K-ras Mutations In Pancreatic Cancer Research Articles

Abstract C047: Autophagy-mediated proline-supply is important for cell survival in pancreatic cancers

Abstract Oncogenic KRAS mutations present in 90–95% of pancreatic cancer patients. KRAS mutation in pancreatic cancer increased the dependency on glutamine and consumption of proline. Proline is a non-essential amino acid but it is important for the synthesis of proteins such as collagen, and also for precursors of antioxidant molecules. Several enzymes such as P5CS(ALDH18A1),PYCR1 and PYCR2 are involving proline synthesis. The effects of proline synthesis enzymes on pancreatic cancer cells are relatively less studied than that of enzymes catalyzing proline. Here, we examined the impact of proline synthesis enzymes on pancreatic cancer cell survival and proliferation. We first examined that exogenous proline promotes cell survival under low glutamine conditions in various pancreatic cancer cells. Exogenous proline supplementation increased some pancreatic cell survival even when glutamine was not sufficiently available, whereas proline supplement is dispensible for certain cell types of pancreatic cancer. Next, to investigate the importance of proline synthesis enzymes in cell survival, we measured the cell proliferation of pancreatic cancer with knock-down the proline synthesis enzymes. The knockdown of ALDH18A1 and PYCR1,2, which are proline biosynthesis enzyme genes, reduced cell survival in pancreatic cancer cells, suggesting that in PDAC cells, the enzymes that synthesize proline from glutamine are crucial for cancer cell survival and growth. To test the effect of proline-sysnthesis enzymes on autophagy, we measured autophagy flux in the cell harboring GFP-RFP-LC3 or GFP-LC3 as a reporter. Knockdown of proline synthesis enzymes, particularly ALDH18A1 or PYCR1 led to an increase in autophagy levels, indicating that proline-independent cells can bypass proline synthesis by autophagy induction in the absence of proline synthesis genes. Furthermore gemcitabine-resistant cells showed reduced levels of proline synthesis enzymes. In pancreatic cancer cell lines, proline promotes cancer cell survival, generated by both biosynthesis from glutamine and starvation-induced autophagy, which also critically important for supporting cellular drug resistance. Taken together, these results indicate that proline synthesis is mechanistically associated with autophagy induced by glutamine-starvation, which is pivotal for supplying proline in pancreatic cancer cells. Citation Format: Ji hyeon Kim, Heesun Cheong. Autophagy-mediated proline-supply is important for cell survival in pancreatic cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C047.

Targeting KRAS mutations in pancreatic cancer: opportunities for future strategies.

Targeting the RAS pathway remains the holy grail of precision oncology. In the case of pancreatic ductal adenocarcinomas (PDAC), 90-92% harbor mutations in the oncogene KRAS, triggering canonical MAPK signaling. The smooth structure of the altered KRAS protein without a binding pocket and its affinity for GTP have, in the past, hampered drug development. The emergence of KRASG12C covalent inhibitors has provided renewed enthusiasm for targeting KRAS. The numerous pathways implicated in RAS activation do, however, lead to the development of early resistance. In addition, the dense stromal niche and immunosuppressive microenvironment dictated by oncogenic KRAS can influence treatment responses, highlighting the need for a combination-based approach. Given that mutations in KRAS occur early in PDAC tumorigenesis, an understanding of its pleiotropic effects is key to progress in this disease. Herein, we review current perspectives on targeting KRAS with a focus on PDAC.

1646P Genomic and prognostic differences in patients with different KRAS mutations in pancreatic cancer

1646P Genomic and prognostic differences in patients with different KRAS mutations in pancreatic cancer

KRAS mutations in pancreatic cancer: could we talk about a risk factor for the development of thromboembolic phenomena?

The oncogenic KRAS mutation is associated with increased tissue factor expression and thus hypercoagulability. In this regard, numerous studies published in the last decade have shown that KRAS mutations are an important risk factor for the development of thromboembolic phenomena in neoplasms of the digestive tract, such as colorectal cancer. On the other hand, some recently published studies suggest that KRAS mutations are also associated with an increased risk of developing thromboembolic phenomena in pancreatic cancer. Based on these premises, we have conducted a single-centre retrospective study on a cohort of patients with pancreatic cancer. Our aim is to demonstrate whether there is an association between the presence of KRAS mutations in our cohort of pancreatic cancer patients and an increased risk of developing thromboembolic phenomena.

Multiplex Digital Polymerase Chain Reaction on a Droplet Array SlipChip for Analysis of KRAS Mutations in Pancreatic Cancer.

Pancreatic cancer is a terminal disease with high mortality and very poor prognosis. A sensitive and quantitative analysis of KRAS mutations in pancreatic cancer provides a tool not only to understand the biological mechanisms of pancreatic cancer but also for diagnosis and treatment monitoring. Digital polymerase chain reaction (PCR) is a promising tool for KRAS mutation analysis, but current methods generally require a complex microfluidic handling system, which can be challenging to implement in routine research and point-of-care clinical diagnostics. Here, we present a droplet-array SlipChip (da-SlipChip) for the multiplex quantification of KRAS G12D, V, R, and C mutant genes with the wild-type (WT) gene background by dual color (FAM/ROX) fluorescence detection. This da-SlipChip is a high-density microwell array of 21,696 wells of 200 pL in 4 by 5424 microwell format with simple loading and slipping operation. It does not require the same precise alignment of microfeatures on the different plates that are acquired by the traditional digital PCR SlipChip. This device can provide accurate quantification of both mutant genes and the WT KRAS gene. We collected tumor tissue, paired normal pancreatic tissue, and other normal tissues from 18 pancreatic cancer patients and analyzed the mutation profiles of KRAS G12D, V, R, and C in these samples; the results from the multiplex digital PCR on da-SlipChip agree well with those of next-generation sequencing (NGS). This da-SlipChip moves digital PCR closer to the practical point-of-care applications not only for detecting KRAS mutations in pancreatic cancer but also for other applications that require precise nucleic acid quantification with high sensitivity.

Diagnostic and Prognostic Values of KRAS Mutations on EUS-FNA Specimens and Circulating Tumor DNA in Patients With Pancreatic Cancer.

INTRODUCTION:The ability of carbohydrate antigen 19-9 (CA19-9) to differentiate pancreatic cancer from other benign pancreatic lesions is unsatisfactory. This study explored the diagnostic value of KRAS gene mutations and plasma circulating tumor DNA (ctDNA) in patients with pancreatic cancer.METHODS:The prospective cohort study comprised 149 consecutive patients with solid pancreatic lesions who underwent endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). KRAS subtype mutations were analyzed by digital droplet PCR (ddPCR) in EUS-FNA histopathology tissue samples, and blood samples were sent for plasma ctDNA analysis. The final diagnosis was based on surgical resection pathology or follow-up for at least 2 years.RESULTS:Adding KRAS mutation ddPCR increased the sensitivity and accuracy of EUS-FNA from 71.4% to 91.6% (P < 0.001) and 75.8% to 88.6% (P < 0.001), respectively. By comparison, the sensitivities of circulating biomarkers ctDNA and CA19-9 were only 35.2% and 71.2%. The area under the curve of the receiver operating characteristic curve (AUC) of EUS-FNA and KRAS ddPCR combination was >0.90 for distinguishing pancreatic cancer from benign lesions, whereas the AUC of EUS-FNA and CA19-9 combination was 0.83. The median survival time was significantly shorter in patients with G12D KRAS mutations than that in patients with other mutations (180 vs 240 days, P < 0.001).DISCUSSION:FNA tissue sample KRAS mutation analysis in tissues significantly improves the diagnostic accuracy of cyto/histopathological evaluation in EUS-FNA samples. The combination of EUS-FNA and tissue sample KRAS ddPCR provided a more accurate method for pancreatic cancer diagnosis, superior to the combination of EUS-FNA and CA19-9/ctDNA. G12D KRAS mutations in pancreatic cancer were independently associated with poor overall survival.

Phase I study of mesenchymal stem cell (MSC)-derived exosomes with KRASG12D siRNA in patients with metastatic pancreatic cancer harboring a KRASG12D mutation.

TPS633 Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with few effective therapeutic options. Over 90% of patients with PDAC harbor activating mutations in KRAS, a known oncogenic driver of tumor growth, cancer cell survival and metastasis thus making for an attractive therapeutic target. However, targeting the most common KRAS mutations in pancreatic cancer (KRASG12D and KRASG12V) remains a pharmacological challenge. Exosomes are extracellular nano-vesicles that are efficiently internalized by target cells and are under investigation as a drug-delivery vehicle for various therapeutic payloads, including nucleic acids such as small interfering RNA (siRNA). Previously published pre-clinical data demonstrate effective delivery of exosomes loaded with siRNA targeting KRASG12D leading to tumor control in various murine models of PDAC. Methods: This is a single arm, single institution, phase I trial evaluating treatment with KRASG12D-siRNA loaded exosomes. Large-scale production of KRASG12D-siRNA loaded exosomes from mesenchymal stromal cells will be performed at the MD Anderson Cancer Center using pre-specified GMP-compliant protocols. The primary endpoints of this study are to determine a maximum tolerated dose (MTD) of KRASG12D-loaded exosomes and to identify dose-limiting toxicities (DLT). Key secondary endpoints include the pharmacokinetics of circulating exosomes, overall response rate, disease control rate (defined as partial responses and patients with stable disease), median progression-free survival (PFS) and median overall survival (OS). Key inclusion criteria include histologically confirmed metastatic pancreatic ductal adenocarcinoma, documented progression on one or more lines of systemic therapy, and documented presence of a KRASG12D mutation. Selected correlative studies include measurement of circulating siRNA and KRASG12D DNA using PCR. This trial will enroll up to 28 patients and will follow a 3+3 design for dose escalation. This trial is actively accruing and has enrolled six patients at the time of submission. Clinical trial information: NCT03608631.

33P Prognostic values of different subtypes of KRAS mutations in pancreatic cancer

Mutated KRAS is considered as a negative prognostic marker for survival in pancreatic ductal adenocarcinoma (PDAC). However, prognostic values of individual mutations (G12D, G12V or G12R) are still controversial. The aim of current study was to evaluate the survival of pancreatic cancer patients with different subtypes of KRAS mutations.

Abstract PO-064: Inhibition of RAS signaling and tumorigenesis through novel targeting novel vulnerabilities

Abstract RAS GTPases are important mediators of oncogenesis with nearly 20% of human tumors harboring mutant RAS proteins. However, pharmacological inhibition of RAS has proven challenging. We have employed Monobody technology to discover novel vulnerabilities in RAS that can be exploited to inhibit RAS signaling and tumorigenesis. Monobodies are single-domain synthetic binding proteins that achieve levels of affinity and selectivity similar to antibodies. In contrast to antibodies, Monobodies are fully functional in the reducing environment of the cytoplasm and thus are particularly suitable as genetically encoded “tool biologics”. We previously developed the NS1 Monobody that inhibited RAS by targeting the α4-α5 allosteric lobe to prevent RAS self-association and nanoclustering, and NS1 has become a widely used tool in the RAS research community. Following on this success, we sought to identify additional vulnerabilities in RAS. Based on our discovery that nucleotide-free RAS (apoRAS) inhibits PIK3C2B function, we assessed the feasibility of selectively targeting this state of RAS as an approach to inhibit RAS. Here, we have developed several Monobodies and extensively characterized one of them, R15. Although NS1 was agnostic to the nucleotide state of RAS, R15 bound exclusively to the apo state of all three RAS isoforms but did not interact with nucleotide-loaded RAS. When expressed in cells, R15 inhibited the signaling and transforming activity of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., “fast exchange mutants”), such as G13D and Q61L, but was ineffective against slow exchange mutants such as G12V. Surprisingly, R15 also bound and inhibited RAS(G12D), the most common oncogenic KRAS mutation in pancreatic cancer. Biochemical studies demonstrated that R15 captured RAS from cell lysates in the nucleotide-free state, suggesting that R15 traps apoRAS and prevents nucleotide reloading. Disruption of SOS binding by mutation of D69N further enhanced R15 binding to these fast exchange RAS mutants. Finally, inducible expression of R15 selectively inhibited the tumor forming capacity of tumor lines and patient derived xenografts driven by fast exchange RAS mutants, including RAS(G12D), but not RAS(G12V). Thus, in contrast to conventional wisdom, our approach has established a new opportunity to selectively inhibit the tumorigenic activity of certain RAS mutants by targeting the apo state of RAS with drug-like molecules. Citation Format: Imran Khan, Akiko Koide, Mariyam Zuberi, Eric Denbaum, Gayatari Ketavar, Kai-Wen Teng, Matthew Rhett, Russell Sepncer-Smith, Ernest R. Camp, Shohei Koide, John O’Bryan. Inhibition of RAS signaling and tumorigenesis through novel targeting novel vulnerabilities [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-064.

A Protocol for Cancer-Related Mutation Detection on Exosomal DNA in Clinical Application.

BackgroundRecently, some genomic mutations in exosomal DNA have been found to be related to disease progress and clinical outcomes of patients in several cancers. Unfortunately, the methods for exosome isolation and exosomal DNA analysis are still lack of relevant research to ensure their optimal performance and the comparability. Here we aim to establish a protocol for cancer-related mutation detection on exosomal DNA in clinical application.MethodsTaking KRAS mutation in pancreatic cancer as an example, we tested whether the types of blood samples, the potential factors in the courses of exosome isolation and exosomal DNA preparation, as well as the detail in mutation detection by droplet digital PCR (ddPCR) could influence the exosomal DNA analysis.ResultsWe found that the concentration of exosomal DNA from serum was higher than that from plasma, whereas the mutant allele fraction (MAF) of KRAS in serum-derived exosomal DNA was obviously lower. The membrane-based method for exosome isolation showed no evident difference in both exosomal DNA yield and KRAS MAF from the classical ultracentrifugation method. DNase I pretreatment on exosomes could remove the wild-type DNA outside of exosomes and increase the KRAS MAF. PBS might interfere with the effect of DNase I and should not be recommended as resuspension buffer for exosomes if the subsequent experiments would be done with exosomal DNA. Besides, the denaturation of exosomal DNA before droplet generation during ddPCR could effectively improve the total KRAS copy number and mutation-positive droplet number.ConclusionThis study provides some methodological evidences for the selection of the optimal experimental conditions in exosomal DNA analysis. We also suggest a protocol for mutation detection on exosomal DNA, which might be suitable for the clinical testing and could be helpful to the comparison of results from different laboratories.

Abstract C27: Association of mutant KRAS isoforms with weight loss in pancreatic cancer

Abstract Background: Patients with pancreatic cancer often develop progressive weight loss and sarcopenia, described as cancer cachexia syndrome. Despite evidence showing that pancreatic cancer cachexia is associated with a poor prognosis, reduced tolerance to therapy, and diminished quality of life, no predictive biomarkers have been identified to date. Certain mutant KRAS isoforms are associated with significant alterations to tumor metabolism and have been correlated with changes in weight and survival. However, the clinical implications resulting from specific KRAS mutations in pancreatic cancer have yet to be fully explored. Methods: A retrospective analysis was performed to compare weight changes and survival in 150 pancreatic cancer patients with various KRAS alterations identified by next-generation sequencing (Foundation Medicine, Inc., Cambridge, MA). Survival data and weights documented between 6 months prior to diagnosis and the date of death or the last follow-up visit were abstracted from the medical record and evaluated using statistical analyses, including functional principal component analysis for sparse functional data, Ward cluster, logistic regression, analysis of variance, and Cox regression. Using derivative principal component scores, we separated patients into two groups demonstrating either constant or decreasing weight over time. Results: 77% of evaluated patients had a KRAS mutation, and 21% had KRAS wild-type. Of patients with mutant KRAS, 84% had an alteration at G12, 10% at Q61, and 3% at G13. While there was no significant difference in weight changes between KRAS wild-type patients and patients with a Q61 mutation, patients with non-Q61 mutations were 2.5 times more likely (p = 0.05) than wild-type patients to demonstrate weight loss over time. Furthermore, KRAS mutants had worse survival when compared to KRAS wild-type patients. Irrespective of mutational status, patients whose weight did not decrease were 69% less likely to die (p = 0.039). Conclusion: We demonstrated that mutant KRAS isoforms G12 and G13 mutations correlate with weight loss, whereas Q61 KRAS mutations and wild-type KRAS do not. Furthermore, weight loss in all patients increased their risk of death. These findings suggest that KRAS subtypes may have unique genotype-to-phenotype features, particularly with weight loss that could warrant tailored therapeutic strategies in at-risk subgroups. Future studies of larger scope, and ideally prospective in nature, are warranted in patients stratified by KRAS isoform. Citation Format: Haesoo Kim, Camille Ng, Marcio A. Diniz, Michel H. Montoril, Michelle Guan, Victor Brodsky, Eric Vail, Richard Tuli, Edik Blais, Sungjin Kim, Emanuel Petricoin III, Jun Gong, Veronica R. Placencio-Hickok, Andrew E. Hendifar. Association of mutant KRAS isoforms with weight loss in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C27.

Antitumor activity of sorafenib plus CDK4/6 inhibitor in pancreatic patient derived cell with KRAS mutation

KRAS mutation has been known as crucial marker for growth and maintenance of pancreatic cancer (PC) and targeting the KRAS is inevitable component for realizing precision medicine to PC. We established patient-derived tumor cells (PDCs) from patient with KRAS G12R mutant PC. Through the PDC, we investigated the therapeutic impact of sorafenib alone, LEE001 alone and the combination of sorafenib and LEE001 in KRAS mutant PC. For the validation, we also tested a cell viability assay for sorafenib, LEE001, and sorafenib plus LEE001 in KRAS G12R transfected HEK293T cells. Based on MTT proliferation assays using PDCs, values of IC50 were 6.07 uM to sorafenib and > 10.00 uM to LEE001, respectively. The value of IC50 of the combination (sorafenib plus LEE001) was 3.19 uM. Cell proliferation of PDC was significantly inhibited by sorafenib plus LEE001, as compared to sorafenib monotherapy and LEE001 monotherapy. In the validation through KRAS G12R transfected HEK293T cells, consistent to findings in PDCs, combinations of sorafenib plus LEE001 had most effective inhibitory effect in KRAS G12R transfected HEK293T cells. Furthermore, on analyzing the regulation of targeted downstream pathways upon exposure to sorafenib, LEE001, and sorafenib plus LEE001 by immunoblot assay using KRAS G12R transfected HEK293T cells, AKT phosphorylation was distinctively decreased in KRAS G12R transfected HEL293 cells after only sorafenib plus LEE001. This study suggests that the combination of RAF and CDK4/6 inhibitors might be a novel treatment strategy for KRAS G12R mutant pancreatic cancer. The antitumor effect of RAF plus CDK4/6 inhibitors also needs to be evaluated in other subtypes of KRAS mutation in pancreatic cancer.

The prognostic value of KRAS mutation by cell-free DNA in cancer patients: A systematic review and meta-analysis.

KRAS mutation has been found in various types of cancer. However, the prognostic value of KRAS mutation in cell-free DNA (cfDNA) in cancer patients was conflicting. In the present study, a meta-analysis was conducted to clarify its prognostic significance. Literature searches of Cochrane Library, EMBASE, PubMed and Web of Science were performed to identify studies related to KRAS mutation detected by cfDNA and survival in cancer patients. Two evaluators reviewed and extracted the information independently. Review Manager 5.3 software was used to perform the statistical analysis. Thirty studies were included in the present meta-analysis. Our analysis showed that KRAS mutation in cfDNA was associated with a poorer survival in cancer patients for overall survival (OS, HR 2.02, 95% CI 1.63–2.51, P<0.01) and progression-free survival (PFS, HR 1.64, 95% CI 1.27–2.13, P<0.01). In subgroup analyses, KRAS mutation in pancreatic cancer, colorectal cancer, non-small cell lung cancer and ovarian epithelial cancer had HRs of 2.81 (95% CI 1.83–4.30, P<0.01), 1.67 (95% CI 1.25–2.42, P<0.01), 1.64 (95% CI 1.13–2.39, P = 0.01) and 2.17 (95% 1.12–4.21, p = 0.02) for OS, respectively. In addition, the ethnicity didn’t influence the prognostic value of KRAS mutation in cfDNA in cancer patients (p = 0.39). Prognostic value of KRAS mutation was slightly higher in plasma than in serum (HR 2.13 vs 1.65), but no difference was observed (p = 0.37). Briefly, KRAS mutation in cfDNA was a survival prognostic biomarker in cancer patients. Its prognostic value was different in various types of cancer.

Abstract A32: GFAT: The crossroads of glucose and glutamine metabolism in pancreatic cancer

Abstract Due to a lack of early detection methods and effective treatment strategies, pancreatic cancer is slated to become the second leading cause of cancer related death in the United States. Pancreatic ductal adenocarcinoma (PDA), the most common type of pancreatic cancer, is initiated almost universally by mutations in the Kras oncogene. Kras mutations rewire metabolism in the cancer cells by enhancing nutrient capture and facilitating metabolic pathways that support survival and proliferation in the austere tumor microenvironment. Among these pathways, we have previously demonstrated that mutant Kras drives glucose flux into the hexosamine biosynthetic pathway (HBP) through upregulation of the rate limiting enzyme, glutamine fructose-6-phosphate transaminase (GFAT). The HBP plays a well-established role in the generation of the precursor substrates in glycosylation. In addition to this, GFAT has a less well appreciated facet, as it requires input from both glucose and glutamine (Gln). Specifically, GFAT deamidates the terminal nitrogen on Gln and releases glutamate, which can serve as an anaplerotic carbon source for the TCA cycle. Accordingly, GFAT sits at a nexus in the metabolism of the two canonical nutrients used by proliferating cells. Previous work from our group demonstrated that GFAT1 knockdown in mouse PDA cells results in the inhibition of colony formation and decreased growth of subcutaneous xenografts in mice. These results combined with the nearly ubiquitous nature of Kras mutations in pancreatic cancer, strongly indicate that targeting the HBP by inhibition of GFAT could offer a powerful new avenue of treatment for PDA. Utilizing a Cas9/CRISPR approach to modulate GFAT expression in human PDA cells, we have generated cell lines which require the addition of downstream HBP metabolites to maintain cell viability. We have also found several cell lines in which the loss of GFAT expression appears to have no impact on cell viability, suggesting novel mechanisms of glucose and Gln metabolism in these cells. In addition to validating GFAT as a therapeutic option in PDAC, we have also explored the utility of the GFAT reaction as an imaging tool. To accomplish this, we have developed a method to monitor the conversion of Gln to glutamate by magnetic resonance spectroscopy using hyperpolarized glutamine. We further assessed the importance of GFAT in this conversion utilizing our GFAT-/- cell lines. Taken together, these results highlight the benefit of working to understand the complex nature of metabolic rewiring in pancreatic cancer cells, where a further understanding of these pathways may lead to more effective early detection and therapeutic options. Citation Format: Christopher J. Halbrook, Daniel M. Kremer, Lucia Salamanca-Cardona, Kayvan R. Keshari, Costas A. Lyssioitis.{Authors}. GFAT: The crossroads of glucose and glutamine metabolism in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A32.

K-Ras mutation detection in liquid biopsy and tumor tissue as prognostic biomarker in patients with pancreatic cancer: a systematic review with meta-analysis.

K-Ras gene mutations have been found in most pancreatic cancers; however, conflicting data on the prognostic value of K-Ras mutations in pancreatic cancer have been published. We conducted a meta-analysis to assess its prognostic significance. Literature searches of PubMed, EMBASE, Cochrane Library, Web of Science and Google Scholar were performed through December 2015 to identify publications exploring the association of K-Ras mutation with overall survival. Forty eligible studies involving 3427 patients with pancreatic cancer were included in the present meta-analysis. Our analysis showed a hazard ratio (HR) of negative association with survival of 1.61 [95% confidence interval (CI) 1.36-1.90; p<0.01] in K-Ras mutant pancreatic cancer patients. In subgroup analyses, K-Ras mutations detected in tumor tissues and in liquid biopsies had HRs of 1.37 (95% CI 1.20-1.57; p<0.01) and 3.16 (95% CI 2.1-4.71; p<0.01), respectively. In addition, the HR was higher when K-Ras mutations were detected in fresh frozen samples (HR=2.01, 95% CI 1.28-3.16, p=0.002) than in formalin-fixed, paraffin-embedded (FFPE) samples (HR=1.29, 95% CI 1.12-1.49, p<0.01). Though K-Ras alterations are more frequent among non-East Asian individuals than East Asian individuals, there were no significant differences in HRs of survival between the two ethnic subgroups. In conclusion, this meta-analysis suggests that K-Ras mutations are associated with a worse overall survival in pancreatic cancer patients, especially when mutations are detected in liquid biopsies or fresh frozen tumor tissue samples.

Prognostic significance of K-ras mutations in pancreatic cancer: a meta-analysis.

BackgroundK-ras gene mutations are common in patients with pancreatic cancer (PC); however, their prognostic value for PC remains inconclusive. This meta-analysis was performed to quantitatively evaluate the association between K-ras mutations and survival in patients with pancreatic cancer.MethodsWe performed a comprehensive search of electronic sources including MEDLINE (via PubMed), Web of Science, and the Cochrane Library. The search covered a publication period from inception to November 2015.ResultsSeventeen studies with a total of 2249 patients with pancreatic cancer were included in the tissue detection of this study. The meta-analysis indicated a significant association between mutant K-ras genes and overall survival (OS) (HR = 1.51, 95 % CI 1.32–1.72, P < 0.001). Moreover, further subgroup analyses by ethnicity, publication year, therapy method, cancer resectability, and gene detection method all revealed that pancreatic cancer patients with the K-ras mutation had significantly poorer OS (P < 0.05). And results from four studies with 225 patients focused on plasma K-ras mutations enhanced such association (HR = 2.23, 95 % CI 1.69–2.95, P < 0.001).ConclusionsAs a prediction of poor prognosis, the detection of K-ras mutations may be a useful prognostic factor for pancreatic cancer patients.

ERK kinase phosphorylates and destabilizes the tumor suppressor FBW7 in pancreatic cancer.

F-box and WD repeat domain-containing 7 (FBW7) is the substrate recognition component of the Skp1-Cul1-F-box (SCF) ubiquitin ligase complex and functions as a major tumor suppressor by targeting various oncoproteins for degradation. Genomic deletion or mutation of FBW7 has frequently been identified in many human cancers but not in pancreatic ductal adenocarcinoma. Thus it is important to know how the tumor suppressive function of FBW7 is impaired in pancreatic cancer. In this study, we first observed that low FBW7 expression correlated significantly with ERK activation in pancreatic cancer clinical samples, primarily due to KRAS mutations in pancreatic cancer. We further showed that ERK directly interacted with FBW7 and phosphorylated FBW7 at Thr205, which sequentially promoted FBW7 ubiquitination and proteasomal degradation. Furthermore, the phospho-deficient T205A FBW7 mutant is resistant to ERK activation and could significantly suppress pancreatic cancer cell proliferation and tumorigenesis. These results collectively demonstrate how the oncogenic KRAS mutation inhibits the tumor suppressor FBW7, thus revealing an important function of KRAS mutations in promoting pancreatic cancer progression.

Nanoparticle delivery of an AKT/PDK1 inhibitor improves the therapeutic effect in pancreatic cancer.

The K-ras mutation in pancreatic cancer can inhibit drug delivery and increase drug resistance. This is exemplified by the therapeutic effect of PH-427, a small molecule inhibitor of AKT/PDK1, which has shown a good therapeutic effect against a BxPC3 pancreatic cancer model that has K-ras, but has a poor therapeutic effect against a MiaPaCa-2 pancreatic cancer model with mutant K-ras. To increase the therapeutic effect of PH-427 against the MiaPaCa-2 pancreatic cancer model with mutant K-ras, we encapsulated PH-427 into poly(lactic-co-glycolic acid) nanoparticles (PNP) to form drug-loaded PH-427-PNP. PH-427 showed a biphasic release from PH-427-PNP over 30 days during studies in sodium phosphate buffer, and in vitro studies revealed that the PNP was rapidly internalized into MiaPaCa-2 tumor cells, suggesting that PNP can improve PH-427 delivery into cells harboring mutant K-ras. In vivo studies of an orthotopic MiaPaCa-2 pancreatic cancer model showed reduced tumor load with PH-427-PNP as compared with treatment using PH-427 alone or with no treatment. Ex vivo studies confirmed the in vivo results, suggesting that PNP can improve drug delivery to pancreatic cancer harboring mutant K-ras.

Adjusting serum concentrations of organochlorine compounds by lipids and symptoms: A causal framework for the association with K-ras mutations in pancreatic cancer

In clinically aggressive diseases, patients experience pathophysiological changes that often alter concentrations of lipids and environmental lipophilic factors; such changes are related to disease signs and symptoms. The aim of the study was to compare the effects of correcting for total serum lipids (TSL) and other clinical factors on the odds of mutations in the K-ras oncogene by organochlorine compounds (OCs), in logistic models, in 103 patients with exocrine pancreatic cancer (EPC) using a causal directed acyclic graph (DAG) framework. Results and likelihood of bias were discussed in the light of possible causal scenarios. The odds of K-ras mutated EPC was associated with some TSL-corrected OCs, including p,p′-DDT (p-value: 0.008) and polychlorinated biphenyl 138 (p-trend: 0.024). When OCs were not corrected by TSL, the OR of a K-ras mutation was significant for p,p′-DDT (p-trend: 0.035). Additionally adjusting for cholestatic syndrome increased the ORs of TSL-corrected OCs. When models were adjusted by the interval from first symptom to blood extraction (ISE), the ORs increased for both TSL-corrected and uncorrected OCs. Models with TSL-corrected OCs and adjusted for cholestatic syndrome or ISE yielded the highest ORs. We show that DAGs clarify the covariates necessary to minimize bias, and demonstrate scenarios under which adjustment for TSL-corrected OCs and failure to adjust for symptoms or ISE may induce bias. Models with TSL-uncorrected OCs may be biased too, and adjusting by symptoms or ISE may not control such biases. Our findings may have implications as well for studying environmental causes of other clinically aggressive diseases.

535. In Vivo Study of a Bi-Functional Short-Hairpin RNA (bi-shRNA) Targeting Multiple Single-Nucleotide KRAS Mutations in Pancreatic Cancer

535. In Vivo Study of a Bi-Functional Short-Hairpin RNA (bi-shRNA) Targeting Multiple Single-Nucleotide KRAS Mutations in Pancreatic Cancer