Ion AmpliSeq Cancer Panel Research Articles

Discordance of the PAM50 Intrinsic Subtypes Compared with Immunohistochemistry-Based Surrogate in Breast Cancer Patients: Potential Implication of Genomic Alterations of Discordance.

PurposeWe aimed to analyze the discordance between immunohistochemistry (IHC)-based surrogate subtyping and PAM50 intrinsic subtypes and to assess overall survival (OS) according to discordance.Materials and MethodsA total of 607 patients were analyzed. Hormone receptor (HR) expression was evaluated by IHC, and human epidermal growth factor receptor 2 (HER2) expression was analyzed by IHC and/or fluorescence in situ hybridization. PAM50 intrinsic subtypes were determined according to 50 cancer genes using the NanoString nCounter Analysis System. We matched concordant tumor as luminal A and HR+/HER2–, luminal B and HR+/HER2+, HR–/HER2+ and HER2–enriched, and triple-negative breast cancer (TNBC) and normal- or basal-like. We used Ion Ampliseq Cancer Panel v2 was used to identify the genomic alteration related with discordance. The Kaplan-Meier method was used to estimate OS.ResultsIn total, 233 patients (38.4%) were discordant between IHC-based subtype and PAM50 intrinsic subtype. Using targeted sequencing, we detected somatic mutation–related discordant breast cancer including the VHL gene in the HR+/HER2– group (31% in concordant group, 0% in discordant group, p=0.03) and the IDH and RET genes (7% vs. 12%, p=0.02 and 0% vs. 25%, p=0.02, respectively) in the TNBC group. Among the luminal A/B patients with a discordant result had significantly worse OS (median OS, 73.6 months vs. not reached; p < 0.001), and among the patients with HR positivity, the basal-like group as determined by PAM50 showed significantly inferior OS compared to other intrinsic subtypes (5-year OS rate, 92.2% vs. 75.6%; p=0.01).ConclusionA substantial portion of patients showed discrepancy between IHC subtype and PAM50 intrinsic subtype in our study. The survival analysis demonstrated that current IHC-based classification could mislead the treatment and result in poor outcome. Current guidelines for IHC might be updated accordingly.

KRAS mutation is predictive of outcome in patients with pulmonary sarcomatoid carcinoma.

Pulmonary sarcomatoid carcinoma (PSC) is a poorly differentiated non-small-cell lung carcinoma (NSCLC) with aggressive behaviour. This study aimed to evaluate the prognostic clinicopathological and genetic characteristics of PSCs. Fifty-three cases of surgically treated PSCs were selected, 23 of which were subjected to mutation and copy number variation analysis using the 50-gene Ion AmpliSeq Cancer Panel. The majority of the patients were male (32 of 53, 60.3%) and smokers (51 of 53, 96.2%). Overall, 25 (47.1%) patients died within 2-105months (mean=22.7months, median=15months) after diagnosis, and 28 were alive 3-141months (mean=38.7months, median=21.5months) after diagnosis. Five-year overall survival was 12.5%. KRAS codon 12/13 mutation in adenocarcinomas (P=0.01), age more than 70years (P=0.008) and tumour size ≥4.0cm (P=0.02) were associated strongly with worse outcome. TP53 (17 of 23, 74.0%) and KRAS codon 12 of 13mutations (10 of 23, 43.4%) were the most common genetic alterations. Potentially actionable variants were identified including ATM (four of 23, 17.3%), MET, FBXW7 and EGFR (two of 23, 8.7%), AKT1, KIT, PDGFRA, HRAS, JAK3 and SMAD4 (one of 23, 4.3%). MET exon 14 skipping and missense mutations were identified in two (11.1%) cases with adenocarcinoma histology. Copy number analysis showed loss of RB1 (three of 23, 13%) and ATM (two of 23, 8.7%). Copy number gains were seen in EGFR (two of 23, 13.0%) and in one (4.3%) of each PIK3CA, KRAS, MET and STK11. Potentially targetable mutations can be identified in a subset of PSC, although most tumours harbour currently untargetable prognostically adverse TP53 and KRAS mutations.

Next-Generation Sequencing Approach to Non-Small Cell Lung Carcinoma Yields More Actionable Alterations.

- Different testing algorithms and platforms for EGFR mutations and ALK rearrangements in advanced-stage lung adenocarcinoma exist. The multistep approach with single-gene assays has been challenged by more efficient next-generation sequencing (NGS) of a large number of gene alterations. The main criticism of the NGS approach is the detection of genomic alterations of uncertain significance. - To determine the best testing algorithm for patients with lung cancer in our clinical practice. - Two testing approaches for metastatic lung adenocarcinoma were offered between 2012-2015. One approach was reflex testing for an 8-gene panel composed of DNA Sanger sequencing for EGFR, KRAS, PIK3CA, and BRAF and fluorescence in situ hybridization for ALK, ROS1, MET, and RET. At the oncologist's request, a subset of tumors tested by the 8-gene panel was subjected to a 50-gene Ion AmpliSeq Cancer Panel. - Of 1200 non-small cell lung carcinomas (NSCLCs), 57 including 46 adenocarcinomas and NSCLCs, not otherwise specified; 7 squamous cell carcinomas (SCCs); and 4 large cell neuroendocrine carcinomas (LCNECs) were subjected to Ion AmpliSeq Cancer Panel. Ion AmpliSeq Cancer Panel detected 9 potentially actionable variants in 29 adenocarcinomas that were wild type by the 8-gene panel testing (9 of 29, 31.0%) in the following genes: ERBB2 (3 of 29, 10.3%), STK11 (2 of 29, 6.8%), PTEN (2 of 29, 6.8%), FBXW7 (1 of 29, 3.4%), and BRAF G469A (1 of 29, 3.4%). Four SCCs and 2 LCNECs showed investigational genomic alterations. - The NGS approach would result in the identification of a significant number of actionable gene alterations, increasing the therapeutic options for patients with advanced NSCLCs.

Genetic alterations in Japanese extrahepatic biliary tract cancer.

Biliary tract cancer (BTC) is one of the most devastating types of malignant neoplasms worldwide. However, the mechanisms underlying the development and progression of BTC remain unresolved. BTC includes extrahepatic bile duct carcinoma (EBDC), gallbladder carcinoma (GBC) and ampulla of Vater carcinoma (AVC), named according to the location of the tumor. Although genetic alterations of intrahepatic cholangiocarcinoma have been investigated, those of EBDC, GBC and AVC have not yet been fully understood. The present study analyzed somatic mutations of 50 cancer-associated genes in 27 Japanese BTC cells, including: 11 EBDC, 14 GBC and 2 AVC. Next-generation sequencing using an Ion AmpliSeq Cancer Panel identified a total of 44 somatic mutations across 14 cancer-associated genes. Among the 44 mutations, 42 were judged as pathological mutations. Frequent mutations were identified in tumor protein 53 (TP53) (14/27), SMAD family member 4 (SMAD4) (6/27), phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α (PIK3CA) (6/27), and Kirsten rat sarcoma (KRAS) (6/27); no significant differences were identified between EBDC and GBC tissues. Notably, the frequency of the PIK3CA mutation was higher when compared with previous reports. This result may suggest that the activation of the PIK3CA-protein kinase B signaling pathway, in addition to the abrogation of p53, SMAD4 and RAS mitogen-activated protein kinase may have a crucial role in the carcinogenesis of Japanese BTC. These findings may be useful for the development of personalized therapies for BTC.

Treatment inferred from mutations identified using massive parallel sequencing leads to clinical benefit in some heavily pretreated cancer patients.

Molecular portraits of numerous tumors have flooded oncologists with vast amounts of data. In parallel, effective inhibitors of central pathways have shown great clinical benefit. Together, this promises potential clinical benefits to otherwise end-stage cancer patients. Here, we report a clinical service offering mutation detection of archived samples using the ion Ampliseq cancer panel coupled with clinical consultation.A multidisciplinary think tank consisting of oncologists, molecular-biologists, genetic counselors, and pathologists discussed 67 heavily pretreated, advanced cancer patient cases, taking into account mutations identified using ion Ampliseq cancer panel, medical history, and relevant literature.The team generated a treatment plan, targeting specific mutations, for 41 out of 64 cases. Three patients died before results were available. For 32 patients, the treating oncologists chose not to include the panel recommendation in the treatment plan for various reasons. Nine patients were treated as recommended by the panel, 5 with clinical benefit, and 4 with disease progression.This study suggests that routine use of massive parallel tumor sequencing is feasible and can judiciously affect treatment decisions when coupled with multidisciplinary team-based decision making. Administration of personalized based therapies at an earlier stage of disease, expansion of genetic alterations examined, and increased availability of targeted therapies may lead to further improvement in the clinical outcome of metastatic cancer patients.

Association of PIK3CA H1047R and STK11 F354L mutations with response to neoadjuvant chemoradiotherapy and survival in esophageal cancer.

55 Background: Neoadjuvant chemoradiotherapy is the standard treatment for locally advanced esophageal cancer. However, its efficacy is limited in non-responders and there is no reliable clinical parameter in predicting treatment response. We aim to identify chemoradiation-resistant cancers and avoid unnecessary treatments in non-responders with mutation analysis. Methods: Genomic DNA was extracted from diagnostic biopsy specimens of 20 patients (10 with good and 10 with poor response) with esophageal squamous cell carcinoma after neoadjuvant chemoradiotherapy and esophagectomy. Using next-generation sequencing, we screened mutation hotspots in 46 cancer-related genes (Ion AmpliSeq Cancer Panel). Potential predictive genetic variations were validated in another cohort of patients. Chemoradiotherapy response was based on pathological evaluation of surgically resected specimens. The prognostic significance of genetic variations was evaluated with Cox regression survival analysis. Results: In the first cohort, we discovered PIK3CA H1047R and STK11 F354L as potential targets to predict chemoradiation resistance. Next, we investigate these genetic variations in another 54 patients. Altogether, 18.9% (14/74) and 6.8% (5/74) of patients had PIK3CA H1047R and STK11 F354L, respectively. In patients with poor response to chemoradiotherapy, 45.8% (11/24) had either PIK3CA H1047R or STK11 F354L; compared to 12.0% (6/50) in patients with good response ( p = 0.001). In other words, 64.7% of patients with either one genetic variation responded poorly to chemoradiotherapy. In survival analysis, the 5-yr survival rates were 7.7% and 43.7% in patients with either one genetic variation and those without, respectively ( p = 0.003). In multivariable analysis, the presence of either one genetic variation was a significant prognostic factor for overall survival (Hazard ratio: 2.43, 95% confidence interval: 1.195-4.944, p= 0.014). Conclusions: PIK3CA H1047R and STK11 F354L mutations are associated with poor response to neoadjuvant chemoradiotherapy and worse prognosis in esophageal cancer. It may potentially be biomarkers to guide treatment decisions.

Genomic profiling of ampullary adenocarcinoma (AA): Insights from a comparative analysis of pancreatic and intestinal adenocarcinoma and opportunities for targeted therapies use.

300 Background: Ampullary adenocarcinoma (AA) is a rare entity. AA can originate from either intestinal or pancreaticobiliary ductal epithelium, and patients are often managed as those with pancreaticobiliary carcinomas. The study objectives were the genetic profiling of AA and the identification of specific molecular profiles according to these 2 pathological types. Methods: AA patients included in the AGEO retrospective multicenter cohort who underwent surgical resection of their tumor between 1999 and 2010 were selected. Formalin-fixed, paraffin-embedded (FFPE) archival tissue blocks were collected. Next generation sequencing (NGS) using a 50 gene panel (Ion AmpliSeq Cancer panel) on tumor DNA, and immunohistochemistry (IHC) panel including CK7, CK20, MUC1, MUC2 and CDX2, on tumor sections, were performed. Results: NGS was performed on 101 tumors from 6 hospitals, with 1 technical failure. In total, the most frequent gene mutations were: KRAS (45%), TP53 (40%), APC (15%), PIK3CA (12%), SMAD4 (9%), BRAF (8%), CDKN2A (6%). No mutation was found in 21% of tumors. According to IHC, the most common histological type was intestinal (51%), followed by pancreaticobiliary type (42%) and undetermined (7%). BRAF mutation was significantly associated with intestinal type (8 vs 0, p = 0.017). According to Cosmic database, similarities of molecular profiles exist between AA with intestinal type and colorectal adenocarcinoma, and between AA with pancreaticobiliary type and pancreas adenocarcinoma, respectively. Conclusions: This study shows that AA is a heterogeneous entity and that a large proportion of AA presents a molecular profile that is more similar to that of colorectal adenocarcinoma, compared to pancreatic adenocarcinoma. This important information could be interesting to guide treatment decision in patients with this rare disease.

88P - Treatment inferred from mutations identified using massive parallel sequencing leads to clinical benefit in some heavily pretreated cancer patients

Molecular portraits of numerous tumors have flooded oncologists with vast amounts of data. In parallel, effective inhibitors of central pathways have shown great clinical benefit. Together, this promises potential clinical benefits to otherwise end-stage cancer patients. Here, we report a clinical service offering mutation detection of archived samples using the ion Ampliseq cancer panel coupled with clinical consultation.A multidisciplinary think tank consisting of oncologists, molecular-biologists, genetic counselors, and pathologists discussed 67 heavily pretreated, advanced cancer patient cases, taking into account mutations identified using ion Ampliseq cancer panel, medical history, and relevant literature.The team generated a treatment plan, targeting specific mutations, for 41 out of 64 cases. Three patients died before results were available. For 32 patients, the treating oncologists chose not to include the panel recommendation in the treatment plan for various reasons. Nine patients were treated as recommended by the panel, 5 with clinical benefit, and 4 with disease progression.This study suggests that routine use of massive parallel tumor sequencing is feasible and can judiciously affect treatment decisions when coupled with multidisciplinary team-based decision making. Administration of personalized based therapies at an earlier stage of disease, expansion of genetic alterations examined, and increased availability of targeted therapies may lead to further improvement in the clinical outcome of metastatic cancer patients.

Comparison between circulating tumor DNA and tumor tissue multiple gene detection in non-small cell lung cancer patients by targeted sequencing with the Ion PGM and AmpliSeq Cancer Panel.

8544Background: Circulating tumor DNA (ctDNA) is a noninvasive assessment that can be used as an alternative method to detect gene mutations in lung cancer patients. However, previous studies have ...

Early onset esophageal adenocarcinoma: a distinct molecular entity?

Esophageal adenocarcinoma (EAC) is typically diagnosed in elderly with a median age of 68 years. The incidence of EAC has been rising over the last decades, also among young adults. The aim of the study was to investigate whether early onset EAC is a distinct molecular entity. To identify early onset EACs, the nationwide network and registry of histo- and cytopathology in the Netherlands (PALGA) was searched. Twenty-eight tumors of patients aged ≤40 years were selected and matched with 27 tumors of patients aged ≥68 years. DNA was isolated from surgically resected specimen and sequenced on the Ion Torrent Personal Genome Machine with the Ion AmpliSeq Cancer Panel. No differences in mutational load between early onset and conventional EACs were observed (P=0.196). The most frequently mutated genes were TP53 (73%) and P16 (16%). Additional mutations in early onset EACs occurred exclusively in: APC, CDH1, CTNNB1, FGFR2, and STK11. In the conventional EACs additional mutations were exclusively identified in: ABL1, FBXW7, GNA11, GNAS, KRAS, MET, SMAD4, and VHL. Additional mutations besides TP53 and P16 seem to occur in different genes related to cell fate pathways for early onset EACs, while the additional mutations in conventional EACs are related to survival pathways.

Mutational profiling of brain metastasis from breast cancer: matched pair analysis of targeted sequencing between brain metastasis and primary breast cancer

Although breast cancer is the second most common cause of brain metastasis with a notable increase of incidence, genes that mediate breast cancer brain metastasis (BCBM) are not fully understood. To study the molecular nature of brain metastasis, we performed gene expression profiling of brain metastasis and matched primary breast cancer (BC). We used the Ion AmpliSeq Cancer Panel v2 covering 2,855 mutations from 50 cancer genes to analyze 18 primary BC and 42 BCBM including 15 matched pairs. The most common BCBM subtypes were triple-negative (42.9%) and basal-like (36.6%). In a total of 42 BCBM samples, 32 (76.2%) harbored at least one mutation (median 1, range 0-7 mutations). Frequently detected somatic mutations included TP53 (59.5%), MLH1 (14.3%), PIK3CA (14.3%), and KIT (7.1%). We compared BCBM with patient-matched primary BC specimens. There were no significant differences in mutation profiles between the two groups. Notably, gene expression in BCBM such as TP53, PIK3CA, KIT, MLH1, and RB1 also seemed to be present in primary breast cancers. The TP53 mutation frequency was higher in BCBM than in primary BC (59.5% vs 38.9%, respectively). In conclusion, we found actionable gene alterations in BCBM that were maintained in primary BC. Further studies with functional testing and a delineation of the role of these genes in specific steps of the metastatic process should lead to a better understanding of the biology of metastasis and its susceptibility to treatment.

Molecular profiles of high-grade and low-grade pseudomyxoma peritonei.

Pseudomyxoma peritonei (PMP) is a rare disease exhibiting a distinct clinical feature caused by cancerous cells that produce mucinous fluid in the abdominal cavity. PMPs originate most frequently from the appendix and less frequently from the ovary. This disease can range from benign to malignant, and histologically, PMP is classified into two types: disseminated peritoneal adenomucinosis (DPAM) representing the milder phenotype, and peritoneal mucinous adenocarcinomas (PMCA) representing the aggressive phenotype. Although histological classification is clinically useful, the pathogenesis of PMP remains largely unknown. To elucidate the molecular mechanisms underlying PMP, we analyzed 18 PMP tumors comprising 10 DPAMs and 8 PMCAs. DNA was extracted from tumor and matched non‐tumorous tissues, and was sequenced using Ion AmpliSeq Cancer Panel containing 50 cancer‐related genes. Analysis of the data identified a total of 35 somatic mutations in 10 genes, and all mutations were judged as pathological mutations. Mutations were frequently identified in KRAS (14/18) and GNAS (8/18). Interestingly, TP53 mutations were found in three of the eight PMCAs, but not in the DPAMs. PIK3CA and AKT1 mutations were also identified in two PMCAs, but not in the DPAMs. These results suggested that KRAS and/or GNAS mutations are common genetic features of PMP, and that mutations in TP53 and/or genes related to the PI3K‐AKT pathway may render malignant properties to PMP. These findings may be useful for the understanding of tumor characteristics, and facilitate the development of therapeutic strategies.

Identification of Driving ALK Fusion Genes and Genomic Landscape of Medullary Thyroid Cancer.

The genetic landscape of medullary thyroid cancer (MTC) is not yet fully understood, although some oncogenic mutations have been identified. To explore genetic profiles of MTCs, formalin-fixed, paraffin-embedded tumor tissues from MTC patients were assayed on the Ion AmpliSeq Cancer Panel v2. Eighty-four sporadic MTC samples and 36 paired normal thyroid tissues were successfully sequenced. We discovered 101 hotspot mutations in 18 genes in the 84 MTC tissue samples. The most common mutation was in the ret proto-oncogene, which occurred in 47 cases followed by mutations in genes encoding Harvey rat sarcoma viral oncogene homolog (N = 14), serine/threonine kinase 11 (N = 11), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (N = 6), mutL homolog 1 (N = 4), Kiesten rat sarcoma viral oncogene homolog (N = 3) and MET proto-oncogene (N = 3). We also evaluated anaplastic lymphoma kinase (ALK) rearrangement by immunohistochemistry and break-apart fluorescence in situ hybridization (FISH). Two of 98 screened cases were positive for ALK FISH. To identify the genomic breakpoint and 5’ fusion partner of ALK, customized targeted cancer panel sequencing was performed using DNA from tumor samples of the two patients. Glutamine:fructose-6-phosphate transaminase 1 (GFPT1)-ALK and echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusions were identified. Additional PCR analysis, followed by Sanger sequencing, confirmed the GFPT1-ALK fusion, indicating that the fusion is a result of intra-chromosomal translocation or deletion. Notably, a metastatic MTC case harboring the EML4-ALK fusion showed a dramatic response to an ALK inhibitor, crizotinib. In conclusion, we found several genetic mutations in MTC and are the first to identify ALK fusions in MTC. Our results suggest that the EML4-ALK fusion in MTC may be a potential driver mutation and a valid target of ALK inhibitors. Furthermore, the GFPT1-ALK fusion may be a potential candidate for molecular target therapy.

Abstract 2014: MultiOmyxTM: A multiplexed immunofluorescent assay capable of profiling protein expression and phosphorylation, in combination with next-generation sequencing from a single FFPE tissue section

Abstract A comprehensive signaling pathway profile in combination with mutational analysis may be a critical guide for selecting effective clinical strategies for targeted drugs in combinations or in sequential regimens. In the United States, colorectal cancer (CRC) is the third most common cancer and the third leading cause of tumor associated death in men and women. CRC is a heterogeneous disease defined by different receptor tyrosine kinase (RTK) activation, signaling through phosphatidylinositol 3-kinase (PI3K)/AKT and RAS/MAP2K pathways. In addition, activating mutations in RTK and/or activating or loss-of-function mutations in downstream intracellular signaling proteins, can alter the efficacy of targeted drugs resulting in an ineffective treatment. GE Healthcare, through its affiliate Clarient Diagnostic Services Inc., has developed a novel hyperplexed multi-omic technology, MultiOmyx, to enable visualization and characterization of multiple biomarkers across multiple assays on a single 4μm tissue section. MultiOmyx protein immunofluorescence (IF) assays utilize a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining and deactivation for up to 60 protein biomarkers. The same protein IF processed slide is then used to perform DNA FISH assay, followed by DNA extraction using laser capture microdissection (LCM) from region(s) of interest for next generation sequencing. Herein, we report an analysis of the key receptor tyrosine kinases (EGFR, HER2, HER3, and cMET) and their downstream signaling proteins (PI3K, phospho AKT, and phospho ERK1/2) in 10 colon tumor samples using the MultiOmyx technology. Mutational analysis was performed on LCM extracted tumor and tumor adjacent regions, and sequenced using the Ion AmpliSeq cancer panel, consisting of 50 targeted genes. Protein IF staining revealed heterogeneous expression and activation across different samples. With high EGFR and HER3 expression correlating with activation of AKT, possibly through EGFR:HER3 dimer, to high expression of EGFR correlating with positive staining for phospho ERK1/2. Additionally, intra-tumor heterogeneity was observed within the same tumor tissue, with varied expression of EGFR, HER2, and HER3. Preliminary mutational analysis also detected BRAF G469A known to increase kinase activity of BRAF and downstream activation of pERK1/2 in tumor but not in tumor-adjacent region. The MultiOmyx assay can be utilized for clinical samples testing with limited size and availability. A comprehensive mutational and signaling protein analysis can guide oncologists tailored either a single or combinational targeted therapy to patient's individual tumor profile. Citation Format: Qingyan Au, Maoyong Fu, Alexander Bordwell, Tripathi Pinky, Michael Lazare, Nam Tran, Nicholas Hoe. MultiOmyxTM: A multiplexed immunofluorescent assay capable of profiling protein expression and phosphorylation, in combination with next-generation sequencing from a single FFPE tissue section. [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 2014. doi:10.1158/1538-7445.AM2015-2014

Abstract 4825: Association between genetic profiles and histological classifications of 18 Japanese pseudomyxoma peritonei

Abstract Pseudomyxoma peritonei (PMP) is a rare disease occurring in 1-2 / 1000,000 and shows a distinct clinical feature caused by cancerous cells that produce mucin or gelatinous fluid in the abdominal cavity. Tumor cells are the most frequently originated from appendiceal mucinous cancer, and less frequently from mucinous tumors of the ovary. PMP is histologically classified into two types; disseminated peritoneal adenomucinosis (DPAM) showing milder phenotype, and peritoneal mucinous adenocarcinomas (PMCA) exhibiting more aggressive phenotype. Several molecular studies have shown frequent mutations in the KRAS and GNAS genes. However molecular profiles associated with different histological and/or clinical characteristics remain largely unknown. To elucidate molecular mechanisms underlying PMP, we analyzed 18 Japanese PMP tumors comprising of 10 DPAMs and 8 PMCAs. DNA was extracted from tumor tissues and their matched normal samples, and was subjected to amplicon sequencing using Ion AmpliSeq Cancer Panel v2 (Life Technologies) containing 49 cancer-related genes. In the 18 tumors, we identified a total of 42 somatic mutations (34 missense, 1 deletion, 1 insertion, 2 intron, 1 splice site mutations, and 3 synonymous mutations) in 13 genes. Of the 42 mutations, 35 were judged as pathogenic, and the remaining seven as non-pathogenic by the search of public databases and reports, or by in silico analysis. KRAS and GNAS mutations were found in 14 (77.8%) and 8 (44.4%) tumors, respectively. Interestingly, no mutations were detected in APC or CTNNB1. Notably, TP53 mutation was found in 3 of the 8 PMCAs, but not in the 10 DPAMs. Two of the 8 PMCAs carried mutation in either PIK3CA or AKT1. These data suggest that KRAS and/or GNAS mutations play a vital role in the development of both types of PMP, and that inactivation of Wnt signaling pathway may not be involved in the tumorigenesis. Furthermore, mutations in TP53 and/or genes related to PIK3CA-AKT pathway may be associated with malignant properties of PMCA. Further studies will facilitate the elucidation of molecular mechanism underlying PMP, and contribute to the development of effective therapeutic and preventive strategies to this disease. Citation Format: Rei Noguchi, Hideaki Yano, Yoshimasa Gohda, Ryuichiro Suda, Toru Igari, Yasunori Ohta, Naohide Yamashita, Kiyoshi Yamaguchi, Yumi Terakado, Tsuneo Ikenoue, Yoichi Furukawa. Association between genetic profiles and histological classifications of 18 Japanese pseudomyxoma peritonei. [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 4825. doi:10.1158/1538-7445.AM2015-4825

The impact of concomitant genomic alterations on treatment outcome for trastuzumab therapy in HER2-positive gastric cancer.

Clinical benefit from trastuzumab and other anti-human epidermal growth factor receptor-2 (HER2) therapies in patients with HER2-positive gastric cancer (GC) remains limited by primary or acquired resistance. We aimed to investigate the impact of concomitant molecular alterations to HER2 amplification on the clinical outcome of trastuzumab-treated patients. Using immunohistochemistry (IHC), copy number variations (CNVs), and Ion Ampliseq Cancer Panel, we analyzed the status of concomitant alterations in 50 HER2-positive advanced GC patients treated with trastuzumab in combination with other chemotherapeutic agents. The percentage of tumor samples with at least one concomitant alteration was 40% as assessed by IHC, 16% by CNVs, and 64% by Ampliseq sequencing. Median progression-free survival (PFS) was 8.0 months (95% confidence interval, 4.8–11.3). Patients were divided into two subgroups according to PFS values with a cutoff point of 8 months; results show that concomitant genomic alterations do not correlate with trastuzumab response. However, CNVs of CCNE1 significantly correlated (p < 0.05) with a shorter survival time. Our findings indicate that additional alterations implemented for prediction of clinical benefit from HER2-targeting agents in GC remained unclear. Further studies will be needed to elucidate the role of each specific biomarker and to optimize therapeutic approaches.

Towards Personalized Therapy in Pediatric Acute Lymphoblastic Leukemia; Ras Mutations and Prednisolone Resistance

Background:The 5-year event-free survival of pediatric precursor-B acute lymphoblastic leukemia (BCP-ALL) has currently reached 80-90%. Targeted drugs are warranted to cure the remaining 10-20%. Prednisolone has been shown a pivotal drug and resistance remains an adverse prognostic factor in BCP-ALL. This study aimed to find new druggable targets that may modulate prednisolone resistance.Methods:Leukemic cells from children with newly diagnosed ALL were isolated from bone marrow aspirates, only samples with ≥ 90% leukemic blasts were used. Protein expression of patient leukemic cells before and after exposure to prednisolone was analyzed by reverse phase protein arrays. Deep sequencing of 26 BCP-ALL patients was performed on the Ion Torrent PGM using the Ion AmpliSeq Cancer Panel. Cytotoxicity of primary patients’ cells to Trametinib, Sorafenib, Crizotinib and AS1517499 in combination with or without prednisolone was determined by the MTT assay.Results:We compared phosphorylation levels of 18 key-signaling proteins in leukemic cells between in vitroprednisolone sensitive and resistant ALL patients. Basal phosphorylation levels of these proteins did not differ between prednisolone resistant and sensitive patients’ cells. Exposure to prednisolone for 48h induced discrepant changes in 7 out of 18 proteins between prednisolone resistant and sensitive cases. Exposure to prednisolone decreased phosphorylation of the RAS-RAF-MEK pathway, STAT6 and c-Met in prednisolone sensitive patients cells but not in resistant patients. These findings prompted us to investigate the presence of RAS-pathway and c-Met activating mutations. We investigated hotspot regions of mutations in the protein coding domains of BRAF, NRAS, HRAS, KRAS, PTPN11, FLT3 and c-Met with Ion Torrent deep sequencing (1000x read depth) in pediatric BCP-ALL patients. We observed that 42% of the BCP-ALL patients tested carried activating NRAS and/or KRAS, FLT3 or PTPN11 mutations, of which 6 cases harbored ≥2 RAS-mutations predominantly associated with presence of multiple minor clones (<30%). Interestingly, we detected more often RAS activating mutations in prednisolone resistant compared to sensitive patients (62% vs. 23%). We next evaluated the prednisolone sensitizing properties of inhibitors directed against MEK1/2 (Trametinib), BRAF (Sorafenib), STAT6 (AS1517499) and cMET (Crizotinib) in primary BCP-ALL patients samples. Remarkably, Trametinib was impressively potent in reversing prednisolone resistance eradicating all leukemic cells of the NRAS/KRAS-mutated BCP-ALL patients. In one NRAS/KRAS-mutated case Sorafenib but not Trametinib sensitized to prednisolone significantly.Conclusion:We observed a relative high frequency of RAS-pathway mutations in children with newly diagnosed BCP-ALL (42%). These mutations were more frequently found in prednisolone resistant cases compared to sensitive cases. Highly resistant leukemic patients’ cells harbouring K/N-RAS mutations were sensitized to prednisolone in the presence of the MEK1/2 inhibitor Trametinib or the BRAF inhibitor Sorafenib. Our data suggest that BCP-ALL patients with RAS-activating mutations may benefit from treatment with Trametinib/Sorafenib and strongly argue to implement these inhibitors in combination with prednisone. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1200: Characterization of a novel radiation-induced sarcoma cell line

Abstract Background: Radiation-induced sarcoma (RIS) is a potential complication of cancer treatment. No widely available cell line models currently exist to facilitate studies of RIS. Methods: We derived a primary human cell line, UACC-SARC1, from a RIS. Short tandem repeat (STR) genotyping was used to confirm that this cell line was propagated from the tumor. Further characterization of this cell line involved comparing 24 markers using immunocytochemistry (ICC) to immunohistochemistry (IHC) of the tumor, a Matrigel invasion assay, karyotyping of the cell line, comparative genomic hybridization (CGH), DNA sequencing using the Ion AmpliSeq Cancer panel and in vivo mouse xenografts after subcutaneous injection of UACC-SARC1 in immunodeficient mice. Results: STR profiling of UACC-SARC1 was virtually identical to its parental tumor. IHC analysis of the tumor and ICC analysis of UACC-SARC1 revealed shared expression of vimentin, osteonectin, CD68, Ki67 and PTEN but tumor-restricted expression of the histiocyte markers α1-antitrypsin and α1-antichymotrypsin. Karyotyping of the tumor demonstrated aneuploidy. CGH provided direct genetic comparison between the tumor and UACC-SARC1. Sequencing of 740 mutation hotspots revealed no mutations in UACC-SARC1 nor in the tumor. SCID mice xenografts exhibited tumor formation but resulting tumors failed to metastasize. Further xenografts with NOD SCID gamma (NSG) mice are planned. The doubling time of UACC-SARC1 was 28.3 hours. Conclusion: Our novel RIS strain constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. Citation Format: Julie E. Lang, Brandon Nokes, Grishma Sheth, Petr Novak, Laura Fuchs, George S. Watts, Bernard W. Futscher, Neal Mineyev, Weizhu Zhu, Lauren LeBeau, Ray Nagle, Lee Cranmer. Characterization of a novel radiation-induced sarcoma cell line. [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 1200. doi:10.1158/1538-7445.AM2014-1200

Abstract 4284: Evaluation of cancer-related mutations in tumorgraft models

Abstract Personalized TumorGraft models are generated from tumor fragments resected from patients with refractory advanced cancers that are implanted and propagated in immunodeficient mice. They maintain the genetic characteristics of the original tumor and can potentially be used for individualized therapy selection. We used samples from 120 TumorGrafts to analyze genetic changes (353 mutations and 31 copy number assays) with a customized Qiagen qBiomarker Somatic mutation PCR array. We chose the alterations to be analyzed based on the recent and classic literature data on significant known somatic mutations in cancer, either with biological or therapy response relevance in different cancer types (prioritizing lung and colorectal tumors). Our models consisted of: 39 colorectal cancer samples, 30 pancreatic cancers, 25 non-small cell lung cancers (NSCLC), 11 melanomas, 7 ovarian cancers, 4 breast cancers, and 4 small cell lung cancers. Among our 120 samples, 22% (26) did not show any of the analyzed mutations or deletions, 78% (94) showed at least one mutation, and of those: 56 had 2 or more mutations (44%). In colorectal tumors, the most frequently observed mutation was Kras, followed by p53, and PIK3CA. Among NSCLC: p53, PIK3CA, deletion of p16 and mutation of Kras. In melanoma: Braf and deletion of p16. All 30 cases of pancreatic cancer showed mutations, being Kras the most frequent, followed by p16 deletion and p53 mutation. On the other tumor types, not more than three samples displayed the same mutation. A subgroup of the samples (95) was validated for mutations with other two techniques: conventional Sanger sequencing and/or IonAmpliseq Cancer panel (a panel of key cancer related genes, Life Technologies). 56/69 (81%) cases had the same mutation(s) detected by two or three techniques simultaneously. Three samples were concordantly wild type for the evaluated genes by three or two techniques. Comparison of the genetic alterations with the demographic and clinicopathological data available for these samples (including tumor stage, therapy response, patient's gender and age) is underway to evaluate possible correlations among these parameters and the molecular changes. With the recent massive data produced by next generation sequencing, and with our growing knowledge on the applicability of genetic alterations in the clinic, it is important to have cost-effective, easy straight- forward to perform and to analyze techniques in order to profile the tumors for relevant mutations on the era of personalized medicine. In the present work, we assess a quick and simple, real time PCR based method with selected targets for mutation detection and compare with two other widely used methods. Moreover we aim to profile tumors and uncover their possible genetic targets to best utilize our powerful investigational method of TumorGrafts. Further studies with larger cohorts and alternative mutation detection techniques are needed to validate and expand the present results. Citation Format: Mariana Brait, Luciane T. Kagohara, Evgeny Izumchenko, Samuel Long, Tin Khor, Elizabeth Bruckheimer, David Sidransky. Evaluation of cancer-related mutations in tumorgraft models. [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 4284. doi:10.1158/1538-7445.AM2014-4284

Abstract 1523: Molecular typing of Chinese gastrointestinal stromal tumors using a multigene next generation sequencing panel

Abstract Identifying mutations in individual tumor is critical to improve the efficacy of cancer therapy by matching targeted drugs to specific mutations. Gastrointestinal stromal tumor (GIST) is a stromal or mesenchymal subepithelial neoplasm affecting the gastrointestinal tract and it frequently contains an activating mutation in either the KIT or platelet-derived growth factor A (PDGFRA) gene. Although GIST is highly responsive to several selective tyrosine kinase inhibitors, combined use of inhibitors targeting other mutations is needed to further prolong survival. In this study, we aim to screen and identify genetic mutations in GIST for targeted therapy using the new Ion Torrent next-generation sequencing platform. By utilizing the Ion Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes from DNA extracted from formalin-fixed and paraffin-embedded (FFPE) samples of 125 human gastrointestinal stromal tumors, set up stringent parameters for reliable variant calling by filtering out potential raw base calling errors, and identified frequent mutations in PDGFRA, KIT, APC, MLH1, MET, RET, and PIK3CA. Moreover, frequent missense mutations were detected in MLH1 (12%), MET (16.80%), KIT (33.60%), and PIK3CA (41.60%) genes. The mutations in MLH1 gene were frequently detected in the exons 2, while MET gene was frequently mutated in the exon 12. In addition, we identified missense mutations in other genes, including FLT3, KRAS, PDGFRA, and STK11, at lower frequencies. This study demonstrates the utility of using Ion Torrent sequencing to efficiently identify human cancer mutations. With the finding of the frequent mutations in MLH1 and MET genes, in addition to KIT, PDGFRA and PIK3CA, in GISTs, this study provides a molecular basis for clinically developing new drugs targeting these gene mutations for GIST therapy. Citation Format: Zhi Xu, Zhibin Hu, Xinying Huo, Chuanning Tang, Si-Yi Chen, Jinfei Chen. Molecular typing of Chinese gastrointestinal stromal tumors using a multigene next generation sequencing 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 1523. doi:10.1158/1538-7445.AM2014-1523