KRAS Gene Amplification Research Articles

Abstract NG12: Altered calcium signaling in cancer: PTHrP-KRAS collateral amplification governs pancreatic cancer metastasis, cachexia, and immunosuppression

Abstract Purpose: Metastasis is the leading cause of cancer-related death in pancreatic ductal adenocarcinoma (PDAC), yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Whole-genome sequencing has established that the squamous/quasi-mesenchymal/basal-like PDAC subtype, which is characterized by its high metastatic proclivity, is annotated by KRAS gene amplification. Here, we report that the squamous lineage gene parathyroid hormone-related protein (PTHrP encoded by the PTHLH gene) is located directly adjacent to KRAS and is co-amplified as part of the KRAS amplicon in metastatic PDAC patients. We hypothesize that this collateral amplification of PTHrP, and downstream activation of calcium signaling, exerts its own oncogenic and pro-metastatic phenotypes beyond KRAS and set out to determine if this will confer a novel therapeutic vulnerability. Finally, we postulate that PTHrP-mediated Calcium signaling may be responsible for driving other non-cell autonomous PDAC-associated phenotypes, including cancer cachexia and the organization of an immunosuppressive microenvironment. Methods: We generated a novel genetically engineered mouse model whereby we deleted the cytokine PTHrP in the autochthonous KPCY model of pancreatic cancer (mutant Kras and mutant p53 activated in the pancreatic epithelium). To functionally demonstrate the pro-metastatic and cachexia-inducing roles of PTHrP, we further employed genetic deletion and pharmacological inhibition in orthotopic injection and mouse hospital pre-clinical trials. Results: In silico analysis established that PTHLH (the gene encoding the PTHrP protein) is co-amplified along with KRAS in TCGA and correlates with significantly decreased overall survival. We generated KPCY-PthlhCKO mice and showed that they have significantly reduced primary and metastatic tumor burden and dramatically increased overall survival relative to KPCY controls. In parallel experiments, we treated mice with an anti-PTHrP neutralizing monoclonal antibody, which similarly reduced primary and metastatic tumor growth. Mechanistic studies revealed that intracellular calcium was responsible for turning on an epithelial-to-mesenchymal (EMT) program that could be impeded by inhibiting PTHrP, thus blocking tumor cells from entering the metastatic cascade. Additionally, both genetic deletion and pharmacological inhibition of PTHrP in vivo led to a profound reduction in cachexia-related adipose tissue wasting and muscle atrophy. Re-introduction of PTHrP into a PDAC cell line with low cachexia-inducing potential (and low baseline PTHrP) dramatically increased the degree of cachexia and metastasis observed upon orthotopic implantation. Thus, PTHrP is both necessary and sufficient to induce cachexia and metastasis. Additionally, mice implanted with PTHrP overexpressing tumor cells had a dramatic reduction in overall survival. Finally, we demonstrated that PTHrP is responsible for organizing a pro-tumor immunosuppressive microenvironment by recruiting Myeloid Derived Suppressive Cells (MDSCs). Blocking PTHrP signaling reduces the degree of local immune suppression allowing for the enhanced recruitment of CD8+ T cells. Thus, PTHrP is a pleiotropic molecule that governs many pancreatic cancer-associated disease phenotypes. Conclusions: This work has demonstrated the importance of the previously unappreciated roles of PTHrP signaling in driving diverse pancreatic cancer phenotypes from metastasis to cachexia to immunosuppression, and future studies will look to translate anti-PTHrP therapy into clinical trials. Citation Format: Jason Robert Pitarresi. Altered calcium signaling in cancer: PTHrP-KRAS collateral amplification governs pancreatic cancer metastasis, cachexia, and immunosuppression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr NG12.

Molecular and genetic features of colorectal carcinoma: pathomorphological demonstration of clinical cases and literature review

Background. Colorectal carcinoma (CRC) is one of the most widespread malignancies worldwide; its morbidity rate is on the third place amidst the most common cancers. According to present data, such molecular markers as CyD1, HIF1α, LC3B, p21, p53 as well as the detection of KRAS gene amplification could be used for evaluation of tumor grade, its invasiveness, risk of metastases, sensitivity to anti-EGFR treatment, and further prognosis for patient’s survive. Objective. The purpose of this study was to make a literature review of the CRC molecular diagnostic approaches and demonstrate 5 cases of colorectal carcinoma in patients of Dnipro-city region in purpose to reveal their molecular-genetic features. Methods. Five formalin fixed paraffin embedded specimens of colorectal carcinoma from patients of Dnipro-city region were evaluated pathomorphologically with histological, immunohistochemical and fluorescence in situ hybridization methods. Results. Case study revealed increased level of p53, p21, and LC3B expression, minor elevation of CyD1 and HIF1α expression in demonstrated samples. Amplification of KRAS gene was not found. Conclusion. Our data analysis had revealed multiple studies dedicated to CRC molecular diagnostics with controversial results, what could be explained by both variable methodological approaches and epidemiological peculiarities of CRC in different sites of the globe. Therefore, there is a necessity in empirical identification of the molecular-genetic features of colorectal carcinomas in patients of our region that could improve current state of the art in CRC diagnostic and treatment approaches. Achieved data are the result of the trial and our research of this issue is ongoing.

Abstract PR-010: Collateral amplification of the KRAS linked gene PTHLH governs pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability

Abstract Purpose: Metastasis is the leading cause of cancer-related death in PDAC, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Whole-genome sequencing has established that the squamous/quasi-mesenchymal/basal-like PDAC subtype, which is characterized by its high metastatic proclivity, is annotated by KRAS gene amplification. Here, we report that the squamous lineage gene parathyroid hormone-related protein (PTHrP encoded by PTHLH) is located directly adjacent to KRAS and is co-amplified in metastatic PDAC patients. We hypothesize that this collateral amplification of PTHrP may exert its own oncogenic and pro-metastatic phenotype beyond KRAS and set out to determine if this will confer a novel therapeutic vulnerability. Methods: We generated a novel genetically engineered mouse model whereby we deleted the cytokine Pthlh in the autochthonous KPCY model. To functionally demonstrate the oncogenic and pro-metastatic roles of PTHrP, we further employed genetic deletion and pharmacological inhibition in orthotopic injection, tail vein metastasis assays, mouse hospital pre-clinical trials, and patient-derived 3D organoid models. Results: In silico analysis established that PTHLH is co-amplified along with KRAS in TCGA, is specifically enriched in metastatic patients from the COMPASS trial, and correlates with significantly decreased overall survival in both cohorts. Further examination revealed that PTHLH is a squamous/quasi-mesenchymal/basal-like lineage marker. We generated KPCY-PthlhCKO mice and showed that they have significantly reduced primary and metastatic tumor burden and dramatically increased overall survival relative to KPCY controls. In parallel experiments, we treated mice with an anti-PTHrP neutralizing monoclonal antibody, which similarly reduced primary and metastatic tumor growth. Finally, RNA-seq revealed a downstream mechanism whereby PTHrP is important for metastatic competency through induction of EMT, thus facilitating entry into the metastatic cascade. Loss of PTHrP reduced the ability of tumor cells to undergo EMT both in vivo and in vitro, resulting in a nearly complete elimination of disseminating cells in KPCY-PthlhCKO mice. Thus, KPCY-PthlhCKO tumors are locked in a well-differentiated epithelial state and are unable to initiate the metastatic process. Conclusions: This work has demonstrated the importance of the previously unappreciated role for PTHrP signaling in pancreatic cancer cell plasticity and metastasis, and future studies will look to translate anti-PTHrP therapy into clinical trials. In a broader sense, we establish a new paradigm of collateral amplification, where an assumed passenger gene (PTHLH) is co-amplified along with a known oncogene (KRAS) and endows the evolving tumor with its own oncogenic and pro-metastatic phenotype. Citation Format: Jason R. Pitarresi, Robert J. Norgard, Anna M. Chiarella, Kensuke Suzuki, Richard Kremer, Ben Z. Stanger, Anil K. Rustgi. Collateral amplification of the KRAS linked gene PTHLH governs pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-010.

Abstract 2861: Collateral amplification KRAS-PTHrP drives pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability

Abstract Purpose: Metastasis is the leading cause of cancer-related death in PDAC, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Whole-genome sequencing has established that the squamous/quasi-mesenchymal/basal-like PDAC subtype, which is characterized by its high metastatic proclivity, is annotated by KRAS gene amplification. Here, we report that the squamous lineage gene parathyroid hormone-related protein (PTHrP encoded by PTHLH) is located directly adjacent to KRAS and is co-amplified in metastatic patients. We hypothesize that this collateral amplification of PTHrP may exert its own oncogenic and pro-metastatic phenotype beyond KRAS and set out to determine if this will confer a novel therapeutic vulnerability. Methods: We generated a novel genetically engineered mouse model whereby we deleted the cytokine Pthlh in the autochthonous KPCY model. To functionally demonstrate the oncogenic and pro-metastatic roles of PTHrP, we further employed genetic deletion and pharmacological inhibition in orthotopic injection, tail vein metastasis assays, mouse hospital pre-clinical trials, and patient-derived 3D organoid models. Results: In silico analysis established that PTHLH is co-amplified along with KRAS in TCGA, is specifically enriched in metastatic patients from the COMPASS trial and correlates with significantly decreased overall survival in both cohorts. Further examination revealed that PTHLH is a squamous/quasi-mesenchymal/basal-like lineage marker. We generated KPCY-PthlhCKO mice and showed that they have significantly reduced primary and metastatic tumor burden and dramatically increased overall survival relative to KPCY controls. In parallel experiments, we treated mice with an anti-PTHrP neutralizing monoclonal antibody, which similarly reduced primary and metastatic tumor growth. Finally, RNA-seq revealed a downstream mechanism whereby PTHrP is important for metastatic competency through induction of EMT, thus facilitating entry into the metastatic cascade. Loss of PTHrP reduced the ability of tumor cells to undergo EMT both in vivo and in vitro, resulting in a nearly complete elimination of disseminating cells in KPCY-PthlhCKO mice. Thus, KPCY-PthlhCKO tumors are locked in a well-differentiated epithelial state and are unable to initiate the metastatic process. Conclusions: This work has demonstrated the importance of the previously unappreciated role for PTHrP signaling in pancreatic cancer cell plasticity and metastasis, and future studies will look to translate anti-PTHrP therapy into clinical trials. In a broader sense, we establish a new paradigm of collateral amplification, where an assumed passenger gene (PTHLH) is co-amplified along with a known oncogene (KRAS) and endows the evolving tumor with its own oncogenic and pro-metastatic phenotype. Citation Format: Jason Robert Pitarresi, Robert J. Norgard, Anna M. Chiarella, Richard Kremer, Ben Z. Stanger, Anil K. Rustgi. Collateral amplification KRAS-PTHrP drives pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2861.

KRAS mutation effects on the 2-[18F]FDG PET uptake of colorectal adenocarcinoma metastases in the liver

BackgroundDeriving individual tumor genomic characteristics from patient imaging analysis is desirable. We explore the predictive value of 2-[18F]FDG uptake with regard to the KRAS mutational status of colorectal adenocarcinoma liver metastases (CLM).Methods2-[18F]FDG PET/CT images, surgical pathology and molecular diagnostic reports of 37 patients who underwent PET/CT-guided biopsy of CLM were reviewed under an IRB-approved retrospective research protocol. Sixty CLM in 39 interventional PET scans of the 37 patients were segmented using two different auto-segmentation tools implemented in different commercially available software packages. PET standard uptake values (SUV) were corrected for: (1) partial volume effect (PVE) using cold wall-corrected contrast recovery coefficients derived from phantom spheres with variable diameter and (2) variability of arterial tracer supply and variability of uptake time after injection until start of PET scan derived from the tumor-to-blood standard uptake ratio (SUR) approach. The correlations between the KRAS mutational status and the mean, peak and maximum SUV were investigated using Student’s t test, Wilcoxon rank sum test with continuity correction, logistic regression and receiver operation characteristic (ROC) analysis.These correlation analyses were also performed for the ratios of the mean, peak and maximum tumor uptake to the mean blood activity concentration at the time of scan: SURMEAN, SURPEAK and SURMAX, respectively.ResultsFifteen patients harbored KRAS missense mutations (KRAS+), while another 3 harbored KRAS gene amplification. For 31 lesions, the mutational status was derived from the PET/CT-guided biopsy. The Student’s t test p values for separating KRAS mutant cases decreased after applying PVE correction to all uptake metrics of each lesion and when applying correction for uptake time variability to the SUR metrics. The observed correlations were strongest when both corrections were applied to SURMAX and when the patients harboring gene amplification were grouped with the wild type: p ≤ 0.001; ROC area under the curve = 0.77 and 0.75 for the two different segmentations, respectively, with a mean specificity of 0.69 and sensitivity of 0.85.ConclusionThe correlations observed after applying the described corrections show potential for assigning probabilities for the KRAS missense mutation status in CLM using 2-[18F]FDG PET images.

Abstract PO-057: Collateral amplification of the PTHrP gene drives pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability

Abstract Purpose: Metastasis is the leading cause of cancer-related death in PDAC, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Whole-genome sequencing has established that the squamous/quasi-mesenchymal/basal-like PDAC subtype, which is characterized by its high metastatic proclivity, is annotated by KRAS gene amplification. Here, we report that the squamous lineage gene parathyroid hormone-related protein (PTHrP encoded by PTHLH) is located directly adjacent to KRAS and is co-amplified in metastatic PDAC patients. We hypothesize that this collateral amplification of PTHrP may exert its own oncogenic and pro-metastatic phenotype beyond KRAS and set out to determine if this will confer a novel therapeutic vulnerability. Methods: We generated a novel genetically engineered mouse model whereby we deleted the cytokine Pthlh in the autochthonous KPCY model. To functionally demonstrate the oncogenic and pro-metastatic roles of PTHrP, we further employed genetic deletion and pharmacological inhibition in orthotopic injection, tail vein metastasis assays, mouse hospital pre-clinical trials, and patient-derived 3D organoid models. Results: In silico analysis established that PTHLH is co-amplified along with KRAS in TCGA, is specifically enriched in metastatic patients from the COMPASS trial and correlates with significantly decreased overall survival in both cohorts. Further examination revealed that PTHLH is a squamous/quasi-mesenchymal/basal-like lineage marker. We generated KPCY-PthlhCKO mice and showed that they have significantly reduced primary and metastatic tumor burden and dramatically increased overall survival relative to KPCY controls. In parallel experiments, we treated mice with an anti-PTHrP neutralizing monoclonal antibody, which similarly reduced primary and metastatic tumor growth. Finally, RNA-seq revealed a downstream mechanism whereby PTHrP is important for metastatic competency through induction of EMT, thus facilitating entry into the metastatic cascade. Loss of PTHrP reduced the ability of tumor cells to undergo EMT, resulting in a nearly complete elimination of disseminating cells in KPCY-PthlhCKO mice. Thus, KPCY-PthlhCKO tumors are locked in a well-differentiated epithelial state and are unable to initiate the metastatic process. Conclusions: This work has demonstrated the importance of the previously unappreciated role for PTHrP signaling in pancreatic cancer cell plasticity and metastasis, and future studies will look to translate anti-PTHrP therapy into clinical trials. In a broader sense, we establish a new paradigm of collateral amplification, where an assumed passenger gene (PTHLH) is co-amplified along with a known oncogene (KRAS) and endows the evolving tumor with its own oncogenic and pro-metastatic phenotype. Citation Format: Jason R. Pitarresi, Robert J. Norgard, Anna M. Chiarella, Richard Kremer, Ben Z. Stanger, Anil K. Rustgi. Collateral amplification of the PTHrP gene drives pancreatic cancer growth and metastasis and reveals a new therapeutic vulnerability [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-057.

PIK3CA and KRAS Amplification in Esophageal Adenocarcinoma and their Impact on the Inflammatory Tumor Microenvironment and Prognosis

Gene amplifications of PIK3CA or KRAS induce a downstream activation of the AKT-mTOR or RAF-ERK-pathways. Interactions of the active AKT pathway have been implicated in the inflammatory tumor microenvironment. Nothing is known about these interactions or prognostic power in esophageal adenocarcinoma (EAC). We retrospectively analyzed a large cohort of 685 EAC considering KRAS and PIK3CA gene amplification using fluorescence in situ hybridization (FISH) and immunohistochemistry. These results were correlated with clinical and molecular data as well as the inflammatory tumor microenvironment. Amplifications of KRAS were seen in 94 patients (17.1%), PIK3CA amplifications in 23 patients (5.0%). KRAS amplifications significantly correlated with nodal positive patients and poorer overall survival (OS) in the subgroup without neoadjuvant treatment (p = 0.004), coamplifications of Her2 (p = 0.027), and TP53 mutations (p = 0.016). PIK3CA amplifications significantly correlated with a high amount of tumor infiltrating T cells (p = 0.003) and showed a tendency to better OS (p = 0.068). A correlation with checkpoint makers (PD-L1, LAG3, VISTA, TIM3, IDO) could not be revealed. Our findings are the first to link the KRAS amplified genotype with lymphonodal positivity and poor prognosis and the PIK3CA-amplified genotype with a T cell–rich microenvironment in EAC. Future studies must show whether these two genotype subgroups can be therapeutically influenced. A dual inhibition of MEK and SHP2T could be effective in the subgroup of KRAS amplified EACs and an immune checkpoint blockade may prove to be particularly promising in the subgroup of PIK3CA-amplified EACs.

Clinical Benefit of Sorafenib Combined with Paclitaxel and Carboplatin to a Patient with Metastatic Chemotherapy-Refractory Testicular Tumors.

Testicular cancer is one of the few tumor types that have not yet benefited from targeted therapy. Still no new active agents for treating this cancer have been identified over the past 15 years. Once patients are refractory to cisplatin-based chemotherapy, they will be expected to die from testicular cancer. This report describes a 21-year-old man who was refractory to chemotherapy and immunotherapy. Whole exome sequencing and low-depth whole genome sequencing confirmed the KRAS gene amplification, which may lead to the tumor cells' progression and proliferation. After discussion at the molecular tumor board, the patient was offered paclitaxel, carboplatin, and sorafenib (CPS) based on a phase III clinical trial of melanoma with KRAS gene copy gains. After treatment with CPS, the patient achieved excellent curative effects. Because of a nearly 50% frequency of KRAS amplification in chemotherapy-refractory testicular germ cells, CPS regimen may provide a new therapy, but it still warrants further validation in clinical studies. KEY POINTS: Chemotherapy-refractory testicular cancer has a very poor prognosis resulting in a lack of effective targeted therapies. KRAS gene amplification occurs in nearly 20% of testicular cancer and 50% of chemotherapy-refractory testicular cancer. KRAS amplification may activate the MAPK signaling pathway, and inhibition of MAPK by sorafenib combined with paclitaxel and carboplatin could be a viable option based on a phase III clinical trial of melanoma.To the authors' knowledge, this is the first report of response to sorafenib-based combination targeted therapy in a patient with chemotherapy-refractory testicular cancer.Clinical genomic profiling can confirm copy number variation of testicular cancer and provide insights on therapeutic options.

Response and acquired resistance to savolitinib in a patient with pulmonary sarcomatoid carcinoma harboring MET exon 14 skipping mutation: a case report.

BackgroundPulmonary sarcomatoid carcinoma (PSC) is a rare and poorly differentiated type of non-small cell lung cancer (NSCLC) with specific characteristics, which usually presents a challenge in clinical practice. Mesenchymal–epithelial transition (MET) gene has been identified as a promising target for treatments in the past few years. Here, we report a case of a patient with PSC harboring MET exon 14 mutation, who responded to a novel MET inhibitor – savolitinib.Case presentationA 75-year-old male patient with symptoms of cough, dyspnea and intermittent chest pain was diagnosed with sarcomatoid carcinoma. The tumor involved the right lung, the right hilum and multiple lesions in the right pleura, indicating a clinical disease stage IV. Next-generation sequencing of lung biopsy specimen indicated a MET exon 14 skipping mutation (NM_000245:c.3028+3A>G), with a variant allele frequency of 73.9%. The patient achieved a rapid and durable partial response with the initiation of savolitinib administration (600 mg, orally, once daily). The progression-free survival in this patient was 36 weeks. There were no ≥grade 3 adverse events reported and there was no dose reduction during treatment. Following savolitinib treatment, the allele frequency of MET exon 14 mutation in plasma circulating tumor DNA decreased with the reduction in tumor size. At the time of disease progression, fibroblast growth factor receptor 1 (FGFR1), EGFR and KRAS gene amplification were newly identified in tumor biopsy sample.ConclusionThis patient with PSC harboring MET exon 14 skipping mutation achieved significant clinical benefit with savolitinib treatment. Emergence of FGFR1, EGFR and KRAS gene amplification at the time of disease progression was likely responsible for the resistance to savolitinib in this case.

Prognostic impact of KRAS mutant type and MET amplification in metastatic and recurrent gastric cancer patients treated with first-line S-1 plus cisplatin chemotherapy.

Receptor tyrosine kinase (RTK)-related genes, including HER2, EGFR, MET, FGFR2 and KRAS, are target molecules that are clinically beneficial in gastric cancer (GC). We investigated the correlation between RTK-related genes and the curative effect of first-line S-1 plus cisplatin (SP) combination chemotherapy in metastatic and recurrent GC. We enrolled 150 patients with histopathologically confirmed metastatic and recurrent GC treated with SP. KRAS mutation was detected using direct sequencing. DNA copy number was measured by real-time PCR. Formalin-fixed paraffin-embedded specimens were examined immunohistochemically for HER2, EGFR, FGFR2 and MET. Among 144 patients, KRAS mutation was detected in five (3.5%) at codon 12 and one (0.7%) at codon 13. FGFR2, EGFR, HER2, MET and KRAS gene amplification was suggested in 4.4%, 5.9%, 9%, 3.7% and 10.3% of patients, respectively. KRAS mutation, but not KRAS amplification, was associated with significantly shorter overall and progression-free survival. MET membranous overexpression was associated with a significantly higher tumor response. MET amplification was associated with significantly shorter overall survival. We show for the first time that KRAS mutation and MET amplification are promising predictive markers in metastatic and recurrent GC patients treated with SP. KRAS status may be a useful prognostic marker in patients treated with SP.

KRAS gene amplification to define a distinct molecular subgroup of gastroesophageal adenocarcinoma.

74 Background: KRAS mutation is rare ( < 5%) in gastroesophageal cancer (GEC). However, the incidence of KRAS gene amplification (amp+), consequent protein levels, and prognostic and/or therapeutic implications are unknown. Methods: 410 GEC samples and 30 cell lines were assessed for KRAS gene copy number (GCN) by fluorescence in situ hybridization (FISH) (n = 90), Kras expression by selected reaction monitoring mass spectrometry (Kras-SRM-MS) (n = 393), and Kras-SRM level evaluated for correlation with KRAS amp+ status (n = 73). Survival analysis was performed comparing KRAS amp+ versus non-amp+ patients. When possible, concurrent 315 gene next-generation sequencing was also performed. Four KRAS-amplified xenograft lines (CAT-2,12,14,15) were established from malignant effusions. Tumorigenic activity of KRAS amp+ lines (CAT lines, MKN-1) were assessed using MTT and soft agar assays in vitro and subcutaneous xenograft models, compared to non-amp+ lines. Inhibitory assays were performed using KRAS siRNA and CRIPSR, and commercial inhibitors targeting downstream effectors MEK and/or PIK3CA. Results: KRAS FISH revealed clustered gene amp+ in 28.9% (26/90); these patients had worse prognosis than non-amp+ patients. GCN significantly correlated with Kras expression. All KRAS amp+ cell lines significantly overexpressed Kras protein and were tumorigenic in xenograft subcutaneous models. KRAS siRNA and KRAS CRISPR of KRAS amp+ cell lines demonstrated inhibition in MTT viability and soft agar assays, compared to appropriate controls, and demonstrated significant and durable xenograft growth reduction. Conversely, inhibition using MEK and/or PI3K inhibitors demonstrated only transient growth reduction in vivo. Conclusions: KRAS gene amp+ was observed in a large subset (26%) of GEC patients, which correlated with extreme expression by mass spectrometry. Established xenograft lines serve as models to investigate therapeutic strategies for KRAS amp+ patients. Inhibition using MEK/PIK3CA inhibitors provided transient benefit for KRAS amp+ tumors while durable inhibition was observed with Kras protein knockdown, suggesting potential benefit from novel siRNA therapeutics currently in development.

P114: Intratumor heterogeneity of driver mutations in lung adenocarcinoma

Background Lung cancers are characterized by the genetic alterations that often affect a common group of oncogenic signaling pathways. Identification of biologically significant genetic alterations in lung cancer that lead to activation of oncogenes and inactivation of tumor suppressor genes has the potential to provide further therapeutic opportunities (Cooper, 2013). In this context, it is important that molecular aberrations may act as negative predictors of sensitivity to treatment. The discovery of driver oncogenes, such as activating mutations in the epidermal growth factor receptor gene (EGFR), has made personalized medicine for lung cancer a reality. Despite the high initial results, NSCLC patients acquire resistance to tyrosine kinase inhibitors in the worldwide. There are several findings suggesting that the effect of tyrosine kinase inhibitors could be limited to patients harboring KRAS mutation. To date, a limited information regarding link of morphological portrait of adenocarcinomas with bearing of simultaneously concurrent mutations of KRAS and EGFR genes. The detection of concurrent gene mutation is usually carried out in a mixture of tumor cells. We plan to analyze EGFR and KRAS mutations in all clonal components (or morphological structures) of NSCLC tumors isolated by laser microdissection. So, we suggest that NSCLC clonal components might bear two concurrent EGFR and KRAS mutations simultaneously or different clonal components might bear either EGFR or KRAS mutations. The data obtained can predict resistance to EGFR-TKIs and affect therapeutic decision making. Materials and methods The tumor samples were obtained from archived formalin-fixed paraffin-embedded tissue blocks. Paraffin was removed by xylene extraction, and the sample was subsequently lysed by proteinase K. The region containing the highest percentage of tumor cells (at least 50%) was dissected. Microscopes Axio Scope A1 and Axio Star plus (Carl Zeiss, Germany) was used to perform histological analysis. If necessary, tumour cells were carefully selected and removed from the samples by laser microdissection using a P.A.L.M. microlaser instrument (PALM AdhesiveCaps, P.A.L.M., Bernried, Germany). EGFR (exon 19 deletion and 21 L858R) and KRAS (Gly12Cys, Gly12Ser, Gly12Arg, Gly12Val, Gly12Asp, Gly12Ala, Gly13Asp) gene amplification was based on RT-PCR on CFX96 Real-Time System (Bio-Rad). Results A total of 115 patients with stage (IIIB–IV) of NSCLC with EGFR-TKI- sensitive mutations were eligible for the analysis. A rare coexistence of KRAS and EGFR mutations were observed in 3 patients (2.6%). The first sample was described as nonmucinous solid adenocarcinoma and has deletion of 19 exon of EGFR gene and G12C mutation of KRAS gene. The second sample was described as mucinous lepidic adenocarcinoma and has deletion of 19 exon of EGFR gene and G12D mutation of KRAS gene. The third sample was described as mucinous adenocarcinoma with solid and acinar monoclonal components and also has deletion of 19 exon of EGFR gene and G12C mutation of KRAS gene. Of all the samples, only one had two different monoclonal components. Unfortunately, this sample was not available due to biopsy and few numbers of cells for microdissection. Fiala et al. (2013) reported that KRAS mutations were not only as a negative prognostic factor and also as a biomarker predicting resistance to EGFR-TKIs, especially G12C. Patient with nonmucinous solid adenocarcinoma with deletion of 19 exon and G12C (EGFR and KRAS genes, respectively) is unlikely to benefit from receiving gefitinib or erlotinib. Conclusion The data obtained show that further studies are required to improve personalized therapy for NSCLC patients. The frequency and type of KRAS and EGFR mutations were first assessed in NSCLC patients from West Siberia. This work was supported by a Grant from the OPTEK Company (No 122/2014/51/Nvs ).

Abstract A55: KRAS gene amplification is a distinct molecular subgroup of gastroesophageal adenocarcinoma that may benefit from combined RAS/RAF/MEK/ERK and PI3K/PTEN/AKT/mTOR pathway inhibition

Abstract Background: Given the prevelance of chromosomal instability and gene amplification (amp+) as driver mechanisms in gastroesophageal adenocarcinoma (GEC), we assessed the incidence of KRAS gene amp+ and gene copy number (GCN) along with protein expression, at baseline and after molecular targeted therapy in GEC cell lines/tissues. KRAS undergoes activating mutation in various cancers. KRAS mutation is rare (<5%) in GEC. KRAS is a downstream effector of HER2 and MET tyrosine kinases, and KRAS upregulation may be a resistance mechanism to targeted anti-HER2/anti-MET therapy. We assessed KRAS amp+ in a series of 81 GEC samples and 19 GEC cell lines. We established a KRAS amp+ model to evaluate potential therapeutic strategies. Methods: 81 GEC samples and 19 GEC cell lines were assessed for KRAS GCN by fluorescence in situ hybridization (FISH) using HER2 scoring criteria. KRAS expression was assessed by immunohistochemistry (IHC) and selected reaction monitoring mass spectrometry (SRM-MS) using a novel mulit-plex (GEC-plex) assay for formalin fixed paraffin embedded (FFPE) samples. Patient samples with HER2+ status treated with trastuzuamb as well as a cohort of patients treated with anti-MET therapy were available for pre/post treatment assessment. A KRAS-amplified cell line (Cat2) was established from ascites fluid and grown in vitro and screened for KRAS mutation using deep next-gerenation sequencing to determine if KRAS mutant alleles were present. Tumorigenic activity of Cat2, and another amplified line, MKN1, were assessed using standard proliferation assays in vitro, and an intraperitoneal (IP) murine model assessing time to ascites development (TTA), with/without anti-MEK, anti-PI3K/mTOR, or combinations thereof. Cat2 and MKN1 were also treated with KRAS siRNA and compared to siRNA control in vitro. Results: Assessment of KRAS GCN revealed clustered wild type gene amp+ in 16% (13/81) of FFPE samples, while KRAS mutation was observed in 7% (6/81) (mutually exclusive). KRAS amp+ was found in newly diagnosed metastatic patients (12/55, 22%) more than locally advanced (1/26, 4%) (p=0.06). GCN correlated with expression as determined by IHC and the MS GEC-plex. Patients treated with anti-HER2 or anti-MET therapy were observed to have developed KRAS amp+ in post-treatment biopsies at progressive disease as compared to pre-treatment biopsy. In the cell line, Cat2, KRAS was amp+ (by FISH, mean copies/nucleus: 43.2) and extremely overexpressed by IHC and MS (60 fold over KRAS non-amp+ samples); KRAS was not mutated. In vitro, KRAS siRNA substantially inhibited KRAS amp+ lines versus siRNA control (>80%). We observed diminished KRAS amp+ growth/proliferation both in vitro and in vivo using combined MEK and AKT pathway inhibitors over either alone, and compared to various inhibitor controls (anti-EGFR, anti-MET, anti-HER2). Conclusions: In this series, we observed KRAS wild type gene amp+ to be present in a subset (16%) of GEC patients at diagnosis, correlating with very high protein expression. KRAS amp+ was present after treatment with trastuzumab in HER2+ patients, and also after anti-MET therapy. These data suggest that KRAS amp+ represents a molecular subset with advanced disease at diagnosis. The observation of acquired KRAS amp+ after targeted therapies may be a resistance mechanism to anti-HER and anti-MET inhibitors. Inhibition using combined MEK/AKT pathway inhibitors, and proof-of-principle siRNA, warrants further investigation for KRAS amp+ tumors. Citation Format: Les Henderson, Peng Xu, Brittany Rambo, Wei-Li Liao, Todd Hembrough, Daniel Catenacci. KRAS gene amplification is a distinct molecular subgroup of gastroesophageal adenocarcinoma that may benefit from combined RAS/RAF/MEK/ERK and PI3K/PTEN/AKT/mTOR pathway inhibition. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A55. doi: 10.1158/1557-3125.RASONC14-A55

Circulating tumor cells

The hypothesis of the “liquid biopsy” using circulating tumor cells (CTCs) emerged as a minimally invasive alternative to traditional tissue biopsy to determine cancer therapy. Discordance for biomarkers expression between primary tumor tissue and circulating tumor cells (CTCs) has been widely reported, thus rendering the biological characterization of CTCs an attractive tool for biomarkers assessment and treatment selection. Studies performed in metastatic colorectal cancer (mCRC) patients using CellSearch, the only FDA-cleared test for CTCs assessment, demonstrated a much lower yield of CTCs in this tumor type compared with breast and prostate cancer, both at baseline and during the course of treatment. Thus, although attractive, the possibility to use CTCs as therapy-related biomarker for colorectal cancer patients is still limited by a number of technical issues mainly due to the low sensitivity of the CellSearch method. In the present study we found a significant discordance between CellSearch and AdnaTest in the detection of CTCs from mCRC patients. We then investigated KRAS pathway activating mutations in CTCs and determined the degree of heterogeneity for KRAS oncogenic mutations between CTCs and tumor tissues. Whether KRAS gene amplification may represent an alternative pathway responsible for KRAS activation was further explored. KRAS gene amplification emerged as a functionally equivalent and mutually exclusive mechanism of KRAS pathway activation in CTCs, possibly related to transcriptional activation. The serial assessment of CTCs may represent an early biomarker of treatment response, able to overcome the intrinsic limit of current molecular biomarkers represented by intratumor heterogeneity.

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.

KRAS gene amplification in colorectal cancer and impact on response to EGFR‐targeted therapy

KRAS mutations are the most common oncogenic event in colorectal cancer (CRC) progression and their occurrence is associated with lack of response to anti epidermal growth factor receptor (EGFR) targeted therapies. Using preclinical models and patients' samples we recently reported that the emergence of KRAS mutations but also KRAS amplification is associated with acquired resistance to the EGFR inhibitors cetuximab or panitumumab. We reasoned that KRAS amplification may also be responsible for primary resistance to these agents. Furthermore, while the prevalence of KRAS mutations has been well established in CRC, little is known about the frequency of KRAS amplification in large CRC series. We performed a screening of 1,039 CRC samples to assess the prevalence of KRAS amplification in this tumor type and further evaluated the role of this genetic alteration on the sensitivity to anti EGFR therapies. We detected KRAS amplification in 7/1,039 (0.67%) and 1/102 evaluable CRC specimens and cell lines, respectively. KRAS amplification was mutually exclusive with KRAS mutations. Tumors or cell lines harboring this genetic lesion are not responsive to anti-EGFR inhibitors. Although KRAS amplification is an infrequent event in CRC, it might be responsible for precluding response to anti-EGFR treatment in a small proportion of patients.

KRAS gene amplification and overexpression but not mutation associates with aggressive and metastatic endometrial cancer

Background:Three quarter of endometrial carcinomas are treated at early stage. Still, 15 to 20% of these patients experience recurrence, with little effect from systemic therapies. Homo sapiens v-Ki-ras2 Kirsten rat sarcoma viral oncogenes homologue (KRAS) mutations have been reported to have an important role in tumorigenesis for human cancers, but there is limited knowledge regarding clinical relevance of KRAS status in endometrial carcinomas.Methods:We have performed a comprehensive and integrated characterisation of genome-wide expression related to KRAS mutations and copy-number alterations in primary- and metastatic endometrial carcinoma lesions in relation to clinical and histopathological data. A primary investigation set and clinical validation set was applied, consisting of 414 primary tumours and 61 metastatic lesions totally.Results:Amplification and gain of KRAS present in 3% of the primary lesions and 18% of metastatic lesions correlated significantly with poor outcome, high International Federation of Gynaecology and Obstetrics stage, non-endometrioid subtype, high grade, aneuploidy, receptor loss and high KRAS mRNA levels, also found to be associated with aggressive phenotype. In contrast, KRAS mutations were present in 14.7% of primary lesions with no increase in metastatic lesions, and did not influence outcome, but was significantly associated with endometrioid subtype, low grade and obesity.Conclusion:These results support that KRAS amplification and KRAS mRNA expression, both increasing from primary to metastatic lesions, are relevant for endometrial carcinoma disease progression.

312 MET and KRAS gene amplification mediates acquired resistance to MET tyrosine kinase inhibitors

312 MET and KRAS gene amplification mediates acquired resistance to MET tyrosine kinase inhibitors

Activating K-Ras mutations outwith ‘hotspot’ codons in sporadic colorectal tumours – implications for personalised cancer medicine

Background:Response to EGFR-targeted therapies in colorectal cancer patients has been convincingly associated with Kirsten-Ras (K-Ras) mutation status. Current mandatory mutation testing for patient selection is limited to the K-Ras ‘hotspot’ codons 12 and 13.Methods:Colorectal tumours (n=106) were screened for additional K-Ras mutations, phenotypes compared in transformation and Ras GTPase activating assays and gene and pathway changes induced by individual K-Ras mutants identified by microarray analysis. Taqman-based gene copy number and FISH analyses were used to investigate K-Ras gene amplification.Results:Four additional K-Ras mutations (Leu19Phe (1 out of 106 tumours), Lys117Asn (1 out of 106), Ala146Thr (7 out of 106) and Arg164Gln (1 out of 106)) were identified. Lys117Asn and Ala146Thr had phenotypes similar to the hotspot mutations, whereas Leu19Phe had an attenuated phenotype and the Arg164Gln mutation was phenotypically equivalent to wt K-Ras. We additionally identified a new K-Ras gene amplification event, present in approximately 2% of tumours.Conclusions:The identification of mutations outwith previously described hotspot codons increases the K-Ras mutation burden in colorectal tumours by one-third. Future mutation screening to facilitate optimal patient selection for treatment with EGFR-targeted therapies should therefore be extended to codon 146, and in addition should consider the unique molecular signatures associated with individual K-Ras mutations.

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

BackgroundGastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells.MethodsDGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively.ResultsDGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity.ConclusionOur study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified KRAS as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of KRAS amplification and overexpression.