fluorescent in situ hybridization for HER2 were also performed.ConclusionsSDCa show multiple somatic mutations, some that are amenable to pharmacologic manipulation and others that confer resistance to treatments currently under investigation. These findings emphasize the need to develop testing and treatment strategies for SDCa.

Somatic mutations in salivary duct carcinoma and potential therapeutic targets

// Timothy K. Khoo 1, * , Bing Yu 1, 2, * , Joel A. Smith 3 , Angus J. Clarke 1 , Peter P. Luk 4 , Christina I. Selinger 4 , Kate L. Mahon 1, 5 , Spiridoula Kraitsek 2 , Carsten Palme 1, 3 , Michael J. Boyer 1, 5 , Marcel E. Dinger 6 , Mark J. Cowley 6 , Sandra A. O’Toole 1, 4 , Jonathan R. Clark 1, 3, 7, * and Ruta Gupta 1, 3, 4, * 1 Central Clinical School, The University of Sydney, Australia 2 Department of Medical Genomics, Royal Prince Alfred Hospital, Sydney, Australia 3 The Sydney Head and Neck Cancer Institute, Chris O’Brien Lifehouse, Sydney, Australia 4 Department of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Sydney, Australia 5 The Department of Medical Oncology, Chris O’Brien Lifehouse, Sydney, Australia 6 Kinghorn Cancer Centre and Garvan Institute of Medical Research, Darlinghurst, Sydney, Australia 7 South West Clinical School, University of New South Wales, Sydney, Australia * These authors contributed equally to this work Correspondence to: Ruta Gupta, email: Ruta.Gupta@sswahs.nsw.gov.au Keywords: salivary duct carcinoma, somatic mutation analysis, targeted therapies, androgen receptor, HER2 Received: January 10, 2017 Accepted: March 20, 2017 Published: May 25, 2017 ABSTRACT Background: Salivary duct carcinomas (SDCa) are rare highly aggressive malignancies. Most patients die from distant metastatic disease within three years of diagnosis. There are limited therapeutic options for disseminated disease. Results: 11 cases showed androgen receptor expression and 6 cases showed HER2 amplification. 6 Somatic mutations with additional available targeted therapies were identified: EGFR (p.G721A: Gefitinib), PDGFRA (p.H845Y: Imatinib and Crenolanib), PIK3CA (p.H1047R: Everolimus), ERBB2 (p.V842I: Lapatinib), HRAS (p.Q61R: Selumetinib) and KIT (p.T670I: Sorafenib). Furthermore, alterations in PTEN , PIK3CA and HRAS that alter response to androgen deprivation therapy and HER2 inhibition were also seen. Materials and Methods: Somatic mutation analysis was performed on DNA extracted from 15 archival cases of SDCa using the targeted Illumina TruSeq Amplicon Cancer Panel. Potential targetable genetic alterations were identified using extensive literature and international somatic mutation database (COSMIC, KEGG) search. Immunohistochemistry for androgen receptor and immunohistochemistry and fluorescent in situ hybridization for HER2 were also performed. Conclusions: SDCa show multiple somatic mutations, some that are amenable to pharmacologic manipulation and others that confer resistance to treatments currently under investigation. These findings emphasize the need to develop testing and treatment strategies for SDCa.

Abstract 3044: Patient-derived adenoid cystic carcinoma xenografts to study molecular target modulation of tumor radiosensitivity

Abstract Background: Adenoid cystic carcinoma (ACC) is a relatively rare cancer that typically arises in salivary tissues of the head and neck region. Hallmark characteristics include slow growth rate, peri-neural tumor spread, and a high propensity for late distant metastasis. Surgery and radiation are the mainstays of treatment with no effective systemic agents to date. Due to infrequency, studies of novel therapeutics are not routinely feasible. In addition, whether these tumors can be sensitized to radiation by concurrent chemotherapy is not known. We report here the establishment and examination of ACC patient derived xenografts (PDX) to investigate the efficacy of novel chemotherapies and combinations of chemotherapy and radiation. Methods: PDXs have been established and maintained in NOD-SCID gamma (NSG) mice from both research biopsies and surgical specimens. Common cancer-associated mutations in both the primary patient tumor and PDX were identified using the Illumina TruSeq Amplicon Cancer panel. Well described immunohistochemical markers of ACC were used to compare histological characteristics between the primary tumor and PDX. The ACC PDX was engrafted into the flanks of nude mice and treated with focal radiotherapy (5 Gy x 8 fractions delivered twice weekly), a panel of chemotherapeutic agents, or combination radiochemotherapy. Tumor size was measured over time and comparisons between treatment groups made by the extra-sum-of-squares f test. Results: PDXs established from ACC maintain the histologic and physical characteristics of the primary tumor. Targeted mutational analysis of ACC identified expected alterations based on previously reported large scale sequencing of other human tumors including mutations in the receptor tyrosine kinases(RTKs) cKit and KDR/VEGFR2. Based on identified tumor mutations, several targeted therapies were selected including dovitinib, a multi-RTK inhibitor, BEZ235, a PI3K/mTORC inhibitor, and cetuximab, an EGFR mAB. Treatment with each of these compounds showed varying degrees of growth inhibition without evidence of frank tumor regression. However, combining these drugs with radiation demonstrated significantly improved tumor control in comparison to drug alone. Conclusions: Studies using our PDX model suggest that several molecular targeting agents can significantly augment the impact of radiation on ACC tumor growth. These preliminary data identify the rationale to investigate selected molecular drug/radiation combinations for ACC, particularly when driven by tumor specific genetic biomarkers. Expansion of these ACC studies may be valuable to advance the design of new investigational treatment strategies for this challenging tumor. Citation Format: Prashanth Prabakaran, Kwangok P. Nickel, David T. Yang, Lauryn R. Werner, Justine Y. Bruce, Aaron M. Wieland, Timothy M. McCulloch, Gregory K. Hartig, Paul M. Harari, Adam D. Swick, Randall J. Kimple. Patient-derived adenoid cystic carcinoma xenografts to study molecular target modulation of tumor radiosensitivity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3044.

Mutational analysis of pulmonary tumours with neuroendocrine features using targeted massive parallel sequencing: a comparison of a neglected tumour group

Background:Lung cancer is the leading cause of cancer-related deaths worldwide. The typical and atypical carcinoid (TC and AC), the large-cell neuroendocrine carcinoma (LCNEC) and the small-cell lung cancers (SCLC) are subgroups of pulmonary tumours that show neuroendocrine differentiations. With the rising impact of molecular pathology in routine diagnostics the interest for reliable biomarkers, which can help to differentiate these subgroups and may enable a more personalised treatment of patients, grows.Methods:A collective of 70 formalin-fixed, paraffin-embedded (FFPE) pulmonary neuroendocrine tumours (17 TCs, 17 ACs, 19 LCNECs and 17 SCLCs) was used to identify biomarkers by high-throughput sequencing. Using the Illumina TruSeq Amplicon-Cancer Panel on the MiSeq instrument, the samples were screened for alterations in 221 mutation hot spots of 48 tumour-relevant genes.Results:After filtering >26 000 detected variants by applying strict algorithms, a total of 130 mutations were found in 29 genes and 49 patients. Mutations in JAK3, NRAS, RB1 and VHL1 were exclusively found in SCLCs, whereas the FGFR2 mutation was detected in LCNEC only. KIT, PTEN, HNF1A and SMO were altered in ACs. The SMAD4 mutation corresponded to the TC subtype. We prove that the frequency of mutations increased with the malignancy of tumour type. Interestingly, four out of five ATM-mutated patients showed an additional alteration in TP53, which was by far the most frequently altered gene (28 out of 130; 22%). We found correlations between tumour type and IASLC grade for ATM- (P=0.022; P=0.008) and TP53-mutated patients (P<0.001). Both mutated genes were also associated with lymph node invasion and distant metastasis (P⩽0.005). Furthermore, PIK3CA-mutated patients with high-grade tumours showed a reduced overall survival (P=0.040) and the mutation frequency of APC and ATM in high-grade neuroendocrine lung cancer patients was associated with progression-free survival (PFS) (P=0.020).Conclusions:The implementation of high-throughput sequencing for the analysis of the neuroendocrine lung tumours has revealed that, even if these tumours encompass several subtypes with varying clinical aggressiveness, they share a number of molecular features. An improved understanding of the biology of neuroendocrine tumours will offer the opportunity for novel approaches in clinical management, resulting in a better prognosis and prediction of therapeutic response.

Abstract 628: Determinants of quality of next-generation sequencing output from the strand-specific TruSight Tumor Sequencing Panel in a clinical diagnostic setting

Abstract The use of Next-Generation Sequencing (NGS) technologies is increasingly prevalent within diagnostic labs. As genomic regions are sequenced to greater depth in cancer diagnostics, it is critical to differentiate clinically actionable variants from artifacts arising from sequencing-errors, sample- processing or sample-age, and to identify samples that will be difficult to evaluate. We sought to determine whether strand-specific sequencing approaches, such as the TruSight Tumor Sequencing Panel (Illumina) could enable sample and variant triage in a clinical diagnostic settingTruSight Tumor Sequencing Panel allows for paired-end sequencing of individual strands of DNA and analyzing them either together (Paired) or separately (Pool A and Pool B). Variants identified in one pool, but not the other, are putative artifacts; variants identified in both pools are considered true calls. Combined analysis of both pools was performed in two ways: By summing variant calls across pools, and by informatically determining overlapping variant calls between pools. In a test cohort of 44 FFPE samples of varying age and tumor type, we assessed whether age of sample, strand bias, and fixation impacted the detection of high confidence variants using the TruSight Tumor Sequencing panel. Data were compared to the results of analysis of the same samples using the established Illumina TruSeq Amplicon Cancer Panel and/or Sanger Sequencing.Sample age, tumor cellularity, tumor type and template DNA quality were not found to be associated with quality of NGS output in our study. We also evaluated the overall transition/transversion (Ti/Tv) ratios for variants detected either uniquely in one pool or in combined analysis. Interestingly, for variants detected in both pools, the Ti/Tv ratio was 1.97, compared to 0.52-0.60 for those detected in only 1 pool (p &amp;lt; 0.001). Strikingly, samples that sequenced successfully but gave inconclusive and difficult to interpret variant lists were associated with%G&amp;gt;A:C&amp;gt;T transition &amp;gt; 62.5% and Ti/Tv ratios of &amp;gt; 4.0 (p &amp;lt; 0.001). G&amp;gt;A:C&amp;gt;T transitions were significantly over-represented in these samples. The overall%G&amp;gt;A:C&amp;gt;T transitions were equivalent (44-52%) in individual pools or in paired analysis. However, when inconclusive samples were accounted for, the%G&amp;gt;A:C&amp;gt;T transitions differed between the two analyses: 49.7% (paired) vs. 30.1-32.1% (individual pools). In summary, the Ti/Tv ratio can act as a critical determinant of variant call quality - Ti/Tv ratios ∼0.5 represent sequencing artifacts, while Ti/Tv ratios &amp;gt; 4.0 are indicative of inconclusive sequencing output.We conclude that variant Ti/Tv ratio as well as%G&amp;gt;A:C&amp;gt;T transition in variants detected by the TruSight Tumor Sequencing Panel may be helpful evaluators of quality and clinical utility of sequencing output for FFPE tumor samples tested in a clinical diagnostic setting. Citation Format: Swati Garg, Mahadeo A. Sukhai, Mariam Thomas, Michelle Mah, Tong Zhang, Trevor Pugh, Suzanne Kamel-Reid, Tracey L. Stockley. Determinants of quality of next-generation sequencing output from the strand-specific TruSight Tumor Sequencing Panel in a clinical diagnostic setting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 628. doi:10.1158/1538-7445.AM2015-628

Abstract 3891: Tackling the obstacles facing the implementation of a molecular screening program in an early drug development unit: The Jules Bordet Institute Program for Molecular Profiling of Metastatic Lesions - feasibility (Precision-f)

Abstract Introduction The identification of pathways involved in carcinogenesis, the emergence of high-throughput technologies enabling tumor molecular analysis and the development of targeted therapies have led to the concept of precision medicine. Higher response rates and longer survivals have been achieved in recent genomic-driven clinical trials. However, genomic-driven cancer medicine is hindered by multiple obstacles. Our aim was to assess the feasibility of incorporating real-time targeted gene sequencing (TGS) of DNA derived from metastatic biopsies into daily clinical practice. Experimental procedures Precision-f (NCT01932489) is a pilot trial conducted at the Jules Bordet Institute. Patients with metastatic colorectal cancer (CRC), melanoma or non-small cell lung (NSCLC) cancer were enrolled. Two FFPE blocks and one fresh frozen sample embedded in OCT were collected from newly performed metastatic biopsies as well as one whole blood sample. The FFPE samples were checked for tumor cellularity and 5×5 μm sections were cut and sent to 2 laboratories. Targeted gene sequencing was performed on DNA extracted for the same sample using the Illumina TruSeq Amplicon Cancer Panel performed on a MiSeq Desktop Sequencer, and the Life Technologies Ion AmpliSeq Cancer Hotspot Panel performed on an Ion Personal Genome Machine. Results were reported to the institutional sequencing tumor board for discussion, annotation and treatment assignment. The main objectives were the evaluation of biopsy quality, turnaround time, the presence of “actionable” alterations, technology cross-validity and treatment assignments. Results Thirty-four patients were enrolled between December 2013 and August 2014: 13 NSCLC patients, 11 CRC patients, 10 melanoma patients. Successful molecular results were achieved from 32/34 biopsies (94%). The most frequent site of biopsy was the liver (10) followed by the lung (7), the skin (5) and lymph nodes (5). 27/34 (79%) samples had ≥ 20% tumor cells. 31/34 (91%) of samples had &amp;gt; 10 ng of DNA for TGS. The median turnaround time for results reporting was 15 calendar days [8-22]. “Actionable” mutations were found for 66% (21/32) of patients, 76% (16/21) of which were treated with therapy according to the identified molecular alteration. Reasons for non-targeted therapy were: non-eligibility (2), unavailable drugs (2) and patient refusal (1). The results of the comparison of TGS data across the 2 platforms is ongoing and will be presented at the meeting. Conclusion The precision-f pilot trial has demonstrated the feasibility and clinical relevance of a molecular screening program in a clinical pharmacology unit. A Belgian national initiative will follow in order to enhance patient participation in genomic-driven clinical trials. Citation Format: Philippe G. Aftimos, Marion Maetens, Catherine Sibille, Jean-François Laes, Sylvain Brohée, Thierry Berghmans, Joseph Kerger, Alain Hendlisz, Alexandre Irrthum, Olivier De Henau, Amélie Deleporte, Stylianos Drisis, Denis Larsimont, Jalal Vakili, Christos Sotiriou, Martine Piccart, Ahmad Awada. Tackling the obstacles facing the implementation of a molecular screening program in an early drug development unit: The Jules Bordet Institute Program for Molecular Profiling of Metastatic Lesions - feasibility (Precision-f). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3891. doi:10.1158/1538-7445.AM2015-3891

Abstract 1192: Establishment and characterization of a new patient-derived renal cell carcinoma xenograft panel

Abstract Objective: Patients with advanced kidney cancer have a poor prognosis. The majority of patients develops treatment resistance towards Standard of Care (SoC) drugs within months. The successful development of novel personalized therapies depends on the availability of preclinical models with high clinical relevance. Our aim was to establish a panel of patient-derived renal cell carcinoma xenograft models for translational research. Methods: Specimens from primary and metastatic renal cell carcinoma tumors from consenting patients were collected after surgery and transplanted subcutaneously to immunodeficient mice within 24 hours. Tumor engraftment was followed for an initial period of up to 4 months. Successful engrafted patient-derived tumors were subsequently passaged to further mice. HE stained tumor sections were histopathologically examined to assess morphological concordance between patient tumor and PDX model. Renal cell carcinoma marker expression was determined by immunohistochemistry. Stably growing xenografts were tested for sensitivity towards SoC drugs, such as Avastin, Sunitinib, Sorafenib and Everolimus. Response to treatment was evaluated by comparing growth inhibition of treated in relation to untreated tumors. Primary tumors and the derived xenografts are currently under characterization for common oncogenic mutations using the Illumina TruSeq Amplicon Cancer Panel. Results: Nearly 200 tumor samples were transplanted since 2011. To date, 30% were successfully engrafted in immunodeficient mice and further patient-derived tumors are presently under establishment. Engraftment rates of samples from pT3 and pT4 tumors were higher (34%) than those from pT1 and pT2 tumors (12%). Two tumor samples which were pre-treated before surgery engrafted so far (11%). We show that specific tumor histology and renal cell carcinoma marker expression remained preserved over the passages. The drug sensitivity testings revealed a heterogeneous response to SoC treatments with Sunitinib (79% significant growth inhibition) and Everolimus (64%) as most effective treatments in the PDX models. Avastin showed a significant growth inhibition in 57% whereas Sorafenib was effective in 36% of the RCC models. We are furthermore presenting the individual gene mutation status of each RCC model. Conclusions: Our newly established patient-derived renal cell carcinoma panel provides a novel tool for the investigation of mechanisms of drug resistance and sensitivity in kidney cancer, biomarker research and the development of new therapeutic approaches. Citation Format: Susanne Flechsig, Annika Wulf-Goldenberg, Christian Schmees, Burkhard Jandrig, Jörg Hennenlotter, Jens Bedke, Martin Schostak, Andrew Crockford, Marco Gerlinger, James Larkin, Charles Swanton, Zoltan Szallasi, Iduna Fichtner, Jens Hoffmann. Establishment and characterization of a new patient-derived renal cell carcinoma xenograft panel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1192. doi:10.1158/1538-7445.AM2014-1192

Abstract 3061: A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of circulating tumor cells

Abstract Background: Personalized cancer treatment relies on the accurate detection of actionable genomic aberrations in tumor cells. Circulating tumor cells (CTCs) could provide an alternative genetic resource for diagnosis as “liquid biopsy”; however, attempts to isolate rare CTCs for further molecular analysis remain technically challenging by the currently available enrichment methods. In this study, we aimed to develop an improved capture system for molecular characterization of CTCs based on a novel cell sorting technology. Methods: We have developed a rare cell capture platform with On-Chip Sort (On-Chip Biotechnologies), a novel benchtop cell sorter equipped with a disposable microfluidic chip. Spike-in experiments using lung cancer cell lines with varying EpCAM expression levels (H1975, A549 and H1755) were conducted to assess capture and purification efficiency of our platform. Samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, followed by fixation and labeling with cytokeratin (CK)-FITC, vimentin-PE, CD45-Alexa Fluor 700 antibodies, and/or Hoechst 33342 for nuclei staining. The enriched and labeled samples were then sorted on On-Chip Sort based on CK, vimentin, and CD45 expression. Captured cells were immediately subjected to whole genome amplification (WGA) followed by mutation analysis using deep targeted sequencing. Amplicon libraries were generated using Illumina TruSeq Amplicon Cancer Panel and sequenced on a MiSeq sequencer. Copy number analysis of the WGA samples were performed by quantitative PCR (qPCR). Results: Spike-in experiments revealed excellent overall mean detection, recovery and purity rates of 85.0 ± 9.9%, 70.9% ± 10.6 and 72.9% ± 15.1, respectively (n=27). These results were obtained from samples containing as few as 5 tumor cells per 4 mL of blood, independent of EpCAM expression. A 100% success rate of mutation detection in EGFR, KRAS and BRAF genes from captured cells (n=27) was achieved by pyrosequencing as well as by deep targeted sequencing. The mutant variant detection rates were markedly higher than those obtained with the CellSearch profile kit. qPCR analysis of amplified DNA demonstrated reproducible detection of copy number changes of the EGFR gene n captured tumor cells. Conclusions: Using a novel cell sorter, we established an efficient and convenient platform for the capture of CTCs. Results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients. Further evaluation using clinical samples is warranted. Citation Format: Yasuhiro Koh, Masaru Watanabe, Takeshi Sawada, Hirotsugu Kenmotsu, Masakuni Serizawa, Masayuki Ishige, Kazuo Takeda, Nobuyuki Yamamoto, Toshiaki Takahashi, Fumiaki Koizumi. A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of circulating tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3061. doi:10.1158/1538-7445.AM2014-3061

Abstract 4285: Structural variant breakpoint detection in advanced colorectal cancer

Abstract Introduction Development of colorectal cancer (CRC) is accompanied by genomic alterations that drive tumor initiation and progression. Gains and losses of large chromosome segments result in DNA copy number alterations and subsequently quantitative changes in mRNA and protein expression levels. Interestingly, the accompanying chromosome breakpoints represent structural variants (SV) that may affect gene architecture and thereby normal gene function. Aim The aim of this study was to identify recurrent SV breakpoints in advanced CRC. Methods Previously a series of 352 advanced CRC samples from CAIRO and CAIRO2 clinical studies [Koopman et al. Lancet 2007; Tol et al. N Engl J Med 2009] was characterized for genome-wide DNA copy number alterations. DNA from formalin-fixed paraffin-embedded (FFPE) tumor and patient-matched normal tissue was subjected to high-resolution array-Comparative Genomic Hybridization (Agilent 180K arrays). Using these data, we now determined the prevalence of recurrent breakpoints in genes in CRC by computational analysis. In addition, multiplexed amplicon analysis involving 48 cancer-related genes (Illumina TruSeq Amplicon Cancer Panel) was applied to determine mutation frequencies. Multi-Dendrix was used to identify modules of (mutually exclusively mutated) cancer driver genes. Results We identified 748 recurrent SV breakpoints in genes (FDR&amp;lt;0.1). The highest frequency of recurrent breakpoints was detected in MACROD2, in up to ∼40% of CRC samples. Most recurrent breakpoints occurred in less than 5% of all tumors, and have not been reported before in CRC. Mutation frequencies in APC, TP53 and KRAS were conform expectations (60%, 58%, and 48%, respectively). Multi-Dendrix analysis revealed modules of cancer driver genes that included both the commonly mutated CRC cancer genes as well as genes with recurrent breakpoints, suggesting that several of the SV breakpoint genes are candidate drivers of the carcinogenic process. Conclusions We were able to pinpoint the prevalence of 748 recurrent SV breakpoint regions in genes using array-CGH data from 352 CRC samples. Moreover, our studies revealed several breakpoints in genes that are mutually exclusive with the commonly mutated APC, KRAS, and TP53 genes, and therefore represent novel candidate cancer driver genes. Further studies are required to investigate their functional and clinical significance. Citation Format: E van den Broek, O Krijgsman, D Sie, JC Haan, M Komor, J Traets, DAM Heideman, MA van de Wiel, ID Nagtegaal, CJA Punt, B Carvalho, B Ylstra, GA Meijer, RJA Fijneman. Structural variant breakpoint detection in advanced colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4285. doi:10.1158/1538-7445.AM2014-4285

Abstract 954: Integrated genomic analysis by whole exome and transcriptome sequencing of tumor samples from EGFR-mutant non-small-cell lung cancer patients with acquired resistance to erlotinib

Abstract Background: NSCLC p with EGFR mutations initially respond to EGFR tyrosine kinase inhibitors (TKIs) but ultimately relapse. Sub-genomic molecular studies indicate that the EGFR T790M mutation and the activation of MET, PI3K, AXL, HER2 and MAPK can lead to acquired resistance to EGFR TKIs. To date, no integrated comprehensive genomic investigation of EGFR TKI resistance has been reported. Methods: FFPE biopsies of erlotinib-sensitive and erlotinib-resistant tumors were obtained from 16 EGFR mutant NSCLC patients. The samples were analyzed by whole exome sequencing and whole transcriptome sequencing utilizing the Illumina HiSeq2500 platform. In addition, targeted gene sequencing was performed with the Illumina TruSeq Amplicon-Cancer Panel and run on the MiSeq system. Results: Erlotinib resistant NSCLC specimens harbored known resistance drivers, including EGFR T790M mutations (6/16; 38%), MET amplification (2/16; 13%), and AXL upregulation (3/16; 19%). Differential expression analysis between resistant and pre-treatment states revealed enrichment in the post-resistant tumors of ERBB2, and FGFR signaling pathways. Biopsies from patients that developed an EGFR T790M mutation post resistance exhibited enrichment in pathways associated with cell cycle, meiosis, telomere maintenance and transcriptional regulation. Copy number analysis demonstrated amplifications in the post-resistance setting that correlated significantly with progression free survival, including regions containing NFKBIA, NKX2-1, and TSC-1. There was also strong correlation between the copy number changes observed and the expression mRNA levels of specific genes. Of note, each resistant tumor exhibited greater copy number similarity to the corresponding matched pre-treatment sample compared to other tumors within the resistance cohort. Conclusions: We conducted the first ever comprehensive integrated genomic analysis of EGFR TKI resistant NSCLC patients, and identified both known and potentially novel drivers of EGFR TKI resistance. This study demonstrated the feasibility and utility of comprehensive genomic analysis in the clinical management of NSCLC receiving targeted therapy. Together, our data provide unprecedented insight into the molecular pathogenesis of escape from EGFR oncogene inhibition in NSCLC. We are now conducting a prospective observational study in additional NSCLC patients on targeted therapy. Citation Format: Petros Giannikopoulos, John St. John, Nicholas Hahner, Joel S. Parker, Niki Karachaliou, Carlota Costa, Oscar Westesson, Urvish Parikh, Catherine K. Foo, Aleah F. Cauhlin, Maria D. Lozano, Santiago Viteri, Jose L. Perez-Gracia, Alessandra Curioni, Eloisa Jantus-Lewintre, Carlos Camps, Alain Vergenegre, Radj Gervais, Anne Wellde, Jonathan Barry, George W. Wellde, Rodolfo Bordoni, Rolf Stahel, Andres Felipe Cardona Zorilla, William R. Polkinghorn, Jonathan Weissman, Trever G. Bivona, Rafael Rosell. Integrated genomic analysis by whole exome and transcriptome sequencing of tumor samples from EGFR-mutant non-small-cell lung cancer patients with acquired resistance to erlotinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 954. doi:10.1158/1538-7445.AM2014-954

Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing.

BackgroundClinical specimens undergoing diagnostic molecular pathology testing are fixed in formalin due to the necessity for detailed morphological assessment. However, formalin fixation can cause major issues with molecular testing, as it causes DNA damage such as fragmentation and non-reproducible sequencing artefacts after PCR amplification. In the context of massively parallel sequencing (MPS), distinguishing true low frequency variants from sequencing artefacts remains challenging. The prevalence of formalin-induced DNA damage and its impact on molecular testing and clinical genomics remains poorly understood.MethodsThe Cancer 2015 study is a population-based cancer cohort used to assess the feasibility of mutational screening using MPS in cancer patients from Victoria, Australia. While blocks were formalin-fixed and paraffin-embedded in different anatomical pathology laboratories, they were centrally extracted for DNA utilising the same protocol, and run through the same MPS platform (Illumina TruSeq Amplicon Cancer Panel). The sequencing artefacts in the 1-10% and the 10-25% allele frequency ranges were assessed in 488 formalin-fixed tumours from the pilot phase of the Cancer 2015 cohort. All blocks were less than 2.5 years of age (mean 93 days).ResultsConsistent with the signature of DNA damage due to formalin fixation, many formalin-fixed samples displayed disproportionate levels of C>T/G>A changes in the 1-10% allele frequency range. Artefacts were less apparent in the 10-25% allele frequency range. Significantly, changes were inversely correlated with coverage indicating high levels of sequencing artefacts were associated with samples with low amounts of available amplifiable template due to fragmentation. The degree of fragmentation and sequencing artefacts differed between blocks sourced from different anatomical pathology laboratories. In a limited validation of potentially actionable low frequency mutations, a NRAS G12D mutation in a melanoma was shown to be a false positive.ConclusionsThese findings indicate that DNA damage following formalin fixation remains a major challenge in laboratories working with MPS. Methodologies that assess, minimise or remove formalin-induced DNA damaged templates as part of MPS protocols will aid in the interpretation of genomic results leading to better patient outcomes.

Abstract B179: Targeted next generation sequencing for somatic mutations in human cancer.

Abstract Objective: We have replaced single gene PCR-based methods for the detection of BRAF, KRAS and EGFR variants in routine clinical practice with next generation sequencing assays for the Ion Torrent AmpliSeq Cancer Hotspot Panel v2 (50 genes) and the Illumina TruSeq Amplicon Cancer panel (48 genes). Implementation of these panels requires constant optimization of technical and analytical procedures as reagents and software continue to be developed. In this study we describe somatic mutation findings from our clinical laboratory service. Methods: DNA was extracted from 90 formalin fixed, paraffin embedded tissue specimens that included 50 lung cancer, 24 colon cancer and 16 other cancers. Barcoded libraries were prepared from up to 10ng of extracted DNA and multiplexed on single 318 chips. Data analysis was performed using Golden Helix SVS for the Ion Torrent panel and Variant Studio for the Illumina panel. Variants that remained after the analysis pipeline were individually interrogated using the Integrative Genomics Viewer (IGV). Results: Of the 90 cancer cases sequenced, each panel returned on average 20 variants. Of these, there was 100% concordance between the two panels with respect to the clinically actionable mutations identified. Conclusions: We have implemented next generation sequencing technologies for the detection of somatic mutations in human cancers. Two platforms, the Ion Torrent PGM and the Illumina MiSeq, have been used for the routine analysis of FFPE cancer tissues in a clinical setting with actionable and non-actionable variants reported and archived. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B179. Citation Format: Jason D. Peterson, Francine de Abreu, Torrey L. Gallagher, Paul R. Burchard, Christopher I. Amos, Wendy A. Wells, J. Marc Pipas, Marc S. Ernstoff, Camilo E. Fadul, James R. Rigas, Gregory J. Tsongalis. Targeted next generation sequencing for somatic mutations in human cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B179.

Abstract A34: A preclinical study of a novel FACS-based rare cell capture platform for the detection, capture and molecular characterization of circulating tumor cells.

Abstract Background: Recent advances in molecular targeted cancer therapy offer a wide variety of therapeutic strategies. The presence or absence of particular actionable genomic aberrations has been shown to predict response to molecularly targeted treatments as a companion diagnostics. Conventionally this strategy relies on analysis of primary tumor samples, whereas circulating tumor cells (CTCs) offer an alternative source for the detection of genetic alterations as a “liquid biopsy”. To acquire genetic profile of CTCs, development of reliable platform and workflow to detect and capture a small number of mutation-bearing CTCs from a blood sample is necessary. However, attempts to isolate rare CTCs for further molecular analysis remain technically challenging by the currently available enrichment methods. Methods: We have developed a rare cell capture platform with On-Chip Sort (On-Chip Biotechnologies), a novel benchtop cell sorter equipped with a disposable microfluidic chip. Spike-in experiments using lung cancer cell lines with varying EpCAM expression levels were conducted to assess capture and purification efficiency of our platform. Samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, followed by fixation and labeling with cytokeratin (CK)-FITC, vimentin-PE, CD45-Alexa Fluor 700 antibodies, and/or Hoechst 33342 for nuclei staining. The enriched and labeled samples were then sorted on On-Chip Sort based on CK, vimentin, and CD45 expression. Captured cells were immediately subjected to whole genome amplification (WGA) followed by mutation analysis using deep targeted sequencing. Amplicon libraries were generated using Illumina TruSeq Amplicon Cancer Panel and sequenced on a MiSeq sequencer. Copy number analysis of the WGA samples were performed by quantitative PCR (qPCR). Results: Spike-in experiments revealed excellent overall mean detection, recovery and purity rates of 85.0 ± 9.9%, 70.9% ± 10.6 and 72.9% ± 15.1, respectively (n=27). These results were obtained from samples containing as few as 5 tumor cells per 4 mL of blood, independent of EpCAM expression. A 100% success rate of mutation detection in EGFR, KRAS and BRAF genes from captured cells (n=27) was achieved with pyrosequencing as well as deep targeted sequencing. qPCR analysis of amplified DNA demonstrated reproducible detection of copy number change of EGFR gene from captured tumor cells. Conclusions: We described an efficient and convenient platform for the capture of CTCs utilizing a novel cell sorter. We also conducted a proof-of-principle preclinical study for a workflow capable of molecular characterization of captured CTC population. Further evaluation using clinical samples is warranted. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A34. Citation Format: Masaru Watanabe, Masakuni Serizawa, Takeshi Sawada, Yuu Fujimura, Fumiaki Koizumi, Yasuhiro Koh. A preclinical study of a novel FACS-based rare cell capture platform for the detection, capture and molecular characterization of circulating tumor cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A34.

Abstract 3229: Validation of Illumina TruSeq Amplicon Cancer Panel with concordance testing using Ion AmpliSeq Cancer Panel and other methods.

Abstract Introduction: The TruSeq Amplicon Cancer Panel (TSACP), a highly multiplexed targeted resequencing assay for use on the Illumina MiSeq platform, is designed for detecting the hotspot mutations in 212 Regions of Interest (ROI) from 48 cancer related genes. Here we report our validation study on the reproducibility, sensitivity and the specificity of detecting single base substitutions and small indels by the TSACP. Methods: We used well characterized cancer cell lines harboring clinically relevant variants as positive controls and HapMap samples NA12878 and NA19240 as wild type control samples. DNA from 8 cancer cell lines was serially diluted into the control DNA NA12878 for validating the Limit of Detection (LOD) of the TSACP assay. A total of 41 FFPE patient specimens representing a variety of cancer types were analyzed in a blinded fashion to evaluate the analytical sensitivity and specificity. DNA quality was assessed using a qPCR assay. With no gold standard available as a reference method to detect mutations with comparable sensitivity, concordance testing was performed using the Ion AmpliSeq Cancer Panel. Variants detected by both panels were considered as true positives. Variants that were only covered by one of the two panels were confirmed by a third method, either Sanger sequencing for variants with frequencies above 10% or a custom Ion TargetSeq Assay for variants with frequencies below 10%. Data were analyzed using MiSeq Reporter software and our proprietary analysis pipeline. In addition to reporting hotspots mutations, we also report “Critical Variants” such as non-synonymous coding mutations and splicing site mutations that fall within the ROI. Results: 95% of our ROIs were sequenced at minimum of 0.2X normalized coverage. A cell line dilution study showed that the LOD of confirmed variants is 5%. DNA extracted from 4 of the 41 FFPE specimens failed the template QC by qPCR and failed in the subsequent sequencing run. A total of 124 unique critical variants, including single base substitution, single- or multi- base (up to 21bp) deletion, one- or two- base insertion, were identified in the cancer cell lines and 37 qualified FFPE samples. The intra-assay and inter-assay reproducibility was ≥96%. Using our proprietary analysis pipeline, the analytical sensitivity and specificity for the FFPE samples were both 99%. One false negative of TSACP was identified by the Ion AmpliSeq Cancer Panel and was further confirmed by Sanger sequencing. This allele drop-off occurred as a result of the capture probe falling on a SNP. Homopolymer indels in KIT and STK11 were accurately identified with the Illumina sequencing chemistry. Conclusions: These studies demonstrate that the TSACP assay is highly specific and sensitive and is suitable for screening patient FFPE tumor specimens for a spectrum of clinically relevant somatic mutations. Citation Format: Peng Fang, Zhenyu Yan, Weihua Liu, Agus Darwanto, Kim Pelak, Kim Anoe, Cynthia Spittle, Sabita Sankar, Chad Galderisi, Jin Li. Validation of Illumina TruSeq Amplicon Cancer Panel with concordance testing using Ion AmpliSeq Cancer Panel and other methods. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3229. doi:10.1158/1538-7445.AM2013-3229