Ras Gene Mutations Research Articles

Capturing the primordial Kras mutation initiating urethane carcinogenesis

The environmental carcinogen urethane exhibits a profound specificity for pulmonary tumors driven by an oncogenic Q61L/R mutation in the gene Kras. Similarly, the frequency, isoform, position, and substitution of oncogenic RAS mutations are often unique to human cancers. To elucidate the principles underlying this RAS mutation tropism of urethane, we adapted an error-corrected, high-throughput sequencing approach to detect mutations in murine Ras genes at great sensitivity. This analysis not only captured the initiating Kras mutation days after urethane exposure, but revealed that the sequence specificity of urethane mutagenesis, coupled with transcription and isoform locus, to be major influences on the extreme tropism of this carcinogen.

Short bowel syndrome and severe skin toxicity as a complication of FOLFOX chemotherapy with panitumumab in a patient with colorectal cancer — a case report

The combination of monoclonal antibodies targeting epidermal growth factor receptor (EGFR) with chemotherapy is the standard treatment in advanced colorectal cancer without mutations in the RAS and BRAF genes. We present a case of a 55-year-old female patient with unacceptable skin toxicity and short bowel syndrome caused by palliative FOLFOX chemotherapy combined with panitumumab. In 2012, after the emergence of an artificial anus due to gastrointestinal obstruction in the course of rectal cancer, the patient underwent inductive chemotherapy, preoperative chemoradiotherapy, and radical surgery. Tubular adenocarcinoma G2, ypT2N0 was diagnosed. In 2013 and 2015 she underwent two additional surgeries including intestinal resection due to obstruction of the gastrointestinal tract and enterovaginal fistula. In February 2018 she was qualified for palliative chemotherapy because of inoperable relapse. Due to very good performance status (PS0) and absence of mutations of RAS and BRAF genes, regardless of being underweight and suffering from loose stools persisting from the time of surgery, FOLFOX chemotherapy with panitumumab was introduced. After the second administration of drugs an acne-like rash, hand-foot syndrome, and diarrhoea appeared. Intensification of symptoms and manifestations of short bowel syndrome were observed afterwards. Topical treatment of skin lesions, doxycycline, and anti-diarrhoeal therapy were introduced, with a mediocre therapeutic effect. Imaging confirmed disease stabilisation, but due to the deterioration of both performance status and life quality, anti-cancer treatment was discontinued. This case draws attention to the necessity of caution while qualifying for potentially toxic combination chemotherapy.

Exploiting RAS Nucleotide Cycling as a Strategy for Drugging RAS-Driven Cancers

Oncogenic mutations in RAS genes result in the elevation of cellular active RAS protein levels and increased signal propagation through downstream pathways that drive tumor cell proliferation and survival. These gain-of-function mutations drive over 30% of all human cancers, presenting promising therapeutic potential for RAS inhibitors. However, many have deemed RAS “undruggable” after nearly 40 years of failed drug discovery campaigns aimed at identifying a RAS inhibitor with clinical activity. Here we review RAS nucleotide cycling and the opportunities that RAS biochemistry presents for developing novel RAS inhibitory compounds. Additionally, compounds that have been identified to inhibit RAS by exploiting various aspects of RAS biology and biochemistry will be covered. Our current understanding of the biochemical properties of RAS, along with reports of direct-binding inhibitors, both provide insight on viable strategies for the discovery of novel clinical candidates with RAS inhibitory activity.

Impact and Outcomes of RAS gene Mutations in Core Binding Factor Acute Myeloid Leukemia

Acute myeloid leukemia (AML) with t(8;21) or inv(16) chromosomal rearrangements are distinct heterogeneous disease entities within AML that are classified together as core binding factor AML (CBF-AML). Given the nature of the chromosomes involved, these rearrangements lead to the production of leukemogenic chimeric transcripts (RUNX1-RUNX1T1 and CBFB-MYH11) which disrupt the physiologic activity of the heterodimeric transcription factor CBF complex. Although CBF-AML patients have a favorable prognosis and good response to treatment compared to other AML subtypes, survival outcomes are not uniform. Indeed, 30-50% of patients with CBF-AML eventually relapse, and the 5-10 yr survival ranges between 55-61% for patients < 60 yr in MRC/NCRI AML trials. Studies have analyzed the clonal architecture of CBF-AML patients and identified cooperating mutations independently of receptor tyrosine kinases (FLT3, KIT) mutations while others have found a 30% occurrence of KIT mutations. Studies of murine models of Runx1 and Cbfb have demonstrated that inactivation of both genes does not lead to leukemia, suggesting that other factors are necessary to recapitulate the leukemia phenotype fully. Although mutations in RAS family of genes (NRAS/KRAS) are among the most frequently observed mutations described in CBF-AML [54% in inv(16) and 30% in t(8;21)], no associations between those mutations and survival outcomes have been found. Because of the lack of association between RASMT and clinical outcomes, their role in CBF-AML is still unknown. Here, we focused on dissecting the impact of RAS mutations (NRAS/KRAS; RASMT) on the clinical characteristics, survival outcomes, and the molecular associations among CBF-AML patients by evaluating the clonal succession of RASMT. In total, 284 CBF-AML patients were identified, in whom inv(16) and t(8;21) represent 61% (n=173) and 39% (n=111) of the cases, respectively. Thirty-five % (99/284) of the patients carried RAS mutations (NRAS=78; KRAS=21) with 8 patients harboring 2 mutations comprising NRAS, KRAS, or both NRAS/KRAS genes. RAS mutations were point mutations affecting known hotspots in NRAS and KRAS. Both RASMT and RASWT had a median age < 60 years (55 (14-83) vs. 49 (7-83) years, P=0.9) and sex was equally distributed among the two groups. Leukopenia, defined as white blood cell count <3.5 x 109/L, was the only significant different hematological parameter between RASMT and RASWT. Fewer RASMT patients had leukopenia (3 vs. 12%; P=0.02). Compared to RASWT patients, RASMT patients had more often +8 (14 vs. 6%; P=0.02) and less -Y (7 vs. 16%; P=0.04) abnormalities. The analysis of 30 genes frequently mutated in myeloid malignancies revealed that RASMT patients had a significantly lower number of additional mutations compared to RASWT patients (n=59 vs. 197; P=0.03). Focusing on molecular associations, there were no differences in the frequency of FLT3 (TKD or ITD) mutations between RASMT and RASWT (15 vs. 18%). Although RASWT were more enriched with ASXL1, DNMT3A, IDH1/2, TET2, and WT1 mutations, no significant difference in frequencies was found when compared to RASMT. KIT mutations have been associated with poor outcomes in CBF-AML. In our cohort, KIT mutations (small deletions, points mutations), were significantly less associated with RASMT than RASWT (8 vs. 59%; P<.0001). RASMT were ancestral events in 58% of the cases (with FLT3 mutations the most common secondary lesions), secondary in 38% (with TET2,ASXL1, RUNX1 and IDH2 mutations represented the most common dominant hits), and codominant in 11% (with KIT mutations). RASMT in CBF-AML was associated with worse OS (HR: 1.520; P=0.04) compared to RASWT. Clonal hierarchy and succession analysis showedno change in OS when RASMT were secondary vs. dominant hits in CBF-AML (HR: 0.8; P=0.5). In sum, our study of the frequency, clonal architecture and impact on survival of RASMT in CBF-AML patients points out the unique characteristics of this specific disease subgroup in which sole RASMT might represent, in addition to KITMT, a mutational lesion cooperating with inv(16) and t(8;21) rearrangements in driving leukemic evolution. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Advani:Glycomimetics: Consultancy, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Kite Pharmaceuticals: Consultancy. Nazha:Abbvie: Consultancy; Incyte: Speakers Bureau; Jazz Pharmacutical: Research Funding; Tolero, Karyopharma: Honoraria; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau; Daiichi Sankyo: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

Pediatric myeloid sarcoma: a single institution clinicopathologic and molecular analysis

Myeloid sarcoma (MS) is a neoplastic condition composed of immature myeloid cells involving an extramedullary site. We investigated underlying chromosomal and molecular alterations to assess potential molecular markers of prognosis and outcome in this rare pediatric disease. We conducted a retrospective review of clinicopathologic and cytogenetic data from 33 pediatric patients with MS (ages 1 month–18 years) at our institution over a 32 year period (1984–2016). Tissue-based cancer microarray and targeted next-generation sequencing analysis were performed on six cases. The median age at diagnosis was 2.8 years with a male-to-female ratio of 2.6:1. MS is commonly presented with concomitant marrow involvement (n = 12, 36.4%) or as a recurrence of acute myeloid leukemia (AML; n = 14, 42.4%). The skin (n = 18, 54.5%) and soft tissue (n = 9, 27.3%) were the most common sites of involvement. Twenty-one of 25 samples (84.0%) harbored chromosomal aberrations; KMT2A alterations (n = 10, 40.0%) or complex cytogenetics (n = 7, 28.0%) were most frequent. Mutations in RAS, tyrosine kinase, cell signaling, and chromatin remodeling genes were detected. When compared to pediatric patients with AML without extramedullary involvement (EMI), inferior overall survival (OS) was observed (18.8 months vs. 89.3 months, p = .008). Pediatric patients with MS with non-favorable cytogenetics [abnormalities other than t(8;21), inv(16)/t(16;16), or t(15;17)] had a significantly lower OS compared to patients with AML with non-favorable cytogenetics and no extramedullary involvement (8.0 months vs. 28.1 months, p < .001). Pediatric MS is a rare disease with diverse clinical presentations. Non-favorable cytogenetics may be a poor prognostic marker for pediatric patients with MS and molecular diagnostics can assist with risk stratification and identify potentially actionable targets.

Mutations of RAS genes in endometrial polyps.

Endometrial polyps are common, yet the molecular mechanisms underlying their formation and progression remain unclear. We examined gene mutations possibly related to the pathogenesis of endometrial polyps, as well as to their clinical features. Four premenopausal patients with endometrial polyps, who were not under drug treatment, were recruited. Whole exomes of endometrial polyps and peripheral blood lymphocytes were analyzed by next-generation sequencing, and somatic mutations were derived by subtraction. Then, 35 samples of endometrial polyps and 12 samples of atypical polypoid adenomyoma were newly recruited to validate the identified mutations by polymerase chain reaction-reverse sequence specific oligonucleotide method. The mutations were also analyzed in separate stromal and glandular components of the polyps after laser-capture microdissection. Whole exome sequencing revealed that KRAS mutations were the only type of mutation detectable in multiple cases (2/4). Targeted mutation analysis revealed that 16 of 35 samples (45.7%) of endometrial polyps harbored RAS mutations. Mutation-positive cases exhibited a significantly higher number of endometrial polyps (3.25±2.70 vs. 1.74±0.87, P=0.045). Laser-capture microdissection in NRAS-mutated endometrial polyps revealed that both stromal and glandular components harbored RAS mutations. There was no RAS mutation in 12 samples of atypical polypoid adenomyoma. This is the first report demonstrating that pathogenic RAS mutations are frequent in non-treated endometrial polyps. RAS mutations may have an important role in tumorigenesis and in the formation of multiple endometrial polyps.

729P - Genomic profiling in Chinese biliary tract cancer patients with PI3K/AKT/mTOR pathway and RAS gene mutations

BackgroundBiliary tract cancers (BTCs) are a group of relatively rare invasive carcinomas including gallbladder carcinoma (GBC), intrahepatic (ICC), hilar (HCCA) and extrahepatic (ECC) cholangiocarcinoma. Clinical trials of PI3K/AKT/mTOR inhibitors have revealed PI3K and PTEN mutations as effective biomarkers. This study aimed to analyze the gene mutations involved in this pathway and other gene mutations in Chinese BTC patients and to analyze the profiling characteristics of each subtype. MethodsA total of 661 (125 ECC, 159 GBC, 47 HCCA, and 330 ICC) BTC patients were included in this study. FFPE and matching blood samples from these patients were collected and sequenced using next-generation sequencing targeting 450 cancer genes. Genomic alterations including single nucleotide variants, insertions, and deletions, copy number variations and fusions were assessed. ResultsA total of 17.9% (118/661) of patients were identified harboring PI3K/AKT/mTOR pathway mutations. In preclinical data, RAS gene (KRAS/HRAS/NRAS) mutations might lead to reduced sensitivity to mTOR inhibitors. There was 11.8% (39/330) in ICC, 7.2% (9/125) in ECC, 12.8% (6/47) in HCCA and 25.2% (40/159) in GBC detected with PI3K pathway mutations and RAS wildtype, where the GBC proportion was statistically significantly higher than any other subtypes. A total of 3.6% (24/661) of patients presented co-occurrence of RAS and PI3K pathway mutations, but this was not found in HCCA. The percentages of co-occurrence in ICC, ECC and GBC were 3.6% (12/330), 4.0% (5/125) and 4.4% (7/159), respectively. The top 3 mutated genes in the PI3K/AKT/mTOR pathway were PIK3CA at 45.8% (54/118), PTEN at 22% (26/118) and TSC2 at 9.3% (11/118). PIK3CA mutations and amplification were discovered in 52 patients and 2 patients, respectively, including 23 ICC and 23 GBC patients, the most frequent being E542K (12/52) and52) mutations./52) mutations. ConclusionsIn Chinese BTC patients, the incidence (3.6%) of RAS and PI3K pathway mutation’s co-occurrence was generally low. Among BTC subtypes, GBC patients had the highest mutation proportion (25.2%) involved in the PI3K/AKT/mTOR pathway with wildtype RAS. Legal entity responsible for the studyDan Liu. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Molecular mechanism of the association and dissociation of Deltarasin from the heterodimeric KRas4B‐PDEδ complex

The formation of the KRas4B-PDEδ complex activates different signaling pathways required for the development and maintenance of cancer. Previous experimental and theoretical studies have allowed researchers to design an inhibitor of the KRas4B-PDEδ complex, "Deltarasin." This inhibitor binds to the prenyl-binding pocket of PDEδ and subsequently inhibits the proliferation of human pancreatic ductal adenocarcinoma cells that depend on oncogenic KRas4B. Nevertheless, structural and energetic information about the inhibitory effects of Deltarasin on the KRas4B-PDEδ complex are not available. In this study, we explore the properties of Deltarasin in inhibiting the formation of wild-type and mutant KRas4B-PDEδ complexes present in different cell lines expressing mutant RAS genes (G12D, G12C, G12V, G13D, Q61L, and Q61R) using 1.7 μs molecular dynamics (MD) simulations in combination with the MMGBSA approach. Our results revealed the energetic and structural mechanisms that suggest a higher affinity of Deltarasin for PDEδ than the farnesylated HVR. Moreover, Deltarasin exerts another dissociative effect by binding to the protein-protein dimeric interface of wild-type KRas4B-PDEδ, whereas associative and dissociative effects were observed for mutant KRas4B-PDEδ, providing a mechanistic explanation for the inhibitory effects of Deltarasin on different cancer cell lines.

Positive Correlation Between Somatic Mutations in RAS Gene and Colorectal Cancer in Telangana Population: Hospital-Based Study in a Cosmopolitan City.

Colorectal cancer (CRC) ranks among the most prevalent cancer types in both men and women. Screening of RAS (Kirsten rat sarcoma viral oncogene homolog (KRAS), neuro-blastoma RAS viral oncogene homolog (NRAS), and v-raf murine sarcoma viral oncogene homolog B1 (BRAF)) somatic mutations is necessary prior to considering anti-epidermal growth factor receptor (EGFR) therapies in CRC patients. Next-generation sequencing studies have confirmed that RAS gene panels could be used while developing treatment strategies for patients with CRC. The present study explored genetic mutations in KRAS, NRAS, and BRAF in CRC patients in the Telangana state of India. Patients with confirmed CRC (n = 100) who visited the Apollo hospitals were evaluated. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissues, and pyrosequencing analysis was performed. Patient DNA samples were screened for 54 different KRAS, NRAS, and BRAF mutations, which revealed 34 somatic mutations. Exon 11 of BRAF possessed 4 mutations with highest individuals documented with G469A mutation. Pyrosequencing, a reliable method for analyzing somatic mutations present in RAS, could aid in taking treatment decisions for patients with CRC.

The Function of RAS Mutation in Cancer and Advances in its Drug Research.

RAS (H-ras, K-ras, and N-ras), as the second largest mutated gene driver in various human cancers, has long been a vital research target for cancer. Its function is to transform the extracellular environment into a cascade of intracellular signal transduction. RAS mutant protein regulates tumor cell proliferation, apoptosis, metabolism and angiogenesis through downstream MAPK, PI3K and other signaling pathways. In KRAS or other RAS-driven cancers, current treatments include direct inhibitors and upstream/downstream signaling pathway inhibitors. However, the research on these inhibitors has been largely restricted due to their escape inhibition and off-target toxicity. In this paper, we started with the role of normal and mutant RAS genes in cancer, elucidated the relevant RAS regulating pathways, and highlighted the important research advancements in RAS inhibitor research. We concluded that for the crosstalk between RAS pathways, the effect of single regulation may be limited, and the multi-target drug combined compensation mechanism is becoming a research hotspot.

Combination chemotherapy with TAS-102 plus bevacizumab in salvage-line treatment of metastatic colorectal cancer: A single-center, retrospective study examining the prognostic value of the modified Glasgow Prognostic Score in salvage-line therapy of metastatic colorectal cancer.

The combination regimen of TAS-102, a novel oral nucleoside antitumor agent containing trifluridine and tipiracil hydrochloride, with bevacizumab (C-TASK FORCE), a selective monoclonal antibody inhibitor of vascular endothelial growth factor-A, as salvage-line therapy for metastatic colorectal cancer (mCRC) was established based on its high clinical effectiveness. The aim of the present study was to evaluate the prognostic accuracy of the modified Glasgow Prognostic Score (mGPS) in patients receiving TAS-102 plus bevacizumab. The study included 17 patients (12 men and 5 women, mean age 60.4±13.4 years) with unresectable mCRC who were confirmed to have wild-type or mutant RAS genes. The patients received salvage-line treatment with TAS-102 plus bevacizumab at the Surgical Oncology Department of Gifu University School of Medicine between March 2016 and August 2018. The study population was heavily pretreated; the majority of the patients (71%) had received ≥4 prior regimens and, in addition to fluoropyrimidine, irinotecan and oxaliplatin, all had received bevacizumab (100%) and either cetuximab or panitumumab (47%). The RAS status was wild-type in 9 (53%) and mutant in 8 (47%) patients. The primary tumor locations included the right-sided colon in 5 patients (29%; cecum in 2 and transverse colon in 3 cases) and left-sided colorectum in 12 patients [71%; sigmoid colon in 4, rectosigmoid (Rs) in 4, and rectum above/below the peritoneal reflection (Ra/b) in 4 cases]. Metastatic sites included the liver in 15 (88%), lung in 13 (76%), lymph nodes in 7 (41%), and peritoneal dissemination in 5 (24%) patients. The number of metastatic sites was 1 in 3 (18%) and >2 in 14 (82%) patients. Their first staging imaging scans (after 2 cycles of therapy) were available for review in all 17 patients. At first evaluation, 5 (29%) patients had progressive disease (PD), 12 (71%) had stable disease, and none had a partial response to TAS-102 plus bevacizumab. The median overall survival (OS) of 14.1 months and progression-free survival (PFS) of 6.8 months were comparable to the 11.2 and 5.6 months, respectively, in the C-TASK FORCE study. Upon considering three groups, namely mGPS 0, mGPS 1 and mGPS 2, the median PFS times were significantly different (mGPS 0 vs. mGPS 2, P=0.02; and mGPS 1 vs. mGPS 2, P=0.06). The median PFS times in the mGPS 0, 1 and 2 groups were 12.1, 4.8 and 2.3 months, respectively. Median OS was also significantly different (mGPS 0 vs. mGPS 2, P=0.01; and mGPS 1 vs. mGPS 2, P=0.04). The median OS times in the mGPS 0, 1 and 2 groups were 14.0, not reached, and 2 months, respectively. The present study demonstrated the efficacy and safety of the TAS-102 plus bevacizumab combination as salvage-line treatment. This combination therapy (the TAS-102 plus bevacizumab) has obtained valid results with PFS OS as well as C-TASK.FORCE study. The results of the present study also confirmed the prognostic accuracy of mGPS in salvage-line treatment of patients with mCRC.

Abstract 345: Novel RAS inhibitor, MCI-062, potently and selectively inhibits the growth of KRAS mutant pancreatic tumor cells by blocking GTP loading of RAS

Abstract Oncogenic mutations in RAS genes result in an intracellular elevation of active RAS and increased cellular propagation through downstream signaling pathways responsible for tumor cell growth and survival. Mutations in KRAS drive over 90% of pancreatic ductal adenocarcinomas (PDACs), presenting promising therapeutic potential for a RAS inhibitor. However, many have deemed RAS “undruggable” as a result of its relatively smooth structure and its high affinity for guanine nucleotide substrates. We have identified a novel series of indene derivatives that potently and selectively inhibits the growth of tumor cells with high levels of active RAS, while having minimal effects on tumor cells lacking constitutively active RAS, or cells derived from normal tissues. Here we report that our lead compound, MCI-062, potently and selectively inhibits the growth of KRAS mutant MIA PaCa-2 PDAC cells harboring oncogenic KRAS with an IC50 value of approximately 5 nM and greater than 300-fold selectivity over BxPC-3 PDAC cells that lack constitutively active RAS. MCI-062 also completely inhibits colony formation of a panel of PDAC cell lines with various KRAS mutations at low nanomolar concentrations. MCI-062 treatment of MIA PaCa-2 cells in basal and EGF-stimulated conditions depletes RAS-GTP levels in a dose-dependent manner. Consequently, MCI-062 treatment leads to inhibition of downstream MAPK and AKT signaling, cell cycle arrest, and induction of apoptosis in MIA PaCa-2 cells. Further investigation of the mechanism of action in cell-free systems demonstrated that this class of compounds inhibits GTP loading of RAS. MCI-062 inhibits binding of MANT-GTP to recombinant K-RAS in a dose-dependent manner. In a functional assay, MCI-062 reduces RAS-RAF-RBD binding when recombinant K-RAS is treated in a nucleotide-free state, but not when K-RAS is treated in a nucleotide-bound state. Testing in 3D spheroid models involving MIA PaCA-2 tumor cells revealed that MCI-062 potently inhibits growth of non-adherent cells. MCI-062 also has anti-tumor activity in a KRAS mutant CT26 mouse tumor xenograft model with no discernable toxicity. Western blotting of tumor lysates from mice treated with MCI-062 showed a reduction of active, GTP-bound RAS when compared to tumor lysates from control mice. These results provide in vitro and in vivo evidence that MCI-062 inhibits RAS-driven tumor cell growth by blocking GTP loading of RAS, supporting further evaluation of this novel class of RAS inhibitors for the treatment of pancreatic cancer, as well as other RAS-driven cancers. Funding provided by NCI grants R01CA131378, R01CA148817, R01CA197147, and R01CA155638. Citation Format: Tyler E. Mattox, Xi Chen, Jacob Valiyaveettil, Yulia Maxuitenko, Bing Zhu, Antonio Ward, Veronica Ramirez-Alcantara, Kristy Berry, Michael Boyd, Adam Keeton, Gary A. Piazza. Novel RAS inhibitor, MCI-062, potently and selectively inhibits the growth of KRAS mutant pancreatic tumor cells by blocking GTP loading of RAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 345.

Abstract 1705: Genetic analysis of thymoma and thymic carcinoma

Abstract [Background] Thymic epidermal tumors (thymoma and thymic cancer) are rare, and its genetic profiles are unclear. Molecular targeted therapy of thymic tumors has not been established. Clarifying a molecular alteration and to identify the novel targets for molecular targeted therapy is important. So, we conducted the next generation sequencing (NGS) analysis with surgically resected thymic tumor tissues. [Methods] From July 2013 to October 2018, thirty patients who were resected thymic epidermal tumors in our hospital were enrolled in this study. We got written informed consent from patients, and NGS analysis of extracted DNA sample was performed in 24 patients to the present time. DNA were extracted from fresh frozen surgically resected tissues (tumors and paired normal tissues) and DNA amplicon sequencing was performed with a custom panel of 53 cancer-related genes based on Ion AmpliSeq™ Cancer Hotspot Panel v2 comprising major oncogenes and tumor suppressor genes (including GTF2I). Sequencing was carried out with an Ion Torrent PGM™. Sequencing data were analyzed using Ion Reporter™. In addition, we collected the patient’s information (age, sex, Masaoka-Koga staging, World Health Organization (WHO) histologic classification, tumor size, and complication such as myasthenia gravis and pure red cell aplasia) from medical records. The study was approved by the Institutional Review Board (IRB) of the Nagasaki University Hospital. [Results] A total of 30 patients were enrolled in this study. The median age was 62 years old (range, 34-84 years old), patients characteristics were 9 male and 21 female, WHO histologic classification (type A, AB, B1, B2, B3, thymic carcinoma) was 1, 11, 4, 10, 2, 2, and Masaoka-Koga staging I, II, III, IV were 12, 6, 9, 3, respectively. Median tumor size was 47mm (range 11-110 mm). Moreover, complications were 7 myasthenia gravis, 4 acetylcholine receptor antibody-positive (do not confirm diagnossis of myasthenia gravis), 1 pure red cell aplasia, and 1 agranulocytosis. In NGS sequencing, the nonsynonymous mutations of HRAS and NRAS (HRAS Q61R, HRAS G13R, and NRAS Q61K) was detected in three patients. These RAS gene mutations were reported to be pathogenic in various malignancies. Low frequently DNMT3A mutation was detected in the other two patients, however this mutation were not validated in other methods. No genetic alterations were detected in the rest 19 patients. [Conclusion] The frequency of genetic alterations in thymic epidermal tumors was very low in this study, however, active mutations of RAS oncogene were detected in three patients. Although the RAS are still not established as treatment targets, it should be one of the interacting molecules. Further investigation is required to establish new therapeutic strategy according to the genetic alteration in thymic tumors. Citation Format: Hiroyuki Yamaguchi, Hiroshi Gyotoku, Hirokazu Taniguchi, Daisuke Sasaki, Midori Shimada, Yosuke Dotsu, Hiroaki Senju, Norihito Kaku, Takaya Ikeda, Minoru Fukuda, Katsunori Yanagihara, Hiroshi Mukae. Genetic analysis of thymoma and thymic carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1705.

Abstract 1262: Relationship between RAS, BRAF, PIK3CA and location of primary tumor in urothelial carcinoma

Abstract Purpose: In urothelial carcinoma (UC), somatic mutations in RAS, BRAF, and PIK3CA genes are suggested for prognostic markers as well as for therapeutic targets. Studies have reported that upper tract (renal pelvis and ureter) and bladder UC differ in factors including prognosis and genetic features. Methods: We investigated the difference in clinicopathological features and oncogenic mutations (RAS, BRAF, and PIK3CA) according to the location of primary disease. In a prospective biomarker study in patients with metastatic or unresectable UC, tumor DNA was analyzed for mutations in KRAS, NRAS, HRAS, BRAF, and PIK3CAusing Ampliseq v2 platform. We further stratified the frequency of the mutations by the location of primary tumor. Results: Among a total of 210 patients, those with upper tract UC and bladder UC were 90 and 120 patients, respectively. Missense mutations in KRAS, NRAS, HRAS, BRAF, and PIK3CA were present in 9 (4.3%), 1 (0.5%), 18 (8.6%), 4 (1.9%), and 32 (15.2%) patients, respectively. PIK3CA mutations were detected in 16 patients with upper tract UC (17.8%) and 16 patients with bladder UC (13.3%). Likewise, HRAS mutations were detected more frequently in upper tract UC (10/90, 11.1%) than bladder UC (8/120, 6.7%). In metastatic UC setting, PIK3CA or HRAS mutations were not associated with different overall survival. Conclusions: PIK3CA and HRAS mutations were more frequently observed in patients with upper tract UC compared to those with bladder UC. The different genetic features need to be studied by further studies. Note: This abstract was not presented at the meeting. Citation Format: Young Saing Kim, Sang-Cheol Lee, In Gyu Hwang, Su Jin Lee, Se Hoon Park. Relationship between RAS, BRAF, PIK3CA and location of primary tumor in urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1262.

RAS mutations in human cancers: Roles in precision medicine

Ras proteins play a crucial role as a central component of the cellular networks controlling a variety of signaling pathways that regulate growth, proliferation, survival, differentiation, adhesion, cytoskeletal rearrangements and motility of a cell. Almost, 4 decades passed since Ras research was started and ras genes were originally discovered as retroviral oncogenes. Later on, mutations of the human RAS genes were linked to tumorigenesis. Genetic analyses found that RAS is one of the most deregulated oncogenes in human cancers. In this review, we summarize the pioneering works which allowed the discovery of RAS oncogenes, the finding of frequent mutations of RAS in various human cancers, the role of these mutations in tumorigenesis and mutation-activated signaling networks. We further describe the importance of RAS mutations in personalized or precision medicine particularly in molecular targeted therapy, as well as their use as diagnostic and prognostic markers as therapeutic determinants in human cancers.

Neurodevelopmental Aspects of RASopathies.

RAS gene mutations are frequently found in one third of human cancers. Affecting approximately 1 in 1,000 newborns, germline and somatic gain-of-function mutations in the components of RAS/mitogen-activated protein kinase (RAS/MAPK) pathway has been shown to cause developmental disorders, known as RASopathies. Since RAS-MAPK pathway plays essential roles in proliferation, differentiation and migration involving developmental processes, individuals with RASopathies show abnormalities in various organ systems including central nervous system. The frequently seen neurological defects are developmental delay, macrocephaly, seizures, neurocognitive deficits, and structural malformations. Some of the defects stemmed from dysregulation of molecular and cellular processes affecting early neurodevelopmental processes. In this review, we will discuss the implications of RAS-MAPK pathway components in neurodevelopmental processes and pathogenesis of RASopathies.

Somatic genetic alterations in a large cohort of pediatric thyroid nodules.

There is a rise in the incidence of thyroid nodules in pediatric patients. Most of them are benign tissues, but part of them can cause papillary thyroid cancer (PTC). The aim of this study was to detect the mutations in commonly investigated genes as well as in novel PTC-causing genes in thyroid nodules and to correlate the found mutations with clinical and pathological data. The cohort of 113 pediatric samples consisted of 30 benign lesions and 83 PTCs. DNA from samples was used for next-generation sequencing to identify mutations in the following genes: HRAS, KRAS, NRAS, BRAF, IDH1, CHEK2, PPM1D, EIF1AX, EZH1 and for capillary sequencing in case of the TERT promoter. RNA was used for real-time PCR to detect RET/PTC1 and RET/PTC3 rearrangements. Total detection rate of mutations was 5/30 in benign tissues and 35/83 in PTCs. Mutations in RAS genes (HRAS G13R, KRAS G12D, KRAS Q61R, NRAS Q61R) were detected in benign lesions and HRAS Q61R and NRAS Q61K mutations in PTCs. The RET/PTC rearrangement was identified in 18/83 of PTCs and was significantly associated with higher frequency of local and distant metastases. The BRAF V600E mutation was identified in 15/83 of PTCs and significantly correlated with higher age of patients and classical variant of PTC. Germline variants in the genes IDH1, CHEK2 and PPM1D were found. In conclusion, RET/PTC rearrangements and BRAF mutations were associated with different clinical and histopathological features of pediatric PTC. RAS mutations were detected with high frequency in patients with benign nodules; thus, our results suggest that these patients should be followed up intensively.

Multi-target, ensemble-based virtual screening yields novel allosteric KRAS inhibitors at high success rate.

RAS mutations account for >15% of all human tumors, and of these ~85% are due to mutations in a particular RAS gene: KRAS. Recent studies revealed that KRAS harbors four druggable allosteric sites. Here, we have (a) used molecular simulations to generate ensembles of wild type and four major oncogenic KRAS mutants (G12V, G12D, G13D, and Q61H); (b) characterized the druggability of each allosteric pocket in each protein; (c) conducted extensive ensemble-based virtual screening using pocket-tailored ligand libraries; (d) prioritized hits through hierarchical postdocking analysis; and (e) validated predicted hits with NMR. Of the 785 diverse potential hits identified by our in silico analysis, we tested 90 for their ability to bind KRAS using NMR and found that nine cause backbone amide chemical shift perturbations of residues near the functionally responsive switch loops, suggesting potential binding. We conducted detailed biophysical analyses on a novel indole-based compound to demonstrate the potential of our workflow to yield lead compounds. We believe the detailed information documented in this work regarding the druggability profile of each allosteric site and the chemical fingerprints of compounds that target them will serve as vital resources for future structure-based drug design efforts against KRAS, a high-value target for cancer therapy.

Rigosertib ameliorates the effects of oncogenic KRAS signaling in a murine model of myeloproliferative neoplasia.

Aberrant signaling triggered by oncogenic or hyperactive RAS proteins contributes to the malignant phenotypes in a significant percentage of myeloid malignancies. Of these, juvenile myelomonocytic leukemia (JMML), an aggressive childhood cancer, is largely driven by mutations in RAS genes and those that encode regulators of these proteins. The Mx1-cre kras+/G12D mouse model mirrors several key features of this disease and has been used extensively to determine the utility and mechanism of small molecule therapeutics in the context of RAS-driven myeloproliferative disorders. Treatment of disease-bearing KRASG12D mice with rigosertib (RGS), a small molecule RAS mimetic that is in phase II and III clinical trials for MDS and AML, decreased the severity of leukocytosis and splenomegaly and extended their survival. RGS also increased the frequency of HSCs and rebalanced the ratios of myeloid progenitors. Further analysis of KRASG12D HSPCs in vitro revealed that RGS suppressed hyperproliferation in response to GM-CSF and inhibited the phosphorylation of key RAS effectors. Together, these data suggest that RGS might be of clinical benefit in RAS-driven myeloid disorders.

Molecular biomarkers and histological parameters impact on survival and response to first- line systemic therapy of metastatic colorectal cancer patients.

BackgroundHistological parameters of primary tumour and nodal metastases are prognostic factors for survival of operable colorectal (CRC) patients, but not predictive for response rate of systemic therapy. KRAS mutations in codons 12 and 13 were first recognized as a predictive factor for resistance to anti-EGFR monoclonal antibodies. Not all patients with wild-type KRAS (wtKRAS) respond to anti-EGFR antibody treatment. Additional mechanisms of resistance may activate mutations of the other main EGFR effectors pathway, such as other mutations in RAS gene, mutations in P13K and PTEN expression.Patients and methodsIn the prospective study prognostic and predictive impact of histological parameters of primary tumour, KRAS and BRAF mutations on overall survival (OS) and objective response (OR) rate of metastatic CRC (mCRC) patients treated with 1st line systemic therapy were analysed. We additionally retrospectively analysed other mutations in RAS genes and their impact on survival and time to progression.ResultsFrom November 2010 to December 2012, we enrolled 154 patients in the study, 95 men and 59 women. Mutations in KRAS gene and V600E BRAF gene were found in 42% and in 3% of patients, respectively. Median OS of the patients with T1, T2 and T3 tumour was 65.4 months (95% CI, 55.7–75.6) while in patients with T4 tumour, lymphangiosis, vascular and perineural invasion it has not been reached yet. Median OS of the patients with G1, G2 and G3 of tumour differentiation was 65.6 (95% CI, 53.7–77.5) and 25.3 months (95% CI, 16.6–34.1), respectively. Median OS of the patients with stage N0, N1 and N2 was 65.6 (95% CI, 56.4–74.8) and 58.0 months (95% CI, 21.9–94.2), respectively. Median OS of wtKRAS and mutated KRAS patients was 56.5 (95% CI, 48.2–64.9) and 58 months (95% CI, 52.6–63.4), respectively. Median OS of mutated codon 12 and codon 13 patients was 57 (95% CI, 50.9–64.4) and 44 months (95% CI, 40.1–48.4), respectively. Median OS of wtBRAF and of mutated BRAF patients was 59.2 (95% CI, 52.5–65.9) and 27.6 months (95% CI, 12.6–42.5), respectively. wtKRAS significantly affected the response to the first systemic therapy (p = 0.028), while other parameters did not affected it, p= 0.07. In 14 patients (17%), additional mutations in NRAS gene, codon 61 and codon 146 were found. Median OS of wtNRAS, codon 61 and 146 patients was 67.1 months (50.3–67.6) while median OS of mutated NRAS patients has not been reached yet (p = 0.072). Median time to progression of wtNRAS, codon 61 and 146 patients was 11.7 months (10.4–14.5) while median time to progression of mutated NRAS patients was 7.9 months (6.1–11.0), (p = 0.025).ConclusionsMutated BRAF, N2 and G3 of primary tumour were poor prognostic factors for OS in mCRC patients. wtKRAS significantly affected the response to the first line systemic therapy. Histological parameters included in the analysis and mutated BRAF did not affect significantly the efficacy of 1st line systemic therapy in mCRC patients.