Antitumor efficacy of XPO1 inhibitor Selinexor in KRAS-mutant lung adenocarcinoma patient-derived xenografts

Gain-of-function Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations occur in 25% of lung adenocarcinomas, and these tumors are challenging to treat. Some preclinical work, largely based on cell lines, suggested KRAS^mut lung cancers are especially dependent on the nuclear export protein exportin-1 (XPO1), while other work supports XPO1 being a broader cancer dependency. To investigate the sensitivity of KRAS^mut lung cancers to XPO1 inhibition in models that more closely match clinical tumors, we treated 10 independently established lung cancer patient-derived tumor xenografts (PDXs) with the clinical XPO1 inhibitor, Selinexor. Monotherapy with Selinexor reduced tumor growth in all KRAS^mut PDXs, which included 4 different codon mutations, and was more effective than the clinical MEK1/2 inhibitor, Trametinib. Selinexor was equally effective in KRAS^G12C and KRAS^G12D tumors, with TP53 mutations being a biomarker for a weaker drug response. By mining genome-wide dropout datasets, we identified XPO1 as a universal cancer cell dependency and confirmed this functionally in two KRAS^WT PDX models harboring kinase drivers. However, targeted kinase inhibitors were more effective than Selinexor in these models. Our findings support continued investigation of XPO1 inhibitors in KRAS^mut lung adenocarcinoma, regardless of the codon alteration.     

Enhanced inflammation and attenuated tumor suppressor pathways are associated with oncogene‐induced lung tumors in aged mice

Aging is often accompanied by a dramatic increase in cancer susceptibility. To gain insights into how aging affects tumor susceptibility, we generated a conditional mouse model in which oncogenic Kras^G12D was activated specifically in lungs of young (3–5 months) and old (19–24 months) mice. Activation of Kras^G12D in old mice resulted in shorter survival and development of higher‐grade lung tumors. Six weeks after Kras^G12D activation, old lung tissues contained higher numbers of adenomas than their young tissue counterparts. Lung tumors in old mice displayed higher proliferation rates, as well as attenuated DNA damage and p53 tumor suppressor responses. Gene expression comparison of lung tumors from young and old mice revealed upregulation of extracellular matrix‐related genes in young tumors, indicative of a robust cancer‐associated fibroblast response. In old tumors, numerous inflammation‐related genes such as Ccl7, IL‐1β, Cxcr6, and IL‐15ra were consistently upregulated. Increased numbers of immune cells were localized around the periphery of lung adenomas from old mice. Our experiments indicate that more aggressive lung tumor formation in older Kras^G12D mice may be in part the result of subdued tumor suppressor and DNA damage responses, an enhanced inflammatory milieu, and a more accommodating tissue microenvironment.     

Reflections on drug resistance to KRASG12C inhibitors and gene silencing/editing tools for targeting mutant KRAS in cancer treatment

KRAS is the most commonly mutated oncogene in human tumors, especially in lung, pancreatic, and colorectal cancers. Small-molecule inhibitors targeting mutant KRAS^G12C demonstrated promising anti-tumor effect in patients with non-small cell lung cancer harboring KRAS^G12C mutation, while the intrinsic and acquired drug resistance occurred frequently and might be inevitable. Unlike the protein-level inhibition approach, gene silencing/editing tools for DNA-level knockout and RNA-level knockdown of mutant KRAS may be advantageous since these approaches directly eliminate the production of mutant KRAS-encoded protein. An in-depth understanding of KRAS biology, drug resistance to KRAS^G12C inhibitors and gene silencing/editing methods applied for anti-KRAS therapy may give new insight into the therapeutic strategy for cancer treatment.     

Autophagy is required for mitochondrial function,lipid metabolism,growth, and fate of KRASG12D-driven lung tumors

Evidence suggests that the role of autophagy in tumorigenesis is context dependent. Using genetically engineered mouse models (GEMMs) for human non-small-cell lung cancer (NSCLC), we found that deletion of the essential autophagy gene, Atg7, in KRAS^G12D-driven NSCLC inhibits tumor growth and converts adenomas and adenocarcinomas to benign oncocytomas characterized by the accumulation of respiration-defective mitochondria. Atg7 is required to preserve mitochondrial fatty acid oxidation (FAO) to maintain lipid homeostasis upon additional loss of Trp53 in NSCLC. Furthermore, cell lines derived from autophagy-deficient tumors depend on glutamine to survive starvation. This suggests that autophagy is essential for the metabolism, growth, and fate of NSCLC.     

KRAS mutations in colorectal cancer from Tunisia: relationships with clinicopathologic variables and data on TP53 mutations and microsatellite instability

Mutations in KRAS gene are among the critical transforming alterations occurring during CRC tumorigenesis. Here we screened 51 primary CRC tumors from Tunisia for mutations in KRAS (codons 12 and 13) using PCR-direct sequencing. Our aim was to analyze tumor mutation frequencies and spectra in Tunisian patients with CRC. KRAS status and mutation site/type were than correlated with familial and clinicopathologic variables and data on TP53 mutations and nuclear protein accumulation and microsatellite instability (MSI). A KRAS somatic mutation has been detected in the CRC tumor of 31.5 % (16/51) of the patients. 81.2 % had a single mutation at codon 12 and 23 % had a single mutation at codon 13. The most common single mutation (50 %) was a G>A transition in codon 12 (c.35G>A; p.Gly12Asp). 81.25 % of the KRAS mutations were transitions and 23 % were transversions. All the mutations in codon 13 were a c.38G>A transition, whereas both G>A transitions and G>T and G>C transversions were found in codon 12. The mutation spectrum was different between MSS and MSI-H tumors and more varied mutations have been detected in MSS tumors. Some amino acid changes were detected only in MSS tumors, i.e. p.Gly12Ser, p.Gly12Cys and p.Gly12Ala. Whereas, the KRAS mutation p.Gly13Asp have been detected only in MSI-H. 43.75 % of the patients harboured combined mutations in KRAS and TP53 genes and the tumor of 71.42 % of them showed TP53 overexpression. In conclusion, the frequency and types of KRAS mutations were as reported for non-Tunisian patients. However, no significant associations have been detected between KRAS mutations and clinicopathologic variables and MSI in Tunisian patients as previously reported.     

Resveratrol inhibits pancreatic cancer stem cell characteristics in human and KrasG12D transgenic mice by inhibiting pluripotency maintaining factors and epithelial-mesenchymal transition

Background

Cancer stem cells (CSCs) can proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which resveratrol inhibits stem cell characteristics of pancreatic CSCs derived from human primary tumors and Kras^G12D transgenic mice.

Methodology/Principal Findings

Human pancreatic CSCs (CD133⁺CD44⁺CD24⁺ESA⁺) are highly tumorigenic and form subcutaneous tumors in NOD/SCID mice. Human pancreatic CSCs expressing high levels of CD133, CD24, CD44, ESA, and aldehyde dehydrogenase also express significantly more Nanog, Oct-4, Notch1, MDR1 and ABCG2 than normal pancreatic tissues and primary pancreatic cancer cells. Similarly, CSCs from Kras^G12D mice express significantly higher levels of Nanog and Oct-4 than pancreatic tissues from Pdx-Cre mice. Resveratrol inhibits the growth (size and weight) and development (PanIN lesions) of pancreatic cancer in Kras^G12D mice. Resveratrol inhibits the self-renewal capacity of pancreatic CSCs derived from human primary tumors and Kras^G12D mice. Resveratrol induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2 and XIAP in human CSCs. Resveratrol inhibits pluripotency maintaining factors (Nanog, Sox-2, c-Myc and Oct-4) and drug resistance gene ABCG2 in CSCs. Inhibition of Nanog by shRNA enhances the inhibitory effects of resveratrol on self-renewal capacity of CSCs. Finally, resveratrol inhibits CSC''s migration and invasion and markers of epithelial-mesenchymal transition (Zeb-1, Slug and Snail).

Conclusions/Significance

These data suggest that resveratrol inhibits pancreatic cancer stem cell characteristics in human and Kras^G12D transgenic mice by inhibiting pluripotency maintaining factors and epithelial-mesenchymal transition. In conclusion, resveratrol can be used for the management of pancreatic cancer.     

Combined Inactivation of MYC and K-Ras oncogenes reverses tumorigenesis in lung adenocarcinomas and lymphomas

Background

Conditional transgenic models have established that tumors require sustained oncogene activation for tumor maintenance, exhibiting the phenomenon known as “oncogene-addiction.” However, most cancers are caused by multiple genetic events making it difficult to determine which oncogenes or combination of oncogenes will be the most effective targets for their treatment.

Methodology/Principal Findings

To examine how the MYC and K-ras^G12D oncogenes cooperate for the initiation and maintenance of tumorigenesis, we generated double conditional transgenic tumor models of lung adenocarcinoma and lymphoma. The ability of MYC and K-ras^G12D to cooperate for tumorigenesis and the ability of the inactivation of these oncogenes to result in tumor regression depended upon the specific tissue context. MYC-, K-ras^G12D- or MYC/K-ras^G12D-induced lymphomas exhibited sustained regression upon the inactivation of either or both oncogenes. However, in marked contrast, MYC-induced lung tumors failed to regress completely upon oncogene inactivation; whereas K-ras^G12D-induced lung tumors regressed completely. Importantly, the combined inactivation of both MYC and K-ras^G12D resulted more frequently in complete lung tumor regression. To account for the different roles of MYC and K-ras^G12D in maintenance of lung tumors, we found that the down-stream mediators of K-ras^G12D signaling, Stat3 and Stat5, are dephosphorylated following conditional K-ras^G12D but not MYC inactivation. In contrast, Stat3 becomes dephosphorylated in lymphoma cells upon inactivation of MYC and/or K-ras^G12D. Interestingly, MYC-induced lung tumors that failed to regress upon MYC inactivation were found to have persistent Stat3 and Stat5 phosphorylation.

Conclusions/Significance

Taken together, our findings point to the importance of the K-Ras and associated down-stream Stat effector pathways in the initiation and maintenance of lymphomas and lung tumors. We suggest that combined targeting of oncogenic pathways is more likely to be effective in the treatment of lung cancers and lymphomas.     

Autophagic reliance promotes metabolic reprogramming in oncogenic KRAS-driven tumorigenesis

Defects in basal autophagy limit the nutrient supply from recycling of intracellular constituents. Despite our understanding of the prosurvival role of macroautophagy/autophagy, how nutrient deprivation, caused by compromised autophagy, affects oncogenic KRAS-driven tumor progression is poorly understood. Here, we demonstrate that conditional impairment of the autophagy gene Atg5 (atg5-KO) extends the survival of KRAS^G12V-driven tumor-bearing mice by 38%. atg5-KO tumors spread more slowly during late tumorigenesis, despite a faster onset. atg5-KO tumor cells displayed reduced mitochondrial function and increased mitochondrial fragmentation. Metabolite profiles indicated a deficiency in the nonessential amino acid asparagine despite a compensatory overexpression of ASNS (asparagine synthetase), key enzyme for de novo asparagine synthesis. Inhibition of either autophagy or ASNS reduced KRAS^G12V-driven tumor cell proliferation, migration, and invasion, which was rescued by asparagine supplementation or knockdown of MFF (mitochondrial fission factor). Finally, these observations were reflected in human cancer-derived data, linking ASNS overexpression with poor clinical outcome in multiple cancers. Together, our data document a widespread yet specific asparagine homeostasis control by autophagy and ASNS, highlighting the previously unrecognized role of autophagy in suppressing the metabolic barriers of low asparagine and excessive mitochondrial fragmentation to permit malignant KRAS-driven tumor progression.     

Detection of KRAS Exon 2 Mutations in Circulating Tumor Cells Isolated by the ISET System from Patients with RAS Wild Type Metastatic Colorectal Cancer

INTRODUCTION: The presence of KRAS mutations in patients with metastatic colorectal cancer (mCRC) predicts poor response to agents targeting the EGFR. Even in patients with RAS wild type (WT) tumors, resistance eventually develops due to multiple mechanisms, including the expansion of previously undetected KRAS mutated clones. In this feasibility study, we aimed to detect KRAS exon 2 mutations in serial samples of circulating tumor cells (CTCs) of RAS WT patients with mCRC captured by the Isolation by Size of Epithelial Tumor cells (ISET) system. METHODS: CTC isolation using the ISET system was performed from prospectively collected blood samples obtained from patients with RAS and BRAF WT mCRC prior to first-line therapy initiation, at first imaging assessment and on disease progression. CTCs were enumerated using hematoxylin & eosin and CD45 double stain on a single membrane spot. DNA was extracted from 5 spots and KRAS exon 2 mutations were detected using a custom quantitative Polymerase Chain Reaction (qPCR) assay. RESULTS: Fifteen patients were enrolled and 28 blood samples were analyzed. In 9 (60%) patients, at least one sample was positive for the presence of a KRAS exon 2 mutation. In 11 out of 28 samples (39.2%) with detectable CTCs a KRAS mutation was detected; the corresponding percentages for baseline and on progression samples were 27% and 37.5%, respectively. The most commonly detected mutations were G13D and G12C (n = 3). The presence of KRAS mutated CTCs at baseline was not prognostic for either PFS (P = .950) or OS (P = .383). CTC kinetics did not follow tumor response patterns. CONCLUSION: The results demonstrate that using a qPCR-based assay, KRAS exon 2 mutations could be detected in CTCs captured by the ISET system from patients with RAS WT primary tumors. However, the clinical relevance of these CTCs remains to be determined in future studies.     

Detection of <Emphasis Type="Italic">KRAS</Emphasis> mutations in tumor cells using biochips

Somatic mutations in the KRAS gene are important markers of some types of tumors, for example, pancreatic cancer, and may be useful in early diagnostics. A biochip has been developed which allows deter-mining most frequent mutations in 12, 13, and 61 codons of the KRAS gene. To increase the sensitivity of the method and to enable the analysis of minor fractions of tumor cells in clinical samples, the method of blocking wild type sequence PCR amplification by LNA-oligonucleotides has been used. The product of LNA-clamp PCR was further hybridized with oligonucleotide probes, immobilized on the biochip. The biochip was tested with 42 clinical DNA samples from patients with pancreatic cancer, mostly duct adenocarcinomas. As reference methods, RFLP analysis and sequencing were used. The developed approach allows detecting somatic mutations in the KRAS gene if the portion of tumor cells with mutation is at least 1% of the whole cell population.     

Loss of Canonical Smad4 Signaling Promotes KRAS Driven Malignant Transformation of Human Pancreatic Duct Epithelial Cells and Metastasis

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in North America. Activating KRAS mutations and Smad4 loss occur in approximately 90% and 55% of PDAC, respectively. While their roles in the early stages of PDAC development have been confirmed in genetically modified mouse models, their roles in the multistep malignant transformation of human pancreatic duct cells have not been directly demonstrated. Here, we report that Smad4 represents a barrier in KRAS-mediated malignant transformation of the near normal immortalized human pancreatic duct epithelial (HPDE) cell line model. Marked Smad4 downregulation by shRNA in KRAS ^G12V expressing HPDE cells failed to cause tumorigenic transformation. However, KRAS-mediated malignant transformation occurred in a new HPDE-TGF-β resistant (TβR) cell line that completely lacks Smad4 protein expression and is resistant to the mito-inhibitory activity of TGF-β. This transformation resulted in tumor formation and development of metastatic phenotype when the cells were implanted orthotopically into the mouse pancreas. Smad4 restoration re-established TGF-β sensitivity, markedly increased tumor latency by promoting apoptosis, and decreased metastatic potential. These results directly establish the critical combination of the KRAS oncogene and complete Smad4 inactivation in the multi-stage malignant transformation and metastatic progression of normal human HPDE cells.     

Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition

The recent development of mutant-selective inhibitors for the oncogenic KRAS^G12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRAS^G12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pK_a of the C12 thiol is depressed (pK_a ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRAS^Y137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRAS^G12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, K_d = 36.0 ± 0.7 μM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pK_a of the C12 thiol, we found that reversible and irreversible oxidation of KRAS^G12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRAS^G12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRAS^G12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRAS^G12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.     

Oncogenic KRAS suppresses store-operated Ca2+ entry and ICRAC through ERK pathway-dependent remodelling of STIM expression in colorectal cancer cell lines

The KRAS GTPase plays a fundamental role in transducing signals from plasma membrane growth factor receptors to downstream signalling pathways controlling cell proliferation, survival and migration. Activating KRAS mutations are found in 20% of all cancers and in up to 40% of colorectal cancers, where they contribute to dysregulation of cell processes underlying oncogenic transformation. Multiple KRAS-regulated cell functions are also influenced by changes in intracellular Ca²⁺ levels that are concurrently modified by receptor signalling pathways. Suppression of intracellular Ca²⁺ release mechanisms can confer a survival advantage in cancer cells, and changes in Ca²⁺ entry across the plasma membrane modulate cell migration and proliferation. However, inconsistent remodelling of Ca²⁺ influx and its signalling role has been reported in studies of transformed cells. To isolate the interaction between altered Ca²⁺ handling and mutated KRAS in colorectal cancer, we have previously employed isogenic cell line pairs, differing by the presence of an oncogenic KRAS allele (encoding KRAS^G13D), and have shown that reduced Ca²⁺ release from the ER and mitochondrial Ca²⁺ uptake contributes to the survival advantage conferred by oncogenic KRAS. Here we show in the same cell lines, that Store-Operated Ca²⁺ Entry (SOCE) and its underlying current, I_CRAC are under the influence of KRAS^G13D. Specifically, deletion of the oncogenic KRAS allele resulted in enhanced STIM1 expression and greater Ca²⁺ influx. Consistent with the role of KRAS in the activation of the ERK pathway, MEK inhibition in cells with KRAS^G13D resulted in increased STIM1 expression. Further, ectopic expression of STIM1 in HCT 116 cells (which express KRAS^G13D) rescued SOCE, demonstrating a fundamental role of STIM1 in suppression of Ca²⁺ entry downstream of KRAS^G13D. These results add to the understanding of how ERK controls cancer cell physiology and highlight STIM1 as an important biomarker in cancerogenesis.     

Loss of p53 Attenuates the Contribution of IL-6 Deletion on Suppressed Tumor Progression and Extended Survival in Kras-Driven Murine Lung Cancer

Interleukin-6 (IL-6) is involved in lung cancer tumorigenesis, tumor progression, metastasis, and drug resistance. Previous studies show that blockade of IL-6 signaling can inhibit tumor growth and increase drug sensitivity in mouse models. Clinical trials in non-small cell lung cancer (NSCLC) reveal that IL-6 targeted therapy relieves NSCLC-related anemia and cachexia, although other clinical effects require further study. We crossed IL-6 ^-/- mice with Kras ^G12D mutant mice, which develop lung tumors after activation of mutant Kras ^G12D, to investigate whether IL-6 inhibition contributes to tumor progression and survival time in vivo. Kras ^G12D; IL-6 ^-/- mice exhibited increased tumorigenesis, but slower tumor growth and longer survival, than Kras ^G12D mice. Further, in order to investigate whether IL-6 deletion contributes to suppression of lung cancer metastasis, we generated Kras ^G12D; p53 ^flox/flox; IL-6 ^-/- mice, which developed lung cancer with a trend for reduced metastases and longer survival than Kras ^G12D; p53 ^flox/flox mice. Tumors from Kras ^G12D; IL-6 ^-/- mice showed increased expression of TNFα and decreased expression of CCL-19, CCL-20 and phosphorylated STAT3 (pSTAT3) than Kras ^G12D mice; however, these changes were not present between tumors from Kras ^G12D; p53 ^flox/flox; IL-6 ^-/- and Kras ^G12D; p53 ^flox/flox mice. Upregulation of pSTAT3 and phosphorylated AKT (pAKT) were observed in Kras ^G12D tumors with p53 deletion. Taken together, these results indicate that IL-6 deletion accelerates tumorigenesis but delays tumor progression and prolongs survival time in a Kras-driven mouse model of lung cancer. However, these effects can be attenuated by p53 deletion.     

Tumor‐Targeting Salmonella typhimurium A1‐R Promotes Tumoricidal CD8+ T Cell Tumor Infiltration and Arrests Growth and Metastasis in a Syngeneic Pancreatic‐Cancer Orthotopic Mouse Model

The present study determined the effect of the tumor‐targeting strain Salmonella typhimurium A1‐R (S. typhimurium A1‐R) on CD8⁺ tumor‐infiltrating lymphocytes (TILs) in a syngeneic pancreatic‐cancer orthotopic mouse model. The effect of tumor‐targeting S. typhimurium A1‐R on CD8⁺ TILs was determined on the Pan02 murine pancreatic‐adenocarcinoma implanted orthotopically in the pancreatic tail of C57BL/6 immunocompromised mice. Three weeks after orthotopic implantation, mice were randomized as follows G1: untreated control group (n = 8); and G2: S. typhimurium A1‐R‐treatment group (n = 8, 1 × 10⁷ colony forming units [CFU]/body, iv, weekly, 3 weeks). On the 22nd day from initial treatment, all mice were sacrificed and tumors were harvested. The tumor‐volume ratio was defined as ratio of tumor volume on the 22nd day relative to the 1st day. The tumor volume ratio was significantly lower in the S. typhimurium A1‐R‐treated group (G2) (3.0 ± 2.8) than the untreated control (G1) (39.9 ± 30.7, P < 0.01). Hematoxylin and easin (H&E) staining on tumor sections was performed to evaluate tumor destruction which was classified according to the Evans grading system and found to be much greater in the S. typhimurium A1‐R‐treated mice (G2). Six mice in G1 had peritoneal dissemination, whereas no mice showed peritoneal dissemination in G2 (P < 0.01). Immunohistochemical staining with anti‐mouse CD8⁺ antibody was performed in order to detect TILs determined by calculating the average number of CD8⁺ cells in three high power fields (200×) in the treated and untreated tumors. The TIL score was significantly higher in G2 (133.5 ± 32.2) than G1 (45.1 ± 19.4, P < 0.001). The present study demonstrates that S. typhimurium A1‐R promotes CD8⁺ T cell infiltration and inhibition of tumor growth and metastasis. J. Cell. Biochem. 119: 634–639, 2018. © 2017 Wiley Periodicals, Inc.     

Somatic Mutations in Exocrine Pancreatic Tumors: Association with Patient Survival

KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09–4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14–2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33–7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application.