KRAS G12A Research Articles

The Genomic Landscape of a Restricted ALL Cohort from Patients Residing on the U.S./Mexico Border.

Next-generation sequencing (NGS) has identified unique biomarkers yielding new strategies in precision medicine for the treatment of Acute lymphoblastic leukemia (ALL). Hispanics show marked health disparities in ALL, often absent in clinical trials or cancer research. Thus, it is unknown whether Hispanics would benefit equally from curated data currently guiding precision oncology. Using whole-exome sequencing, nine ALL patients were screened for mutations within genes known to possess diagnostic, prognostic and therapeutic value. Genes mutated in Hispanic ALL patients from the borderland were mined for potentially pathogenic variants within clinically relevant genes. KRAS G12A was detected in this unique cohort and its frequency in Hispanics from the TARGET-ALL Phase II database was three-fold greater than that of non-Hispanics. STAT5B N642H was also detected with low frequency in Hispanic and non-Hispanic individuals within TARGET. Its detection within this small cohort may reflect a common event in this demographic. Such variants occurring in the MAPK and JAK/STAT pathways may be contributing to Hispanic health disparities in ALL. Notable variants in ROS1, WT1, and NOTCH2 were observed in the ALL borderland cohort, with NOTCH2 C19W occurring most frequently. Further investigations on the pathogenicity of these variants are needed to assess their relevance in ALL.

SO-14 The prognostic impact of KRAS G12C mutation in patients with metastatic colorectal cancer: A multicenter retrospective observational study

KRAS is one of the most frequently mutated oncogenes in colorectal cancer (CRC). Recently, a novel therapy targeting KRAS G12C mutation has demonstrated promising activities for corresponding advanced solid tumors including metastatic CRC (mCRC). However, the prognostic impact of the KRAS G12C mutation remains unclear in patients with mCRC. Therefore, the aim of this study was to evaluate the prognostic impact of KRAS G12C mutation in mCRC in a large cohort of real-world data. We retrospectively reviewed medical records of patients with mCRC who received the first-line chemotherapy between January 2005 and December 2017 at four large oncology facilities in Japan. Patient characteristics and survival outcomes were compared between the patients with KRAS G12C and non-G12C mutations. Among 2,457 patients with mCRC, a total of 1,632 patients met selection criteria. Of these, 696 patients had KRAS exon 2 mutations. In terms of the distribution of KRAS exon 2 mutations, the following six mutations accounted for the majority of them (% of all population [n = 1632]): KRAS G12D (16.0%, n = 261), G13D (9.8%, n = 160), G12V (9.3%, n = 151), G12C (2.8%, n = 45), G12S (2.2%, n = 36), and G12A (1.9%, n = 31). Patient characteristics were not significantly different between patients with the KRAS G12C and non-G12C mutations. At a median follow-up of 64.8 months, patients with the KRAS G12C mutation showed significantly shorter first-line progression-free survival (PFS; median, 9.4 vs. 10.8 months, hazard ratio [HR]: 1.47, 95% confidence interval [CI] 1.08-2.01; P = 0.015) and overall survival (OS; median, 21.1 vs. 27.3 months, HR: 1.50, 95% CI 1.08-2.08, P = 0.015) than those with non-G12C mutations. Multivariate analysis also showed that KRAS G12C mutations were significantly associated with shorter first-line PFS (adjusted HR, 1.43; 95% confidence interval [CI], 1.04–1.96, P=0.030) and OS (adjusted HR, 1.42; 95% CI, 1.01–2.00, P=0.044). We demonstrated that KRAS G12C mutation was significantly correlated with shorter first-line PFS and OS compared to non-G12C mutations. These findings indicate the importance of a stratified treatment targeting KRAS G12C mutation in mCRC.

BMP4 and PHLDA1 are plausible drug-targetable candidate genes for KRAS G12A-, G12D-, and G12V-driven colorectal cancer

Despite the frequent detection of KRAS driver mutations in patients with colorectal cancer (CRC), no effective treatments that target mutant KRAS proteins have been introduced into clinical practice. In this study, we identified potential effector molecules, based on differences in gene expression between CRC patients carrying wild-type KRAS (n = 390) and those carrying KRAS mutations in codon 12 (n = 240). CRC patients with wild-type KRAS harboring mutations in HRAS, NRAS, PIK3CA, PIK3CD, PIK3CG, RALGDS, BRAF, or ARAF were excluded from the analysis. At least 11 promising candidate molecules showed greater than two-fold change between the KRAS G12 mutant and wild-type and had a Benjamini-Hochberg-adjusted P value of less than 1E-08, evidence of significantly differential expression between these two groups. Among these 11 genes examined in cell lines transfected with KRAS G12 mutants, BMP4, PHLDA1, and GJB5 showed significantly higher expression level in KRAS G12A, G12D, and G12V transfected cells than in the wild-type transfected cells. We expect that this study will lead to the development of novel treatments that target signaling molecules functioning with KRAS G12-driven CRC.

K-Ras Peptide Mimotope Induces Antigen Specific Th1 and B-Cell Immune Responses against G12A-Mutated K-Ras Antigen in Balb/c Mice.

KRAS G12A somatic point mutation in adenocarcinomas is categorized clinically as ineligibility criteria for anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies. In this study, a modified G12A-K-ras epitope (139A) with sequence-specific modifications to improve immunogenicity was developed as a potential vaccine against G12A-mutant KRAS cancers. Additionally, coupling of the 139A epitope with a tetanus toxoid (TTD) universal T-cell epitope to improve antigenicity was also reported. To facilitate convenient oral administration, Lactococcus lactis, which possesses innate immunomodulatory properties, was chosen as a live gastrointestinal delivery vehicle. Recombinant L. lactis strains secreting a G12A mutated K-ras control and 139A with and without TTD fusion were generated for comparative immunogenicity assessment. BALB/c mice were immunized orally, and high survivability of L. lactis passage through the gastrointestinal tract was observed. Elevations in B-cell count with a concomitant titre of antigen-specific IgG and interferon-γ secreting T-cells were observed in the 139A treated mice group. Interestingly, an even higher antigen-specific IgA response and interferon-γ secreting T-cell counts were observed in 139A-TTD mice group upon re-stimulation with the G12A mutated K-ras antigen. Collectively, these results indicated that an antigen-specific immune response was successfully stimulated by 139A-TTD vaccine, and a TTD fusion was successful in further enhancing the immune responses.

Impact of KRAS alterations in localized pancreatic cancer (PC).

431 Background: Patients (pts) with localized PC do not routinely undergo comprehensive genomic profiling (CGP) unless they develop recurrent or metastatic disease. KRAS is the most frequently mutated gene in PC, however, the impact of different KRAS mutations in localized PC has not been well characterized. We interrogated our genomic database to analyze the KRAS status in PC pts who presented with localized disease at diagnosis (Dx). Methods: We identified PC pts at our institution who underwent CGP utilizing the Foundation One CDx assay and had localized disease at initial Dx; these pts were categorized into resectable/borderline resectable PC (LPC) and locally advanced PC (LAPC). All pts with LPC and LAPC underwent neoadjuvant chemotherapy and chemoradiation prior to possible surgery (all intended therapy - AIT). Tissue from metastatic sites was used for CGP in pts who developed recurrent/metastatic disease before or after completion of AIT. The primary tumor was used for CGP in pts who completed AIT without subsequent relapse or in the absence of adequate metastatic tissue. Effect of each gene on response and survival outcomes was estimated using proportional odds and Cox regression analysis, respectively, adjusting for stage. Results: 75 pts were identified, median age at Dx was 65 years, 59% were male, 65% had a primary tumor in the pancreatic head. 38 (86%) pts with LPC completed AIT compared to 21 (68%) pts with LAPC (p<0.001). KRAS mutation was detected in 95% (71/75) of pts– 94% (67/71) in codon 12 and 6% (4/71) in codon 61. The various KRAS mutations and their association with completion of AIT is summarized in the table. The likelihood of completing AIT did not differ based on KRAS wildtype (WT) vs mutated status (p =1.00), the mutated codon (codon 12 vs. codon 61; p =1.00) or the individual KRAS point mutations (p = 0.7); however, all patients with G12A (N= 1), G12C (N=1), G12L (N=1) and G12R (N=11) mutations completed AIT. KRAS status (mutated vs. WT) and the individual KRAS mutations were not associated with overall survival (OS) after adjusting for stage (p= 0.13 and p = 0.26 respectively). Median OS for patients with LPC and LAPC, was 39 months (mos) and 29 mos respectively. Conclusions: KRAS status and individual KRAS mutations did not have an impact on completing AIT or mOS; however, these findings need to be interpreted with caution due to the inherent biases involved in such analyses. The clinical significance and functional relevance of KRAS G12A, G12C, G12L and G12R mutations, though relatively rare, needs further characterization as well as mechanistic elucidation. [Table: see text]

Abstract 2705: Visualization of KRAS point mutations in non-small cell lung cancer tumors with morphological context using the BaseScope in situ hybridization assay

Abstract About 25% of non-small cell lung cancer (NSCLC) patients bear one or more KRAS mutations in their tumors, which is correlated with poor prognosis. The precise identification of somatic mutations in tumors is becoming increasingly important for studying tumor progression and developing targeted therapies. While sequencing technologies allow for mutation-profiling, they do not permit direct visualization and association of genetic alterations with cellular morphology. In addition, DNA mutational status does not predict expression of the mutant allele which may provide information connecting genotype to phenotype. Therefore, a technology for mutation detection at the transcript level directly in the tumor context is desirable. To address this need we developed a specialized RNA in situ hybridization (ISH) method known as BaseScope. The BaseScope assay has a unique signal amplification system that allows for highly sensitive and specific detection of single nucleotide point mutations in tissues. BaseScope probes specific for KRAS G12C, G12A, G12V, G12S and wild type KRAS were designed and expression of each point mutation was assessed in a NSCLC tumor microarray with 48 tumor cores with known KRAS mutation status as determined by DNA sequencing. RNA quality and background signal threshold for each tumor core were determined using PPIB (positive) and dapB (negative) control probes. Using the sequencing data as the gold standard, the BaseScope assay demonstrated 83-100% sensitivity and 97-100% specificity for various KRAS mutations [Table 1]. For KRAS G12C, the assay correctly identified all 6 sequencing-positive cores and identified the rest as negatives. For KRAS G12V, the assay detected 5 of 6 mutated cores with 100% specificity. Interestingly, for KRAS G12S and KRAS G12A mutations, the BaseScope assay demonstrated 100% sensitivity and 97% specificity. Furthermore, it was observed that 100% of the KRAS-mutated tumors showed expression for both wild type and mutant KRAS alleles within these NSCLC tumors. In summary, we demonstrate the development of an RNA ISH assay for point mutations detection with morphological context in FFPE tissues. Unlike current sequencing methods that lack spatial information this assay has the unique ability to identify very small subclones whose frequency within the tumor might fall below the detection limit of sequencing. Performance characteristics of BaseScope KRAS assaysKRAS POINT MUTATIONSNo. of cores with specified mutationsNo. of cores without specified mutationsBaseScope sensitivityBaseScope specificityG12C633100% (6/6)100% (33/33)G12A336100% (3/3)97.2% (35/36)G12V63383% (5/6)100% (33/33)G12S138100% (1/1)97.3% (37/38) Citation Format: Anushka Dikshit, Helen Jarnagin, Emerald Doolittle, Courtney Anderson, Bingqing Zhang, Xiao-Jun Ma. Visualization of KRAS point mutations in non-small cell lung cancer tumors with morphological context using the BaseScope in situ hybridization assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2705.

Abstract LB-111: GT19077, a c-Myc/Max protein-protein interaction (PPI) small molecule inhibitor, selectively targets c-Myc-additive B-cell and myeloid malignancies

Abstract c-Myc is a transcriptional factor and an oncogene driver which plays a major role in tumor initiation, progression and poor prognosis in 80% of all tumor types, especially in c-Myc-additive B-cell and myeloid malignancies. c-Myc's critical role as an oncoprotein makes it an attractive target for pharmacological inhibition. However, c-Myc has been considered as “an undruggable” target since it is an intrinsically disordered protein and regulates key physiological functions in normal cells, which can be a liability for effectively pharmacological intervention. Thus, effective and selective c-Myc targeted molecules are highly warranted, but no such inhibitors are now in clinical testing. c-Myc is dependent on the heterodimerization with Max for the stability and activation of transcription. Here we describes a c-Myc/Max PPI small molecule inhibitor, GT-19077, selectively degrades c-Myc and inhibits the proliferation of c-Myc-addictive B-cell and myeloid malignant cells, but spares c-Myc and the cell proliferation in JAK2-STAT5 dependent erythroblast cells. Through the conformation generation/binding site predication, virtual screening of molecules (>200K), optimization and experimental validation, a leading c-Myc binding molecule series was identified. GT19077 was then discovered and optimized as a leading molecule that exhibited a direct c-Myc binding activity in HL-60 myeloid leukemia cells as shown in Cellular Thermal Shift Assay (CETSA). Importantly, GT19077 selectively degraded c-Myc in HL-60 cells with DC50 of 0.40 µM vs. DC50 of 2.31 µM in GM-CSF-JAK2-STAT5 dependent TF-1 erythroblast cells (>5 fold) as measured by ELISA assays. Consistently, GT19077 also selectively inhibited the proliferation of HL-60 cells with the IC50 of 0.34 µM vs. IC50 of 1.6 µM in GM-CSF stimulated TF-1 cells (>5 fold). Profiling on the proliferation of a selected 14 hematologic malignant cell panel with c-Myc overexpression, GT19077 selectively inhibited the proliferation of all seven B-cell malignant cell lines tested, which include three carrying Myc-IGH translocations and two expressing Kras G12A mutations, with IC50s as low as 160 nM. In addition, two myeloblast cell lines and one monoblast cell line were also sensitive to GT19077 with IC50 of 300-700 nM. In contrast, GT19077 showed almost no effect on the proliferation of four leukemia cell lines with known oncogene drivers, including two erythroblasts (JAK2-STAT5 dependent), one macrophage (FGFR1OP2-FGFR1) and one myeloblast (AML-ETO). These results suggest that c-Myc inhibitor GT19077 can selectively target c-Myc-addictive B-cell and myeloid malignancies, but spares the liability on JAK2-STAT5 driven erythroblasts. Moreover, GT19077 has almost no effect on hematologic malignancies with different oncogene drivers that can be used as biomarkers for patient stratification. Further, GT19077 demonstrated a favorable PK profile with the bioavailability of 57.3% in rat by intraperitoneal (IP) administration. Currently GT19077 is being evaluated in B-cell and myeloid malignant animal efficacy models. Since this novel c-Myc inhibitor demonstrates a superior potency and a unique electivity profile, as compared to many experimental c-Myc inhibitors, it has potential for the drug development for targeting c-Myc-addictive B-cell and myeloid malignancies. Citation Format: Liandong Ma, Youzhi Tong, Zhaohui Yang, Qian Xiang Zhou, Jie Chen, Ye Chen, Honglei Bi, Jie Pan, Qi Sun, Ru Xu, Luhua Lai. GT19077, a c-Myc/Max protein-protein interaction (PPI) small molecule inhibitor, selectively targets c-Myc-additive B-cell and myeloid malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-111.

Clinical activity of brigatinib in ROS1-rearranged non-small cell lung cancer.

Brigatinib is a potent ROS1 inhibitor. The existing data on its clinical activity in ROS1-rearranged non-small cell lung cancer (NSCLC) are limited to four cases. Six patients with ROS1-rearranged advanced NSCLC treated with brigatinib were identified through search of the internal databases of four participating cancer centers. Four additional patients were selected by PubMed and Google Scholar search. The objective response rate (ORR), progression-free survival (PFS) (RECIST v.1.1), duration of treatment (DOT), and safety were assessed. Of eight patients evaluable for response assessment (crizotinib naive-1, crizotinib resistant -7), three patients demonstrated a partial response (ORR-37%). One crizotinib-naive patient had an ongoing response at 21.6months. Of seven crizotinib-resistant patients, two patients demonstrated a partial response (ORR-29%), and one patient (14%) had stable disease. PFS, available in four crizotinib-resistant patients, was 7.6 + , 2.9, 2.0, and 0.4months. In crizotinib-resistant patients, DOT was 9.7 + , 7.7 + , 7.6 + , 4.0, 2.0, 1.1, 0.4months, and was not reported in two patients. Genomic profiling in one responder revealed no ROS1 alteration, suggesting that the response was attributable to "off-target" brigatinib activity. In two patients with progressive disease, genomic profiling demonstrated a cMET exon 14 mutation + KRAS G12A mutation in one case, and a persisting ROS1-CD74 fusion + TP53 K139N, FGFR2 E250G, ATM G2695D, and NF1 R2258Q mutations in the other. No grade 3-5 toxicity was observed. Brigatinib demonstrated modest activity in crizotinib-resistant ROS1-rearranged NSCLC. Its intracranial and systemic activity should be assessed in correlation with the underlying molecular mechanism of crizotinib resistance.

KRAS and NRAS mutational gene profile of metastatic colorectal cancer patients in Jordan.

BackgroundA constitutively active RAS protein in the absence of stimulation of the epidermal growth factor receptor (EGFR) is the result of mutations in KRAS and NRAS genes. Mutations in the KRAS exon 2 and outside exon 2 have been found to predict the resistance to anti-EGFR monoclonal therapy. A substantial proportion of metastatic colorectal cancer cases (mCRC) exhibit RAS mutations outside KRAS exon 2, particularly in KRAS exon 3 and 4 and NRAS exons 2 and 3. No data about RAS mutations outside KRAS exon 2 are available for Jordanian patients with mCRC. We aim to study the molecular spectrum, frequency, and distribution pattern of KRAS and NRAS mutations in Jordanian patients with mCRC.MethodsA cohort of 190 Jordanian metastatic colorectal cancer patients were enrolled in the trial. We detected mutations in exon 2 of the KRAS and NRAS gene as well as mutations outside of exon 2 using the StripAssay technique. The KRAS StripAssay covered 29 mutations and 22 NRAS mutations.ResultsMutations were observed in 92 (48.42%) cases, and KRAS exon 2 mutations accounted for 76 cases (83.69%). KRAS G12D was the most common mutation, occurring in 18 cases, followed by KRAS G12A in 16 cases, and G12T in 13 cases. Mutations outside of KRAS exon 2 represented 16.3% of the mutated cases. Among those, 6 cases (6.48%) carried mutations in NRAS exon 2 and 3, and 10 cases (10.87%) in KRAS exon 3 and 4.ConclusionThe frequency of NRAS and KRAS mutations outside of exon 2 appears to be higher in Jordanian patients in comparison with patients from western countries. KRAS mutations outside of exon 2 should be tested routinely to identify patients who should not be treated with anti-EGFR antibodies.

Clonal Competition Models to Understand Progression and Resistance in Myeloma

Background: Progression and relapse in Multiple Myeloma (MM) is induced by changes in the clonal tumor composition. In order to better understand the mechanisms underlying these dynamics, we developed clonal competition models based on the co-culture of fluorescent labelled isogenic MM cells, with or without the alteration under study. Methods: To understand the effect of mono- and bi-allelic TP53 lesions, we use the AMO1 cell line, one of few myeloma cell lines harbouring wild type TP53 (WT). After modification with CRISPR / CAS9, we selected subclones with mono- and/or bi-allelic deletion of TP53. For the characterization of alterations in RAS, we selected OPM2 cells, one of the few lines with the RAS pathway intact. Furthermore, we generated the KRAS WT, G12A and A146T sublines by stable transfection with Sleeping Beauty vectors. To study mutations related to resistance to IMIDs and PIs, we introduced mutations in the target genes IKZF1 (WT, A152T, Q170D or R439H), CUL4B (KO), and PSMB5 (WT or A20T ) in AMO1 and L363, cell lines sensitive to IMiD or PI treatment. In addition, all WT and mutant sublines were also stably transformed with E-GFP or LSS-mkate2-RFP for flow cytometry analysis. Results: We recently demonstrated that lesions in TP53, both mono- and bi-allelic, induce a growth advantage to the affected cells. In the current study, we also observed an increased fitness in KRAS mutated cells (G12A or A146T vs WT) independent of treatment. We co-cultivated KRAS mutant with WT cells at a ratio 1:3 in two independent experiments, with the color labelling switched (red/green wt/mutant and vice-versa). KRAS G12A clone significantly expanded and reached 50% of the cells at day 40. Likewise, A146T clone outcompeted WT cells, but the time required to represent the majority of cells in the coculture was longer. We next explored the effects of resistance mutations and drug exposure. Both the IKZF1 A152T and CUL4B KO mutants outcompete WT cells in the presence of Lenalidomide (LEN). The same effect was observed for the PSMB5 A20T mutant exposed to Bortezomib (BOR). This selection ("Survival Fitness") did not occur without the presence of the drug. Thus, resistance related mutations seem only to provide a fitness advantage under drug exposition. In addition, both the CUL4B KO and PSMB5 A20T mutants were overcome by WT cells when the drug was removed from co-culture, suggesting that these lesions provide a survival disadvantage without the selective pressure of IMiD or PI. This may provide an explanation for the low mutation rate in this gene in recent sequencing publications, as usually samples are not obtained under selective pressure but in treatment free intervals. IKZF1 mutations outside the IMiDs / CRBN binding area (Q170D and R439H) provided no advantage to the cells. Conclusions: Our clonal competition assays provide novel insights on the impact of point mutations on the fitness of affected myeloma subclones, either with or without the selective pressure of therapy. Figure Disclosures Martinez-Lopez: Celgene: Honoraria, Other: Advisory boards and Non-Financial Support ; Amgen: Honoraria, Other: Non-Financial Support ; F. Hoffmann-La Roche Ltd: Honoraria; Janssen: Honoraria, Other: Advisory boards and Non-Financial Support ; BMS: Honoraria, Other: Advisory boards; Incyte: Honoraria, Other: Advisory boards; Novartis: Honoraria, Other: Advisory boards; VIVIA Biotech: Honoraria.

Mutant KRAS Enhances Stress Granules and Resistance to Proteasome Inhibition Via 15-d-PGJ2 in Multiple Myeloma

Introduction: Mutant KRAS leads to activation of the MEK/ERK pathway in approximately 20% of multiple myeloma (MM) patients. Stress granules (SGs) are cytosolic non-membranous structures composed of translational mRNAs, ribosomal proteins, and RNA-binding proteins, which form in response to different stress stimuli and chemotherapeutic treatment. We have previously shown that mutant KRAS upregulates SG formation. The molecular mechanisms underlying this process are unclear. The purpose of this study was to elucidate a) the mechanism by which mutant KRAS upregulates SG formation and b) to provide a molecular rationale for the targeting the cascades for SG formation in MM patients. Methods. We determined whether mutant KRAS is necessary for SG upregulation. To this purpose a panel of MM cell lines including mutant KRAS (KRASM), mutant NRAS (NRASm) and wildtype KRAS (KRASWT) was used. The gain- and loss-of-function of mutant KRAS was generated to suppress (KRASM/KO) in KRASM or switch on by expression of mutant KRAS G12A (KRASG12A) in KRASWT cells by a lentiviral vector system. SG formation was assessed by measuring the cellular distribution of SG resident proteins by immunofluorescence using anti-G3BP and anti-eIF4G antibodies. The quantitative readout for SG formation was analyzed by ImageJ analyze-particle tool as the SG index. Immunoblotting was used to detect inactivation of eIF2a or activity of MAPK. qPCR analysis was employed to detect the mRNA of COX2. MTT assays were used to detect the effect of SGs on the survival of MM cells alone or in combination with diclofenac sodium (DS), or bortezomib (Bzb). Results: We compared the induction of SGs in response to oxidative stress across a panel of MM cells with different Ras mutation status (KRASM, NRASM and KRASWT)to investigate the role mutant KRAS in GS formation. The level of SGs were significantly higher (p<0.01) in six KRASM, compared with four KRASWT cell lines.We assessed SG formation in KRASM, KRASM/KO and KRAS/G12A cells to determine whether mutant KRAS is a causative factor leading to elevated SG levels. SG formation in KRASM/KO cells was significantly attenuated, by approximately 5-fold (p<0.001), compared to KRASM in response to oxidative stress. Conversely, SG levels were 3.5-fold elevated in KRAS/G12A cells (P<0.01), compared to KRASWT cells. Upregulation of SGs by mutant KRAS resulted in a larger average size and number of SGs per cells. Together, these results indicate that mutant KRAS is necessary and sufficient for SG upregulation under oxidative stress condition. We investigated whether 15-d-PGJ2-depedent inactivation of elF4A is involved in SG upregulation to determine the mechanism through which mutant KRAS promotes SG formation. To do this, we assessed the effect of 15-d-PGJ2 on SG formation because that elF4A inactivation is controlled by 15-d-PGJ2, which is one of COX2-derived products. Treatment of KRASM cells with 15-d-PGJ2 significantly induced accumulation of SGs compared with untreated cells (40-fold increase vs control cells, p<0.001). To determine whether COX2, which controls the rate of biosynthesis of 15-d-PGJ2, is regulated by mutant KRAS, we first compared COX2 mRNA of KRASM/KO with KRASM and showed that a decrease in COX2 expression in KRASM/KO (2-fould decrease, P< 0.01). Conversely, increased COX2 mRNA was seen in KRAS/G12A, compared with KRASWT cells. Furthermore, treatment of KRASM cells with DS led to a decrease in SG levels and this effect was rescued by the addition of 15-d-PGJ2. These results indicate that increase in prostaglandin levels by upregulating prostaglandin biosynthesis is essential for mutant KRAS-induced up-regulation of SGs. Finally, we determined if inhibition of COX2 activity by DS was able to overcome Bzb resistance in KRASM or in KRASM co-occurring t(14:16)/MAF. A significant decrease of cell viability (P<0.001) was observed when treatment of the cells with Bzb and DS were combined, compared with DS treatment alone. Taken together, these results indicate that inhibition of COX2 activity enhances mutant KRAS MM cell sensitivity to proteasome inhibition. Conclusion: Our results suggest that mutant KRAS mutant upregulates SG formation by 15-d-PGJ2 and inhibition of COX2 activity enhances KRAS sensitivity to proteasome inhibition and targeting SG formation might be represent an effective strategy to treatment of KRAS mutant myeloma. Disclosures Davies: Janssen, Celgene: Other: Research Grant, Research Funding; Amgen, Celgene, Janssen, Oncopeptides, Roche, Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Consultant/Advisor. Morgan:Amgen, Roche, Abbvie, Takeda, Celgene, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: research grant, Research Funding. Walker:Celgene: Research Funding. van Rhee:Karyopharm Therapeutics: Consultancy; Kite Pharma: Consultancy; Adicet Bio: Consultancy; EUSA: Consultancy; Castleman Disease Collaborative Network: Consultancy; Sanofi Genzyme: Consultancy; Takeda: Consultancy.

Abstract LB-118: Resistance to TRK inhibition mediated by convergent MAP kinase pathway activation

Abstract Background: TRK inhibition is now standard of care for advanced pediatric and adult patients (pts) with TRK fusion solid tumors, regardless of origin. To date, TRK kinase domain mutations are the only known resistance mechanism, and next-generation TRK inhibitors active against these mutations such as LOXO-195 are being developed. We reasoned some pts will develop TRK-independent resistance and hypothesized that these pts will require unique therapeutic approaches. Methods: Paired tumor biopsies and serial cell-free DNA (cfDNA) prospectively collected from pts with TRK fusion-positive cancers treated with first- and next-generation TRK inhibitors before treatment and at progression were sequenced. In parallel, pt-derived and engineered models were analyzed. Results: Alterations involving upstream non-TRK receptor kinases and downstream MAPK pathway members were initially identified in tumors from 3 TRK fusion-positive gastrointestinal (GI) cancer pts who developed resistance to TRK inhibitors. Pt 1 with CTRC-NTRK1 pancreatic cancer developed temporally distinct emergent BRAF V600E and KRAS G12D mutations. Pt 2 with LMNA-NTRK1 colorectal cancer developed temporally distinct KRAS G12A and G12D mutations. Pt 3 with PLEKHA6-NTRK1 cholangiocarcinoma developed focal MET amplification. Phenocopying these clinical observations, pt-derived xenografts and primary cell lines developed BRAF and KRAS mutations following chronic TRK inhibition. Consistently, ectopic expression of these alterations conferred resistance to TRK inhibitors. Given that all 3 index pts had GI cancers, we expanded serial cfDNA sequencing to 5 additional TRK fusion-positive GI disease, identifying 3 with emergent MAPK alterations at progression, bringing the overall frequency of acquired MAPK alterations in GI cancers analyzed to 75% (6/8). To further evaluate whether these emergent alterations induced functional dependence on ERK signaling, pts 1-3 were treated with agents targeting these emergent alterations (dabrafenib + trametinib, LOXO-195 + trametinib, and LOXO-195 + crizotinib, respectively). Pt 1 achieved transient tumor regression, followed by outgrowth of KRAS-mutant disease. Pt 3 achieved a 4.5 months tumor regression. Sequencing at progression in pt 3 identified multiple acquired MET point mutations known to interfere with crizotinib binding. Conclusions: These data suggest that a subset of TRK fusion-positive cancers will develop off-target mechanisms of resistance to TRK inhibition. Relative to other TRK fusion-positive tumors, GI cancers may have a higher propensity for developing these bypass alterations that demonstrate remarkable convergence on ERK signaling. A portion of these mechanisms may be managed with simultaneous targeting of the TRK and MAPK pathways, although additional modeling is required to determine if upfront treatment would confer more durable responses. Citation Format: Emiliano Cocco, Amanda Kulick, Sandra Misale, Rona Yaeger, Pedram Razavi, Helen H. Won, Ryan Ptashkin, Jaclyn F. Hechtman, Eneda Toska, James Cownie, Romel Somwar, Sophie Shifman, Marissa Mattar, S Duygu Selçuklu, Aliaksandra Samoila, Sean Guzman, Brian B. Tuch, Kevin Ebata, Elisa de Stanchina, Rebecca J. Nagy, Richard B. Lanman, Michael F. Berger, Marc Ladanyi, David M. Hyman, Alexander Drilon, Maurizio Scaltriti, Alison M. Schram. Resistance to TRK inhibition mediated by convergent MAP kinase pathway activation [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 LB-118.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA <em>In Situ</em> Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and METΔ14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors α and β in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay.

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and METΔ14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors α and β in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Abstract 3667: Technical advances in plasma genomic biomarkers for mutation detection and monitoring in cancer patients

Abstract A sea-change is imminent for cancer medicine, due to the use of non-invasive genomic biomarkers in blood to inform screening, diagnosis and the selection of treatment. This technology may be used routinely in oncology within five years. Although numerous studies, including work in our laboratory, have shown that genomic analysis of blood can detect the presence and even the type of cancer, researchers have only scratched the surface of what this technology can do. In our laboratory, we are generating new methods to improve the sensitivity and accuracy of such tests. Cell-free DNA from a metastatic colorectal cancer patient has been used to directly compare the sensitivity of mutation detection across three platforms - a custom QiaSeq Targeted amplicon sequencing panel, droplet digital PCR (ddPCR) and UltraSEEK lung panel. UltraSEEK and ddPCR platforms show better sensitivity for detection of the KRAS G12A mutation, compared to the QiaSeq Targeted DNA sequencing panel. This pilot study shows that when looking to specifically detect a small number of known mutations, UltraSEEK or ddPCR gives the highest sensitivity, but when mutations are not limited to a few or are completely unknown, the QiaSeq Targeted DNA panel allows identification of the entire repertoire of mutations in specific cancer-associated genes. Citation Format: Sandra Fitzgerald, Annette Lasham, Cherie Blenkiron, Paula Shields, Ben Lawrence, Cristin Print. Technical advances in plasma genomic biomarkers for mutation detection and monitoring in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3667.

Molecular and clinical characterization of BRAF mutations in pancreatic ductal adenocarcinomas (PDACs).

214 Background: The activation of the RAS/RAF/MEK/ERK pathway is critical for the proliferation, survival, and tumorigenesis of PDACs. Oncogenic mutations in KRAS (90%) or BRAF (3%) are recurrent genomic alterations, but their co-occurrence is not well described. We reviewed BRAF alterations and clinical outcomes in consecutive PDAC patients (pts). Methods: Perthera, Inc. deploys an IRB-approved registry that was utilized in partnership with PanCAN’s “Know Your Tumor” program. Perthera uses CAP/CLIA accredited multi-Omic profiling, including next generation DNA sequencing (NGS, Foundation Medicine) and proteomics/immunohistochemistry (Neogenomics Inc. and Caris Life Sciences, Inc.). We used a Fisher's exact test with multiple testing correction to compare frequencies of mutations and protein over-/under-expression between BRAF-mutated (n = 21) and BRAF wild type pts (n = 745). Results: Of 766 pancreatic cancer pts, 21 pts were identified with BRAF mutations. 18 pts were diagnosed with PDAC, 1 pancreatoblastoma, 1 pancreatic acinar cell carcinoma, and 1 mixed acinar neuroendocrine carcinoma. Amongst the 18 PDAC pts with BRAF mutations, nine variations were found: V600E (5/18), BRAF fusion (4/18), N486_P490del (3/18), T310I (1/18), K601N (1/18), G596R (1/18), exon 2-10 deletions & S467L (1/18), equivocal amplification of BRAF (1/18), and a BRAF inframe deletion (1/18). KRAS was mutated in 6% of BRAF mutated PDACs, compared to 94% in BRAF wild type PDACs (p < 0.001). Amongst KRAS wild type PDACs, 53% of CDKN2A mutated pts had BRAF mutations vs. only 15% of CDKN2A wild type cases (p < 0.05). Two BRAF mutated pts from the database were given BRAF inhibitors. A sustained response to the combination dabrafenib and trametinib was observed in a BRAF V600E mutated PDAC pt, while no response was seen in a pt with concurrent KRAS G12A & BRAF K601N mutations who received trametinib. Conclusions: BRAF mutations are significantly and inversely correlated with KRAS alterations. The most common BRAF alteration, V600E mutation, was found to be mutually exclusive with the KRAS mutation. Clinical trials targeting BRAF alterations in KRAS wild type pancreatic cancer appears warranted.

Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras.

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras-GTP complex, the switch I region undergoes a significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.

Peritoneum as the sole distant metastatic site of lung adenosquamous cell carcinoma: a case report

BackgroundPeritoneum metastasis of lung cancer is a rare event which, in addition to the peritoneum, usually involves multiple metastatic tissues. Here we report a case of a patient with lung adenosquamous cell carcinoma with the peritoneum as the sole distant metastatic site.Case presentationAn 82-year-old Han Chinese man, in the teaching profession, was diagnosed with lung adenosquamous cell carcinoma in the upper lobe of his left lung with the involvement of ipsilateral hilar and mediastinal lymph nodes, and was initially staged as IIIa (cT2N2M0). Molecular testing identified a mutation at KRAS G12A. Due to his poor physical condition, our patient was given gamma knife radiotherapy with a total dose of 28.0 Gy. Two weeks later, our patient was diagnosed as peritoneal metastasis identified by using magnetic resonance imaging and confirmed with ascitic cytology and peritoneal histology. No other distant metastatic sites such as liver, brain, bone, paranephroi, and lungs were found. Subsequently, our patient received palliative intraperitoneal chemotherapy, and died within 2 months.ConclusionsOur patient represented a rare case of lung adenosquamous cell carcinoma harboring the KRAS G12A mutation, which metastasized distantly to the peritoneum only, and progressed rapidly.

Comparative study on driver mutations in primary and metastatic melanomas at a single Japanese institute: A clue for intra- and inter-tumor heterogeneity

BackgroundSearching for driver mutations in melanoma is critical to understanding melanoma genesis, progression and response to therapy. ObjectivesWe aimed to investigate the frequency and pattern of driver mutations in Japanese primary and metastatic melanomas including cases of unknown primary origin, in relation to their clinicopathologic manifestations. MethodsSeventy-seven samples from 60 patients with melanoma were screened for 70 driver mutations of 20 oncogenes by Sequenom MelaCarta MassARRAY, and the results for primary and metastatic melanomas were compared. ResultsOf 77 tissue samples, BRAF V600E was detected in 21 samples (27%), CDK4 R24C in 7, EPHB6 G404S in 6, BRAF V600K in 2, NEK10 E379K in 2, and CDK4 R24H, NRAS Q61K, NRAS Q61R, KRAS G12A, KIT L576P, KIT V559A, ERBB4 E452K, and PDGFRA E996K in one sample each. No driver mutations related to the MAPK cascade including RAS and BRAF were detected in the chronically sun-damaged (CSD) group of melanoma. Dual or triple driver mutations were found in four of 40 (10%) samples from the primary melanomas, and three of 37 (8%) of the metastatic melanomas. Fourteen of 26 (54%) samples of non-CSD melanoma, and 3 of 6 (50%) melanomas of unknown primary origin had the BRAF V600E mutation. Mutations in membrane-bound receptors including KIT, ERBB4 and EPHB6 were detected in 8 of 77 (10%) samples. Of 17 pairs of primary and metastatic melanomas from the same patient, the primary mutation pattern was changed to a novel one in three cases, and only one of the plural mutations in the primary melanoma was found in the metastatic lesions in two cases. ConclusionsBRAF V600E is a predominant mutation in non-CSD melanoma and melanomas of unknown primary origin. Mutational heterogeneity may exist in the primary melanoma (intra-tumor heterogeneity), and between the primary and metastatic lesions (inter-tumor heterogeneity).

Abstract 3172: Comprehensive analysis of intrahepatic cholangiocarcinoma based on viral infections and mutational status in the IDH1/2 and KRAS genes

Abstract Background: Intrahepatic cholangiocarcinoma (ICC) is a primary liver cancer with poor prognosis and limited therapeutic options. Recently comprehensive genetic profiles have elucidated novel fusion genes and somatic mutations. Although somatic mutations in the isocitrate dehydrogenase (IDH) and KRAS gene were frequently found in ICC, the clinical features of this malignancy with mutations in these genes remain unclear. Materials and Methods: A cohort of 49 patients with ICC who underwent curative resection from 2000 to 2013 at Okayama University Hospital were enrolled and analyzed. KRAS (exon2), IDH1 (codon132), and IDH2 (codon172 and codon140) mutations were confirmed by Sanger sequencing. Associations between the mutational profiles of these genes and clinic-pathological features were investigated. Kaplan-Meier curves were plotted, and survival rates were compared using the log-rank analysis. Results: KRAS mutations were observed in twelve ICC patients (24.5%, KRAS-mutant). IDH mutations were found in eight patients (16.3%); five patients harbored IDH1 mutations and three patients harbored IDH2 mutations (IDH1/2-mutant). Among three patients with IDH2 mutations, a rare IDH2 R140L was identified. One patient harbored both KRAS G12A and IDH2 R172K mutations (classified as KRAS-mutant). The rest of 30 ICCs harbored no mutation in either KRAS or IDH1/2 gene, thus categorized as Wild-type. Infection of Hepatitis B (HBV) or C (HCV) was observed in 10 ICCs (6 ICCs were HBV-positive and 4 ICCs were HCV-positive). Of 10 ICCs with viral infection, only two ICCs harbored KRAS mutations (categorized as virus-infected) and eight ICCs with viral infection harbored no mutation in KRAS and IDH1/2 genes. Finally, ICCs were divided into four subsets; viral-infected, KRAS-mutant, IDH1/2-mutant, and ‘others’). With regard to clinic-pathological features, lymph node metastasis was more frequently observed in KRAS-mutant (p = 0.039) compared with the others. IDH1/2 -mutant were more frequently observed in mass-forming type (p = 0.047). By survival analysis, 3-years survival rate was 85.7% in IDH1/2-mutant, 58.3% in the others, 36.3% in viral-infected, and 22.2% in KRAS-mutant, respectively (p = 0.05). Thus, our cohort suggests that IDH1/2, as well as KRAS mutations, have the potential to serve as prognostic biomarkers in ICC. Conclusion: We conclude that not only viral infections, but also IDH1/2 and KRAS mutations could be potential predictive markers for the identification of good and worse prognosis in ICCs. Hence the mutational profiles of these genes provide an attractive rationale for developing a molecular signature for the development of non-invasive screening for ICCs in future. Citation Format: Kazuya Yasui, Takeshi Nagasaka, Yuzo Umeda, Tomokazu Fuji, Fumitaka Taniguchi, Toshiaki Toshima, Keisuke Kimura, Takashi Kawai, Yoshiko Mori, Hiroshi Tazawa, Takahito Yagi, Ajay Goel, Toshiyoshi Fujiwara. Comprehensive analysis of intrahepatic cholangiocarcinoma based on viral infections and mutational status in the IDH1/2 and KRAS genes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3172.