KRAS Amplification Research Articles

PTU-128 Novel insights into metastatic spread of oesophageal adenocarcinoma from whole genome sequencing data

Introduction The evolution of metastatic oesophageal adenocarcinoma (OAC) is poorly understood. Whole genome sequencing (WGS) across multiple metastatic sites provides the unique opportunity to study clonal evolution of OAC. Method 2 cases of junctional OAC were extensively sampled at autopsy within 4 h of death. At presentation case 1 was palliative, case 2 had potentially curative disease but progressed during treatment. 20 fresh frozen tumour samples from primary tumour and multiple metastases (adrenal, liver, pancreas, station 102, left gastric and para-aortic lymph nodes) underwent WGS. Clonal analysis involved estimating copy number status and tumour purity, followed by point mutation clustering based on allele frequency (proportion of tumour cells harbouring a mutation); clusters were referred to as clones and were annotated with driver events of clonal progression (single nucleotide variants (SNVs) and copy number events). Results 11 clones spanned all samples in case 1 compared to 5 in case 2. Median SNVs per clone were 5885 in case 1 (range 1091–29883) and 2149 (range 424–27031) in case 2. The founding clone in both cases contained the bulk of the SNVs and key potential drivers of carcinogenesis (case 1:KRAS and GATA4 amplification, case 2:TP53 mutation). Metastases in case 1 had a branching pattern of evolution; 6 clones were unique to individual metastases and only 1 was shared across sites. Clones were on average 9363 (+/-5827) SNVs distant from the founder clone demonstrating continuing evolution in metastases. Para-aortic nodal metastasis preceded local lymph node metastasis. In contrast case 2 demonstrated linear evolution with just 2 metastatic clones that were on average 1286 (+/-1219) SNVs distinct from the primary tumour; 1 was only in the adrenal, the second was shared across multiple sites suggestive of an embolic pattern of tumour seeding. The liver metastasis was identical to the primary founder clone demonstrating that metastasis to the liver was the initial event, even prior to local lymph node metastasis. Conclusion Metastases can result from contrasting patterns of clonal evolution. Both cases demonstrate the striking phenomenon of early distant metastases, prior to the development of local lymph node metastases thus challenging our traditional algorithms of tumour staging and prognostication. Additional cases are being analysed to support our findings. Disclosure of interest None Declared.

Co-treatment of statins and erlotinib to induce growth arrest and apoptosis in primary TKI-resistant non-small cell lung cancer (NSCLC)-cell lines.

e22181 Background: K-Ras mutations and amplification of MET lead to resistance to tyrosine kinase-inhibitor (TKI) treatment in NSCLC cell lines. HMG-CoA reductase inhibitors are widely prescribed a...

Tensor GSVD of patient- and platform-matched tumor and normal DNA copy-number profiles uncovers chromosome arm-wide patterns of tumor-exclusive platform-consistent alterations encoding for cell transformation and predicting ovarian cancer survival.

The number of large-scale high-dimensional datasets recording different aspects of a single disease is growing, accompanied by a need for frameworks that can create one coherent model from multiple tensors of matched columns, e.g., patients and platforms, but independent rows, e.g., probes. We define and prove the mathematical properties of a novel tensor generalized singular value decomposition (GSVD), which can simultaneously find the similarities and dissimilarities, i.e., patterns of varying relative significance, between any two such tensors. We demonstrate the tensor GSVD in comparative modeling of patient- and platform-matched but probe-independent ovarian serous cystadenocarcinoma (OV) tumor, mostly high-grade, and normal DNA copy-number profiles, across each chromosome arm, and combination of two arms, separately. The modeling uncovers previously unrecognized patterns of tumor-exclusive platform-consistent co-occurring copy-number alterations (CNAs). We find, first, and validate that each of the patterns across only 7p and Xq, and the combination of 6p+12p, is correlated with a patient’s prognosis, is independent of the tumor’s stage, the best predictor of OV survival to date, and together with stage makes a better predictor than stage alone. Second, these patterns include most known OV-associated CNAs that map to these chromosome arms, as well as several previously unreported, yet frequent focal CNAs. Third, differential mRNA, microRNA, and protein expression consistently map to the DNA CNAs. A coherent picture emerges for each pattern, suggesting roles for the CNAs in OV pathogenesis and personalized therapy. In 6p+12p, deletion of the p21-encoding CDKN1A and p38-encoding MAPK14 and amplification of RAD51AP1 and KRAS encode for human cell transformation, and are correlated with a cell’s immortality, and a patient’s shorter survival time. In 7p, RPA3 deletion and POLD2 amplification are correlated with DNA stability, and a longer survival. In Xq, PABPC5 deletion and BCAP31 amplification are correlated with a cellular immune response, and a longer survival.

Clinical Acquired Resistance to RAF Inhibitor Combinations in BRAF-Mutant Colorectal Cancer through MAPK Pathway Alterations.

BRAF mutations occur in approximately 10% of colorectal cancers. Although RAF inhibitor monotherapy is highly effective in BRAF-mutant melanoma, response rates in BRAF-mutant colorectal cancer are poor. Recent clinical trials of combined RAF/EGFR or RAF/MEK inhibition have produced improved efficacy, but patients ultimately develop resistance. To identify molecular alterations driving clinical acquired resistance, we performed whole-exome sequencing on paired pretreatment and postprogression tumor biopsies from patients with BRAF-mutant colorectal cancer treated with RAF inhibitor combinations. We identified alterations in MAPK pathway genes in resistant tumors not present in matched pretreatment tumors, including KRAS amplification, BRAF amplification, and a MEK1 mutation. These alterations conferred resistance to RAF/EGFR or RAF/MEK combinations through sustained MAPK pathway activity, but an ERK inhibitor could suppress MAPK activity and overcome resistance. Identification of MAPK pathway reactivating alterations upon clinical acquired resistance underscores the MAPK pathway as a critical target in BRAF-mutant colorectal cancer and suggests therapeutic options to overcome resistance. RAF inhibitor combinations represent promising approaches in clinical development for BRAF-mutant colorectal cancer. Initial characterization of clinical acquired resistance mechanisms to these regimens identified several MAPK pathway alterations driving resistance by reactivating MAPK signaling, highlighting the critical dependence of BRAF-mutant colorectal cancers on MAPK signaling and offering potential strategies to overcome resistance.

Pancreatic undifferentiated rhabdoid carcinoma: KRAS alterations and SMARCB1 expression status define two subtypes

Pancreatic undifferentiated carcinoma is a heterogeneous group of neoplasms, including pleomorphic giant cell, sarcomatoid, round cell, and rhabdoid carcinomas, the molecular profiles of which have so far been insufficiently characterized. We studied 14 undifferentiated carcinomas with prominent rhabdoid cells, occurring as advanced tumors in seven females and seven males aged 44–96 years (mean: 65 years). Histologically, 10 tumors qualified as pleomorphic giant cell and 4 as monomorphic anaplastic carcinomas. A glandular component, either in the primary or in the metastases, was seen in 5 out of 14 tumors (4 out of 10 pleomorphic giant cell and 1 out of 4 monomorphic anaplastic subtypes, respectively). Osteoclast-like giant cells were absent. Immunohistochemistry revealed coexpression of cytokeratin and vimentin, and loss of membranous β-catenin and E-cadherin staining in the majority of cases. Nuclear SMARCB1 (INI1) expression was lost in 4 out of 14 cases (28%), representing all 4 tumors of the monomorphic anaplastic subtype. FISH and mutation testing of KRAS revealed KRAS amplification in 5 out of 13 (38%) and exon 2 mutations in 6 out of 11 (54%) successfully analyzed cases. A strong correlation was found between KRAS alterations (mutation and/or copy number changes) and intact SMARCB1 expression (7 out of 8; 87%). On the other hand, loss of SMARCB1 expression correlated with the absence of KRAS alterations (3 out of 5 cases; 60%). The results suggest that rhabdoid phenotype in pancreatic undifferentiated rhabdoid carcinomas has a heterogeneous genetic background. SMARCB1 loss is restricted to the anaplastic monomorphic subtype and correlates with the absence of KRAS alterations, whereas the pleomorphic giant cell subtype is characterized by KRAS alterations and intact SMARCB1 expression. Recognition and appropriate subtyping of these rare variants might become necessary for future therapeutic strategies.

Limited genomic heterogeneity of circulating melanoma cells in advanced stage patients

Purpose. Circulating melanoma cells (CMCs) constitute a potentially important representation of time-resolved tumor biology in patients. To date, genomic characterization of CMCs has been limited due to the lack of a robust methodology capable of identifying them in a format suitable for downstream characterization. Here, we have developed a methodology to detect intact CMCs that enables phenotypic, morphometric and genomic analysis at the single cell level. Experimental design. Blood samples from 40 metastatic melanoma patients and 10 normal blood donors were prospectively collected. A panel of 7 chondroitin sulfate proteoglycan 4 (CSPG4)-specific monoclonal antibodies (mAbs) was used to immunocytochemically label CMCs. Detection was performed by automated digital fluorescence microscopy and multi-parametric computational analysis. Individual CMCs were captured by micromanipulation for whole genome amplification and copy number variation (CNV) analysis. Results. Based on CSPG4 expression and nuclear size, 1–250 CMCs were detected in 22 (55%) of 40 metastatic melanoma patients (0.5–371.5 CMCs ml−1). Morphometric analysis revealed that CMCs have a broad spectrum of morphologies and sizes but exhibit a relatively homogeneous nuclear size that was on average 1.5-fold larger than that of surrounding PBMCs. CNV analysis of single CMCs identified deletions of CDKN2A and PTEN, and amplification(s) of TERT, BRAF, KRAS and MDM2. Furthermore, novel chromosomal amplifications in chr12, 17 and 19 were also found. Conclusions. Our findings show that CSPG4 expressing CMCs can be found in the majority of advanced melanoma patients. High content analysis of this cell population may contribute to the design of effective personalized therapies in patients with melanoma.

Activation of RAS family members confers resistance to ROS1 targeting drugs.

The ROS1 tyrosine kinase is activated in lung cancer as a consequence of chromosomal rearrangement. Although high response rates and disease control have been observed in lung cancer patients bearing rearranged ROS1 tumors (ROS1+) treated with the kinase inhibitor crizotinib, many of these patients eventually relapse.To identify mechanisms of resistance to ROS1 inhibitors we generated resistant cells from HCC78 lung cancer cells bearing the SLC34A2-ROS1 rearrangement. We found that activation of the RAS pathway in the HCC78 cell model, due to either KRAS/NRAS mutations or to KRAS amplification, rendered the cells resistant to ROS1 inhibition. These cells were cross-resistant to different ROS1 inhibitors, but sensitive to inhibitors of the RAS signaling pathway. Interestingly, we identified focal KRAS amplification in a biopsy of a tumor from a patient that had become resistant to crizotinib treatment.Altogether our data suggest that the activation of members of the RAS family can confer resistance to ROS1 inhibitors. This has important clinical implications as: (i) RAS genetic alterations in ROS1+ primary tumors are likely negative predictors of efficacy for targeted drugs and (ii) this kind of resistance is unlikely to be overcome by the use of more specific or more potent ROS1 targeting drugs.

428 Clinical acquired resistance to combined RAF/EGFR or RAF/MEK inhibition in BRAF mutant colorectal cancer (CRC) patients through MAPK pathway alterations

Background: BRAF mutations occur in ~10% of CRC and confer poor prognosis. While RAF inhibitor monotherapy leads to response rates of 60−80% in BRAF mutant (BRAFm) melanoma, response rates in BRAFm CRC are poor (~5%). Promising recent studies with RAF inhibitor-based combinations, including combined RAF/EGFR or RAF/MEK inhibition, have produced higher response rates in BRAFm CRC, but a better understanding of acquired resistance mechanisms will be critical to improving therapy. Methods: Mechanisms of acquired resistance to RAF/EGFR or RAF/MEK inhibition were evaluated in BRAFm CRC cell lines and in clinical biopsies obtained from BRAFm CRC patients following disease progression after initial response or prolonged stable disease to either therapeutic combination. BRAFm CRC cell lines were cultured in the presence of combined RAF/EGFR or RAF/MEK inhibitors until resistant clones emerged. Candidate resistance mutations were identified through exome sequencing. Matched pre-treament, post-progression, and normal DNA from BRAFm CRC patients treated with either RAF/EGFR or RAF/MEK combinations were analyzed by whole exome sequencing to identify clinical acquired resistance mechanisms. Results: In resistant clones generated from BRAFm CRC cell lines selected with either RAF/EGFR or RAF/MEK inhibitor combinations, KRAS exon 2 mutations were identified. KRAS mutation led to sustained MAPK pathway activity and cross-resistance to either RAF/EGFR or RAF/MEK inhibitor combinations. Interestingly, the triple combination of RAF/EGFR/MEK inhibition was able to suppress MAPK activity and overcome resistance. In a BRAFm CRC patient with prolonged stable disease on a RAF/EGFR inhibitor combination, whole exome sequencing identified the presence of KRAS amplification in a progressing lesion. RNA sequencing of the same lesion confirmed KRAS transcript overexpression, and ~25-fold amplification of KRAS in the progressing lesion and absence of amplification in the pre-treatment biopsy was confirmed by FISH. In one patient with a minor response to a RAF/MEK inhibitor combination, whole exome sequencing identified amplification of the c-mer oncogene (MERTK) receptor tyrosine kinase as the likely acquired resistance mechanism. In another patient with a minor response to a RAF/MEK inhibitor combination, whole exome sequencing identified subclonal ARAF and MEK1 mutations in the post-progression biopsy, suggesting the presence of heterogeneous acquired resistance mechanisms in this progressing lesion. Conclusions: The identification of alterations affecting the MAPK pathway in BRAFm CRC patients who have developed clinical acquired resistance to RAF/EGFR or RAF/MEK inhibitor regimens underscores the importance of the MAPK pathway in this cancer. Understanding the mechanisms of acquired resistance can lead to novel combination strategies to overcome or delay resistance. Friday 21 November 2014

Chemiluminescence resonance energy transfer imaging on magnetic particles for single-nucleotide polymorphism detection based on ligation chain reaction

A novel ligation chain reaction (LCR) methodology for single-nucleotide polymorphism (SNP) detection was developed based on luminol–H2O2–horseradish peroxidase (HRP)-mimicking DNAzyme–fluorescein chemiluminescence resonance energy transfer (CRET) imaging on magnetic particles. For LCR, four unique target-complement probes (X and X⁎, YG and Y⁎) for the amplification of K-ras (G12C) were designed by modifying G-quadruplex sequence at 3′-end of YG and fluorescein at 5′-end of Y⁎. After the LCR, the resulting products of XYG/X⁎Y⁎ with biotin-labeled X⁎ were captured onto streptavidin-coated magnetic particles (SA-MPs) via specific biotin-SA interaction, which stimulated the CRET reaction from hemin/G-quadruplex-catalyzed luminol–H2O2 CL system to fluorescein. By collecting signals by a cooled low-light CCD, a CRET imaging method was proposed for visual detection and quantitative analysis of SNP. As low as 0.86fM mutant DNA was detected by this assay, and positive mutation detection was achieved with a wild-type to mutant ratio of 10,000:1. This high sensitivity and specificity could be attributed to not only the exponential amplification and excellent discrimination of LCR but also the employment of SA-MPs. SA-MPs ensured the feasibility of the proposed strategy, which also simplified the operations through magnetic separation and separated the reaction and detection procedures to improve sensitivity. The proposed LCR-CRET imaging strategy extends the application of signal amplification techniques to SNP detection, providing a promising platform for effective and high-throughput genetic diagnosis.

Abstract 1228: Clinical relevance of FGFR2 amplification in diffuse gastric cancer

Abstract Diffuse gastric cancer (DGC) is a poorly differentiated adenocarcinoma characterized by infiltration of signet ring cells into the stomach wall and a lack of tumor mass formation. DGC has rapid disease progression with a dismally poor prognosis and is resistant to chemo- and radiotherapy. There is no available targeted therapy, and preclinical models that reliably predict clinical activity of novel compounds are also lacking. In addition, molecular analyses of DGC have been hampered by difficulty in obtaining primary tumors of sufficient tumor cellularity without contaminating normal fibroblasts. To overcome the scarcity of cancer cells and the abundance of surrounding stromal tissue, we isolated cancer cells from malignant ascites of DGC patients. Red blood cells were removed by Ficoll separation and CD45+ hematopoietic cells were depleted using antibody-conjugated magnetic beads. Exome sequencing was performed on cancer cells and peripheral blood cells from the same patient to identify somatic alterations in DGC. We also generated patient-derived xenografts (PDX) using isolated cancer cells to establish preclinical cancer models for DGC. Exome sequencing revealed FGFR2 and KRAS amplifications as well as p53 mutations. Cancer cells isolated from malignant ascites formed subcutaneous tumors that recapitulated the histology of primary DGC tumors with characteristic signet ring cells and intracellular mucin. FGFR2 amplification in PDX tumors was confirmed by fluorescence in situ hybridization (FISH), with correspondingly high levels of FGFR2 transcripts and protein expression. We performed drug treatments on PDX tumors using FGFR2 inhibitor AZD4547 and found that FGFR2 amplification status alone was sufficient to predict the sensitivity of PDX tumors to AZD4547. Tumor volume of FGFR2-amplified tumors decreased or remained the same following AZD4547 treatment compared to vehicle-treated tumors which showed increased tumor volume. In contrast, AZD4547 treatment had little or no effect on tumor growth of non-FGFR2 amplified tumors. Furthermore, these PDX tumors regardless of FGFR2 amplification status responded poorly to cisplatin, a standard drug currently used for treatment of DGC. In conclusion, we report a high prevalence of FGFR2 gene amplification in DGC and the establishment of reliable PDX models that can predict tumor response to targeted therapy. Citation Format: Wen Min Lau, Zhijiang Zang, Eileen Teng, Kakoli Das, Wei Peng Yong, Ming Teh, Tania Chia, Jin Wei Tan, Amy Tay, Asim Shabbir, Koji Kono, Jimmy So, Patrick Tan, Shing Leng Chan. Clinical relevance of FGFR2 amplification in diffuse gastric 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 1228. doi:10.1158/1538-7445.AM2014-1228

Abstract 3711: Erk inhibition as a therapeutic option for the treatment of Raf- and Mek- inhibitor resistant tumors

Abstract The MAPK pathway represents a prime target for therapeutic intervention in cancer. Recent approvals of B-Raf and Mek inhibitors lead to exciting antitumor activities and survival benefits. However the observation of resistance to such Raf and Mek inhibitors has been documented in both clinical and preclinical trials. These phenomena stimulated further research into the regulation of the MAPK pathway and led to the suggestion to target the downstream kinase Erk, as well as to combine its targeting with Raf and Mek inhibitors. Here we report that Erk inhibitors result in efficient growth inhibition of tumor cells that are resistant to Raf and Mek inhibitors. AEZS-134 - a highly potent and selective ATP competitive Erk inhibitor - overcomes the Raf inhibitor induced paradoxical cell activation and the aquired resistance to Mek inhibitors in tumor cells. We demonstrate potent antiproliferative activity of AEZS-134 in B-Raf wildtype, B-RafV600E mutant, Ras wildtype and K-Ras mutant tumor cell lines in comparison to common Raf inhibitors. Furthermore we show that AEZS-134 is efficacious in Mek inhibitor resistant Hct116 and MDA MB231 cells which have been well characterized in terms of Mek F129L allosteric binding pocket mutation, varying degrees of K-Ras amplification, cellular proliferation assays and MAPK pathway phosphorylation studies. Our data provide a rational for new therapeutic opportunities by using Erk inhibitors in oncology. In order to maximize therapeutic benefit for patients and considering that Erk is directly downstream of B-Raf and Mek, targeting Erk may be more effective than targeting Raf or Mek in a variety of MAPK resistance settings. AEZS-134 and related compounds provide potent and selective tools to help address this question. Development of the Erk inhibitors by Æterna Zentaris is integral part of our inhouse kinase research program comprising the investigation of different compounds for single Erk inhibition, single PI3K inhibition and dual kinase inhibitions of Erk and PI3K. All compounds are exclusively synthesized by our medicinal chemistry department. Citation Format: Irene Seipelt, Peter Schmidt, Helene Maerzhaeuser, Matthias Gerlach, Kai Jung, Tilmann Schuster, Michael Teifel. Erk inhibition as a therapeutic option for the treatment of Raf- and Mek- inhibitor resistant tumors. [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 3711. doi:10.1158/1538-7445.AM2014-3711

Mutually exclusive FGFR2, HER2, and KRAS gene amplifications in gastric cancer revealed by multicolour FISH

Gastric cancer (GC) is a major cause of global cancer mortality. Previous genomic studies have reported that several RTK-RAS pathway components are amplified in GC, with individual tumours often amplifying one component and not others (“mutual exclusivity”). Here, we sought to validate these findings for three RTK/RAS components (FGFR2, HER2, KRAS) using fluorescence in situ hybridisation (FISH) on a series of gastric tumours, cell lines and patient-derived xenografts. Applying dual-colour FISH on 137 gastric tumours (89 FFPE surgical resections and 48 diagnostic biopsies), we observed FGFR2 amplification in 7.3% and HER2 amplification in 2.2% of GCs. GCs exhibiting FGFR2 amplification were associated with high tumour grade (p = 0.034). In FISH positive tumours, striking differences in copy number levels between cancer cells in the same tumour were observed, suggesting intra-tumour heterogeneity. Using a multicolour FISH assay allowing simultaneous detection of FGFR2, HER2, and KRAS amplifications, we confirmed that these components exhibited a mutually exclusive pattern of gene amplification across patients. The FISH data were also strongly correlated with Q-PCR levels and at the protein level by immunohistochemistry. Our data confirm that RTK/RAS components are mutually exclusively amplified in GC, and demonstrate the feasibility of identifying multiple aneuploidies using a single FISH assay. Application of this assay to GC samples, particularly diagnostic biopsies, may facilitate enrollment of GC patients into clinical trials evaluating RTK/RAS directed therapies. However, the presence of intra-tumour heterogeneity may require multiple biopsy samples to be obtained per patient before a definitive diagnosis can be attained.

Whole genome gene copy number profiling of gastric cancer identifies PAK1 and KRAS gene amplification as therapy targets.

Gastric cancer is the second leading cause of death from cancer worldwide, with an approximately 20% 5-year survival rate. To identify molecular subtypes associated with the clinical prognosis, in addition to genetic aberrations for potential targeted therapeutics, we conducted a comprehensive whole-genome analysis of 131 Chinese gastric cancer tissue specimens using whole-genome array comparative genomic hybridization. The analyses revealed gene focal amplifications, including CTSB, PRKCI, PAK1, STARD13, KRAS, and ABCC4, in addition to ERBB2, FGFR2, and MET. The growth of PAK1-amplified gastric cancer cells in vitro and in vivo was inhibited when the corresponding mRNA was knocked down. Furthermore, both KRAS amplification and KRAS mutation were identified in the gastric cancer specimens. KRAS amplification was associated with worse clinical outcomes, and the KRAS gene mutation predicted sensitivity to the MEK1/2 inhibitor AZD6244 in gastric cancer cell lines. In summary, amplified PAK1, as well as KRAS amplification/mutation, may represent unique opportunities for developing targeted therapeutics for the treatment of gastric cancer.

KRAS Amplification in a 73-Year-Old Man with Adenocarcinoma of Lung

KRAS Amplification in a 73-Year-Old Man with Adenocarcinoma of Lung

Identification of druggable cancer driver genes amplified across TCGA datasets.

The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 14 cancer subtypes and identified 461 genes that were amplified in two or more datasets. The list was narrowed to 73 cancer-associated genes with potential “druggable” properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 40 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 40 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapter GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts.

Coamplification of receptor tyrosine kinases and downstream targets in Japanese gastric cancers.

41 Background: Amplifications of receptor tyrosine kinases genes (RTKs), EGFR, ERBB2, FGFR2, MET, have been associated with the pathogenesis and progression of gastric cancer (GC). Recent studies have found that coamplifications of RTKs are rare in GC. Two phase III trials using RTK targeted therapy have failed to achieve survival benefit in GC patients. Co-amplification of RTKs and downstream targets genes (DSTs), PIK3CA, KRAS, MYC, CCNE1 may be related to resistance to RTK targeted therapy. We tested the hypothesis whether RTKs and DSTs genes are coamplified in GC. Methods: DNA and RNA were extracted from 221 GC from the Kanagawa Cancer Center Hospital (Yokohama, Japan). Copy number of EGFR, ERBB2, FGFR2, PIK3CA, KRAS, MYC, CCNE1 was investigated by a newly developed multiplex ligation dependent probe amplification (MLPA) assay. RNA expression of the same genes was measured using the NanoString platform and compared to DNA copy number status. The frequency of RTK and DST co-amplifications was established and related to KRAS mutation status. Results: RTK amplification was associated with high levels of RNA expression except MYC (all p<0.05). 68% of GC had no RTK amplification. 9% of GC had RTK amplification and no DST amplification. 10% of GC had RTKs co-amplified. 23% of GC had RTK and DST co-amplified. There was no overlap between KRAS mutations and amplifications of KRAS, PIK3CA, FGFR2 or MET. Conclusions: This is the first study reporting that RTK and DST co-amplification are more frequent than RTK co-amplification in GC. These results indicate that GC patients considered for RTK targeting therapy might require DNA copy number assessment of RTKs as well as key DSTs and KRAS mutation testing in order to identify those patients who benefit most from treatment.

KRAS mutant allele-specific imbalance is associated with worse prognosis in pancreatic cancer and progression to undifferentiated carcinoma of the pancreas

KRAS codon 12 mutations are present in about 90% of ductal adenocarcinomas and in undifferentiated carcinomas of the pancreas. The role of KRAS copy number changes and resulting KRAS mutant allele-specific imbalance (MASI) in ductal adenocarcinoma (n=94), and its progression into undifferentiated carcinoma of the pancreas (n=25) was studied by direct sequencing and KRAS fluorescence in situ hybridization (FISH). Semi-quantitative evaluation of sequencing electropherograms showed KRAS MASI (ie, mutant allele peak higher than or equal to the wild-type allele peak) in 22 (18.4%) cases. KRAS FISH (performed on 45 cases) revealed a trend for more frequent KRAS amplification among cases with KRAS MASI (7/20, 35% vs 3/25, 12%, P=0.08). KRAS amplification by FISH was seen only in undifferentiated carcinomas (10/24, 42% vs 0/21 pancreatic ductal adenocarcinoma, 0%, P=0.0007). In 6 of 11 cases with both undifferentiated and well-differentiated components, transition to undifferentiated carcinoma was associated with an increase in KRAS copy number, due to amplification and/or chromosome 12 hyperploidy. Pancreatic carcinomas with KRAS MASI (compared to those without MASI) were predominantly undifferentiated (16/22, 73% vs 9/97, 9%, P<0.001), more likely to present at clinical stage IV (5/22, 23% vs 7/97, 7%, P=0.009), and were associated with shorter overall survival (9 months, 95% confidence interval, 5–13, vs 22 months, 95% confidence interval, 17–27; P=0.015) and shorter disease-free survival (5 months, 95% confidence interval, 2–8 vs 13 months, 95% confidence interval, 10–16; P=0.02). Our findings suggest that in a subset of ductal adenocarcinomas, KRAS MASI correlates with the progression to undifferentiated carcinoma of the pancreas.

Colorectal carcinomas, KRAS p.G13D mutant allele–specific imbalance, and anti–epidermal growth factor receptor therapy

Woo et al have comprehensively reviewed controversies surrounding anti–epidermal growth factor receptor (anti-EGFR) treatment in metastatic colon cancer, specifically the unresolved issue of anti-EGFR therapy in patients with colorectal carcinomas (CRC) harboring KRAS Gly13Asp (G13D) mutation.1 Even before the report by De Roock et al on the potential benefit of cetuximab in CRC with KRAS p.G13D,2 there were contradicting data on the prognostic significance of this mutation (reviewed in Hartman et al3). The apparently discrepant clinical and cell line data may be reconciled by taking into account the phenomenon of mutant allele–specific imbalance (MASI). Introduced by Soh et al, the term “MASI” summarizes a variety of mechanisms that may lead to the predominance of the mutated allele over the wild-type allele.4 Such an allelic imbalance favoring the mutant allele may result from the deletion of the wild-type allele, and/or copy number gain of the mutant allele. It was shown that KRAS MASI is associated with increased ras guanine triphosphatase activity in cell lines and worse outcome in KRAS-mutated CRC. In a study of 394 patients with KRAS-mutated CRC, KRAS MASI developed by chromosome 12 hyperploidy or KRAS amplification, most frequently in cases with KRAS codon 13 mutations. In a multivariate analysis, KRAS codon 13 MASI represented an adverse prognostic factor independent of clinical stage and tumor site.3 We would like to suggest for future reports on anti-EGFR therapy in KRAS p.G13D-mutated CRC to explicitly control for KRAS MASI. Two scenarios are likely. First, the presence or absence of KRAS MASI may correlate with response to anti-EGFR treatment. Alternatively, anti-EGFR treatment may have no therapeutic effect and the outcome differences attributed to anti-EGFR therapy in some studies may merely reflect the unaccounted effect of KRAS MASI. Practically, KRAS MASI can be estimated from a semiquantitative review of routine sequencing electropherograms or by more sophisticated methods aimed at detecting KRAS allelic imbalances, chromosome 12 hyperploidy, or KRAS amplification. Douglas J. Hartman, MD Simion I. Chiosea, MD Department of Pathology University of Pittsburgh Medical Center Pittsburgh, Pennsylvania

KRAS and MAPK1 Gene Amplification in Type II Ovarian Carcinomas

In this study, we examined the clinical significance of KRAS and MAPK1 amplification and assessed whether these amplified genes were potential therapeutic targets in type II ovarian carcinoma. Using fluorescence in situ hybridization, immunohistochemistry, and retrospectively collected clinical data, KRAS and MAPK1 amplifications were identified in 9 (13.2%) and 5 (7.4%) of 68 type II ovarian carcinoma tissue samples, respectively. Interestingly, co-amplification of KRAS and MAPK1 seemed to be absent in the type II ovarian carcinomas tested, except one case. Active phospho-ERK1/2 was identified in 26 (38.2%) out of 68 type II ovarian carcinomas and did not correlate with KRAS or MAPK1 amplification. There was no significant relationship between KRAS amplification and overall or progression-free survival in patients with type II ovarian carcinoma. However, patients with MAPK1 amplification had significantly poorer progression-free survival than patients without MAPK1 amplification. Moreover, type II ovarian carcinoma cells with concomitant KRAS amplification and mutation exhibited dramatic growth reduction following treatment with the MEK inhibitor PD0325901. These findings indicate that KRAS/MAPK1 amplification is critical for the growth of a subset of type II ovarian carcinomas. Additionally, RAS/RAF/MEK/ERK pathway-targeted therapy may benefit selected patients with type II ovarian carcinoma harboring KRAS/MAPK1 amplifications.

Abstract 58: Differences in genomic alterations revealed by sequencing of 182 genes in recurrent ovarian cancer specimens compared to TCGA analysis: rearrangements in PTCH1 and FLT3; high frequency of RAS pathway alteration.

Abstract Recent comprehensive genomic analyses of advanced stage, high grade serous ovarian carcinomas carried out by TCGA (Nature 274: 609-615, 2011) have revealed their heterogeneous nature and provide an explanation for the failure of molecularly targeted drugs evaluated without consideration of the tumor molecular characteristics of the patients treated. Copy number (CN) alterations or mutations in RB, RAS-MAPK, and PI3K-AKT pathway genes occur in ∼35%, 20%, and 35% of the tumors, respectively. Activation of these pathways may confer sensitivity to drugs that target them (e.g., PI3K or AKT inhibitors, MEK inhibitors, CDK inhibitors) as well as resistance to drugs (e.g., HER2 or EGFR inhibitors) that have failed in ovarian cancer clinical trials. A comprehensive evaluation of the genetic aberrations in targetable pathways can therefore inform the selection of appropriate drugs and combinations for each patient in the context of clinical trials. As part of molecular profiling analyses performed for recurrent ovarian cancer patients to inform selection of their next treatment, the presence of mutations or alterations (e.g., CN changes) in 182 genes that encode proteins that are key mediators in oncogenic and tumor suppressive pathways were determined using a validated exon-capture sequencing platform provided in a CLIA-certified laboratory setting. 20 serous (4 primary/16 recurrent) and 4 recurrent mixed or pure clear cell ovarian tumor samples were analyzed. A median of 2.5 (range 0-7) alterations were detected in the serous histology specimens; frequencies of p53 mutations (16; 80%) and amplifications in CCND1 (1; 5%), CCND2 (1; 5%), CCNE1 (2; 10%) are consistent with those observed in the TCGA tumors. In contrast, more frequent RAS pathway aberrations (45% vs. 20%) corresponding to KRAS amplification (3;15%) and mutations/truncations in NF1 (5; 20%) or NF2 (1; 5%) were observed, possibly reflecting the recurrent nature of these samples (TCGA used primary tumors). Only one PIK3CA amplification was detected (i.e., 5%) in contrast to the 18% observed in the TCGA dataset. Amplifications of IGF1R (3; 15%) and ERBB2 (1;5%) were also detected in the serous histology tumors, while PIK3CA and KRAS mutations were only detected in the tumors with mixed cc and endometrioid histologies. Interestingly, a predicted protein-truncating rearrangement in PTCH1 and a kinase-activating exon duplication in FLT3 were detected in tumors from single patients suggesting drugs targeting the HH pathway and FLT3 as options for ovarian cancer patients. While it is important to increase the number of samples analyzed, these data suggest that selection of patients for clinical trials should be informed by comprehensive molecular characterization of recurrent tumor specimens. Citation Format: Deborah A. Zajchowski, Doron Lipson, Gary Palmer, Laura K. Shawver. Differences in genomic alterations revealed by sequencing of 182 genes in recurrent ovarian cancer specimens compared to TCGA analysis: rearrangements in PTCH1 and FLT3; high frequency of RAS pathway alteration. [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 58. doi:10.1158/1538-7445.AM2013-58