High Frequency Of Ras Mutations Research Articles

Abstract PO-015: A phase Ib/II trial of high dose ascorbic acid (AA) + paclitaxel protein bound (PP) + cisplatin (C) + gemcitabine (G) in patients (pts) with previously untreated metastatic pancreatic cancer (MPC)

Abstract Background: Increasing evidence suggests that high concentrations of AA (vitamin C) can decrease the growth of aggressive tumors, including Ras-mutant tumors. Based on the unmet need in MPC, its high frequency of Ras mutations, and prior work combining PP +C +G in pts with MPC (response rate of 70.5%, median survival 16.4 mos), we explored the addition of AA to that regimen. Pre and post treatment biopsies for biomarkers and digital spacial profiling are also being performed. Methods: This pilot trial utilized a 3 + 3 design for dose escalation of AA. Eligibility criteria included untreated stage IV MPC, ECOG 0-1, and measurable disease. Excluded were pts with a G6PD deficiency, renal oxalate stones, or need of capillary blood glucose monitoring as AA causes false low readings. The primary objective of the phase 1b was to determine the maximum tolerated dose (MTD) of AA in combination with PP +C +G in pts with MPC. Exploratory objectives included analysis of tumor texture on radiologic scans as an imaging biomarker for response; correlation between peak plasma levels of AA and response to treatment; potential biomarkers in the tumor including tumor immune cell infiltration, stromal activation, stem call enumeration; change in numbers of circulating tumor stem cells and macrophage lineage changes. Results: As of 7/2021, the phase Ib enrollment in this study has been completed. Six pts were treated in the cohort of AA 25 gm/m2, 4 pts at AA 37.5 gm/m2, and 7 pts at 56.25 gm/m2. The majority of all reported AEs were grades 1-2. The most common AEs related to study treatment were: platelet count decrease (70.6%), diarrhea (70.6%), hypomagnesemia (52.9%), dysgeusia (47.1%), peripheral sensory neuropathy (47.1%), nausea (35.3%), fatigue (35.3%), neutrophil count decreased (35.3%), and hypokalemia (35.3%). Of the 17 pts enrolled, 15 were evaluable. Response by RECIST 1.1 is 11 PR (73.3%), 2 SD (13.3%), 2 PD (13.3%). Evaluation of median PFS and OS is ongoing. Conclusions: This study has a high response rate of 73%. Most pts in all AA dosing cohorts experienced a brisk response to therapy. AA did not appear to add toxicities compared to historical AE data of PP +C +G and was well tolerated in all dose cohorts. The MTD of AA was not reached in the 56.25 gm/m2 cohort. The protocol allowed for an additional dose escalation of AA to 75 gm/m2. However, all pts in the 56.25 gm/m2 cohort achieved our goal of peak plasma AA levels of > 20 mM so the study was closed to enrollment. Quality of life measures, tumor tissue and blood sample analyses are underway. Supported by SU2C, Cancer Research UK, Lustgarten Foundation, Destroy Pancreatic Cancer & the Young family. Citation Format: Gayle S. Jameson, Erkut H. Borazanci, Daniel D. Von Hoff, Joshua D. Rabinowitz, Michael S. Gordon, Sarah D. LeGrand, Courtney Snyder, Karen Ansaldo, Denise J. Roe, Haiyong Han. A phase Ib/II trial of high dose ascorbic acid (AA) + paclitaxel protein bound (PP) + cisplatin (C) + gemcitabine (G) in patients (pts) with previously untreated metastatic pancreatic cancer (MPC) [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-015.

Primary high-grade non-anaplastic thyroid carcinoma: a retrospective study of 364 cases.

We aimed to study the clinicopathological and molecular features of high-grade non-anaplastic thyroid carcinomas (HGTCs), a carcinoma with a prognosis intermediate between those of well-differentiated carcinoma and anaplastic carcinoma. This study included 364 HGTC patients: 200 patients (54.9%) were diagnosed with poorly differentiated thyroid carcinoma (PDTC), based on the Turin consensus (HGTC-PDTC), and 164 were diagnosed with high-grade features that did not meet the Turin criteria (HGTC-nonPDTC). HGTCs are aggressive: the 3-year, 5-year, 10-year and 20-year disease-specific survival (DSS) rates were 89%, 76%, 60%, and 35%, respectively. Although DSS was similar between HGTC-PDTC and HGTC-nonPDTC patients, HGTC-PDTC was associated with higher rate of radioactive iodine avidity, a higher frequency of RAS mutations, a lower frequency of BRAF V600E mutations and a higher propensity for distant metastasis (DM) than HGTC-nonPDTC. Independent clinicopathological markers of worse outcome were: older age, male sex, extensive necrosis and lack of encapsulation for DSS; older age, male sex and vascular invasion for DM-free survival; and older age, necrosis, positive margins and lymph node metastasis for locoregional recurrence-free survival. The frequencies of BRAF, RAS, TERT, TP53 and PTEN alterations were 28%, 40%, 55%, 11%, and 10%, respectively. TP53, PTEN and TERT were independent molecular markers associated with an unfavourable outcome, independently of clinicopathological parameters. The coexistence of BRAF V600E and TERT promoter mutation increased the risk of DM. The above data support the classification of HGTC as a single group with two distinct subtypes based on tumour differentiation: HGTC-PDTC and HGTC-nonPDTC.

A phase Ib/II trial of high dose ascorbic acid (AA) + paclitaxel protein bound (PP) + cisplatin (C) + gemcitabine (G) with digital spatial profiling (DSP) in patients (pts) with metastatic pancreatic cancer (MPC)

Background: Increasing evidence suggests that high concentration of AA (vitamin C) can decrease the growth of aggressive tumors, including Rasmutant tumors. Based on the unmet medical need in MPC, its high frequency of Ras mutations, and prior work combing PP + C + G with MPC (response rate of 70.5%, median survival 16.4 mos), we are exploring the addition of AA to that regimen. Pre and post-treatment biopsies for biomarkers and DSP are also being performed.

Abstract B27: A phase Ib/II trial of high-dose (HD) ascorbic acid (AA) + paclitaxel protein bound (PP) + cisplatin (C) + gemcitabine (G) in patients (pts) with metastatic pancreatic cancer (MPC)

Abstract Introduction: Based on the unmet medical need in MPC, its high frequency of Ras mutations, and prior work combining PP + C + G demonstrating an objective response rate of 70.5% in 24 pts with MPC with a median survival of 16.4 mos (J Clin Oncol 2017;35 [suppl 4S; abstract 341]), we are exploring the addition of HD AA to PP + C + G (#NCT03410030). Background: Increasing evidence suggests that HD AA (vitamin C) can decrease the growth of aggressive tumors, including Ras-mutant tumors, by inducing oxidative stress (Chen et al., PNAS 2008; Yun et al., Science 2015; Shoenfeld et al., Cancer Cell 2017). Despite the potential to produce toxic radicals, high-dose AA has generally been well tolerated in animals and humans, including in combination with chemotherapy. Methods: This phase Ib/II trial uses a 3 + 3 dose escalation of AA. Eligibility criteria include pts with previously untreated stage IV MPC, ECOG 0-1, and measurable disease. Excluded are pts with a G6PD deficiency, history of renal oxalate stones, and need of frequent capillary blood glucose monitoring as AA causes false low readings. Doses are PP 125 mg/m2, G 1000 mg/m2, C 25 mg/ m2 on days 1 and 8 of each 21-day cycle. AA infusions are given on days 1, 3, 8, 10, 15, and 17 every 21 days. We plan on studying up to 4 dose levels of AA including 25, 37.5, 56.25, and 75 gm/m2, seeking to determine the recommended phase 2 dose (RP2D). Tissue analysis is being done on pre- and optional post-treatment tumor biopsies. Primary objective(s): phase Ib—to determine the maximum tolerated dose (MTD) and RP2D of AA with PP + C + G; phase II—to determine the efficacy (disease control rate of CR+ PR+ SD x 18 weeks) of the combination at the RP2D in pts with MPC. Exploratory objectives include quantitative textural analysis, correlation between peak plasma levels of AA and treatment response, and potential biomarkers in the tumor. Planned sample size is up to 24 pts in phase Ib and up to 21 pts in phase II. Results: As of 6/23/19, 9 pts have been treated. Grade (gr) ≥ 3 treatment-related adverse events are thrombocytopenia, gr 3 (44%), gr 4 (11%) with no serious bleeding events; neutropenia gr 3 (22%); anemia gr 3 (22%); hypokalemia gr 3 (22%); diarrhea gr 3 (11%); hypomagnesemia gr 3 (11%); and hypophosphatemia gr 3 (11%). In the 7 response-evaluable pts (baseline and ≥ 1 follow-up CT scan), all pts have experienced a disease response to therapy with substantial decrease in tumor size and decline in tumor markers. It is too early in the trial to determine complete and partial response rates. Conclusion: Through the first two dose levels of AA we have observed good tolerability and preliminary evidence of high levels of antitumor activity. Dose escalation is continuing. (Supported by SU2C, Cancer Research UK, Lustgarten Foundation & Destroy Pancreatic Cancer.) Citation Format: Gayle S. Jameson, Erkut H. Borazanci, Joshua Rabinowitz, Karen Ansaldo, Sarah LeGrand, David Propper, Courtney Snyder, Michael Gordon, Denise Roe, Daniel D. Von Hoff. A phase Ib/II trial of high-dose (HD) ascorbic acid (AA) + paclitaxel protein bound (PP) + cisplatin (C) + gemcitabine (G) in patients (pts) with metastatic pancreatic cancer (MPC) [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 B27.

Endogenous IQGAP1 and IQGAP3 do not functionally interact with Ras

The Ras family of small GTPases modulates numerous essential processes. Activating Ras mutations result in hyper-activation of selected signaling cascades, which leads to human diseases. The high frequency of Ras mutations in human malignant neoplasms has led to Ras being a desirable chemotherapeutic target. The IQGAP family of scaffold proteins binds to and regulates multiple signaling molecules, including the Rho family GTPases Rac1 and Cdc42. There are conflicting data in the published literature regarding interactions between IQGAP and Ras proteins. Initial reports showed no binding, but subsequent studies claim associations of IQGAP1 and IQGAP3 with K-Ras and H-Ras, respectively. Therefore, we set out to resolve this controversy. Here we demonstrate that neither endogenous IQGAP1 nor endogenous IQGAP3 binds to the major Ras isoforms, namely H-, K-, and N-Ras. Importantly, Ras activation by epidermal growth factor is not altered when IQGAP1 or IQGAP3 proteins are depleted from cells. These data strongly suggest that IQGAP proteins are not functional interactors of H-, K-, or N-Ras and challenge the rationale for targeting the interaction of Ras with IQGAP for the development of therapeutic agents.

Abstract CT163: A Phase Ib/II trial of high dose ascorbic acid (AA) + paclitaxel protein bound (PP) + cisplatin (C) + gemcitabine (G) in patients (pts) with metastatic pancreatic cancer (MPC)

Abstract Background Increasing evidence suggests that AA (vitamin C) can decrease the growth of aggressive tumors, including Ras-mutant tumors, by inducing oxidative stress (Chen et al. PNAS 2008; Yun et al. Science 2015; Shoenfeld et al. Cancer Cell 2017). Despite the potential to produce toxic radicals, high dose AA has generally been well tolerated in animals and humans, including in combination with chemotherapy. The reason for the cancer-specific toxicity of AA is incompletely understood. One possibility is that AA targets cancer cells due to their having larger labile iron pools. Another is that cancer cells selectively take up the oxidized form of AA, dehydroascorbate, via the GLUT1 glucose transporter, causing glutathione (GSH) depletion, intracellular ascorbate accumulation, inactivation of glyceraldehyde 3-phosphate dehydrogenase, and energetic crisis selectively in glycolytic, GLUT1-expressing cancer cells. Based on the unmet medical need in MPC, its high frequency of Ras mutations, and prior work combing PP + C + G showing promising results in 24 pts with MPC with a response rate of 70.5%, median survival 16.4 mos, [J Clin Oncol 35, 2017 (suppl 4S; abstract 341)], here we are exploring the addition of AA to PP + C + G. Methods This Phase Ib/II pilot trial utilizes a 3 + 3 design for dose escalation of AA. Primary objective(s): Phase Ib - to determine the maximum tolerated dose (MTD) of AA with PP + C + G; phase II - to determine the preliminary efficacy (disease control rate of CR+ PR+ SD x 18 weeks) of the combination with AA at MTD in pts with MPC. Exploratory objectives include analysis of tumor texture on radiologic scans as an imaging biomarker for response, biologic, pathologic and outcome measures; correlation between peak plasma levels of AA and response to treatment; potential biomarkers in the tumor including tumor immune cell infiltration, stromal activation, stem cell enumeration; changes in numbers of circulating tumor stem cells and macrophage lineage changes. #NCT03410030 Eligibility criteria include stage IV MPC, no prior treatment for MPC, ECOG 0 -1, and measurable disease. Excluded are pts with a G6PD deficiency, history of renal oxalate stones, or need of frequent capillary blood glucose monitoring as AA causes false low readings. Planned sample size is up to 24 pts in Phase Ib, up to 21 pts in Phase II. Doses are PP 125 mg/m2, G 1000 mg/m2, C 25 mg/ m2 on days 1 & 8 of a 21 day cycle. AA infusions are given on days 1, 3, 8, 10, 15, and 17 of a 21-day cycle. The AA dose for phase II will be determined in the Phase 1b study based on tolerability of AA at 25, 37.5, 56.25 or 75 gm/m2. Tumor tissue analysis will be done on pre and optional post treatment tumor biopsies. Trial opened to enrollment 4/2018. As of 1/2019, 5 pts have been treated in the AA 25 gm/m2 cohort. Supported by SU2C, Cancer Research UK, Lustgarten Foundation & Destroy Pancreatic Cancer Citation Format: Gayle S. Jameson, Erkut H. Borazanci, Joshua D. Rabinowitz, David Propper, Karen Ansaldo, Denise J. Roe, Daniel D. Von Hoff. A Phase Ib/II trial of high dose ascorbic acid (AA) + paclitaxel protein bound (PP) + cisplatin (C) + gemcitabine (G) in patients (pts) with metastatic pancreatic cancer (MPC) [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 CT163.

PS917 SAFE TARGETING OF T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA BY SUBUNIT SELECTIVE GAMMA-SECRETASE INHIBITION

Background: SPI1 fusion genes are recurrently found in Early Thymic Precursor cell Acute Lymphoblastic Leukemia (ETP-ALL) cases and represent a distinct subgroup with high frequency of RAS mutations and dismal prognosis. SPI1 (PU.1) is an important transcription factor in hematopoietic cells and its dysregulation contributes to several types of leukemia. Forced overexpression of SPI1 in T-cell progenitors is known to inhibit T-cell differentiation. Conversely, knock-out of TCF7, which is highly expressed in the earliest thymic progenitors, also leads to a T-cell differentiation block. However, the role of SPI1 fusions in driving and maintaining T-ALL remains unknown. Aims: In this study we aimed to elucidate the role of the TCF7-SPI1 fusion and its potential collaboration with RAS mutation in driving and maintaining T-ALL. Methods: We performed whole genome, transcriptome and nanopore sequencing on a patient sample with the TCF7-SPI1 fusion. Primary mouse pro-T cells were cultured on Dll4-coated plates in the presence of stem cell factor (Scf) and interleukin-7 (Il7). In vivo bone marrow transplant (BMT) assays were performed using hematopoietic stem and progenitor cells (HSPCs) isolated from CD2-Cre C57BL/6J mice and transduced with retroviral vectors for inducible expression of the TCF7-SPI1 fusion and/or NRAS (G12D) mutant within developing T-cells of recipient mice. Results: Combined short read and long read RNA-sequencing characterized the exon structure of the TCF7-SPI1 fusion transcript and confirmed the presence of a NRAS (G12D) mutation in the patient sample. Expression of TCF7-SPI1 and NRAS (G12D) transformed pro-T cells to Scf and interleukin-7 (Il7) independent growth. Using an inducible in vivo BMT model, conditional expression of TCF7-SPI1 and NRAS (G12D) within developing T-cells resulted in an aggressive immature acute T-ALL characterized by hyperleukocytosis (CD4-/CD8-, CD3+, CD117+), splenomegaly but no thymus enlargement. This was in contrast to either conditional TCF7-SPI1 alone that did not develop leukemia, or with NRAS (G12D) alone that developed a mature CD4+/CD8+ lymphoma with normal white blood cell count and no peripheral leukemic invasion of the bone marrow and spleen. Notably, a deletion construct of TCF7-SPI1, lacking the N-terminal beta-catenin binding domain of TCF7 lost its oncogenic properties. Summary/Conclusion: Here, we show that TCF7-SPI1 cooperates with NRAS (G12D) to drive an aggressive immature T-cell leukemia in vivo that is dependent on the N-terminal beta-catenin binding domain of TCF7.

Molecular Profile and Clinicopathologic Features of Follicular Variant Papillary Thyroid Carcinoma.

The non-invasive encapsulated follicular variant of papillary thyroid carcinoma (FVPTC) has an indolent clinical behavior. Recently, it was proposed that this tumor type should be reclassified as non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP). To characterize NIFTPs, we evaluated the molecular and clinicopathologic characteristics of each FVPTC subtype. This study enrolled 29 patients with FVPTC who underwent thyroidectomy between January 2007 and June 2017. They were classified as non-invasive encapsulated FVPTC (NIFTP, n = 10), invasive encapsulated FVPTC (n = 11), and infiltrative FVPTC (n = 8) by two independent pathologists. Genetic alterations were analyzed by targeted next-generation sequencing using formalin-fixed, paraffin-embedded tissue samples and the clinicopathologic characteristics were retrospectively reviewed. There was no difference in preoperative cytologic classification between NIFTPs and invasive encapsulated FVPTCs, whereas infiltrative FVPTC was more likely to be Bethesda class VI than the encapsulated type (50% versus 9.5%; P = 0.033). Lymph node metastasis was not found in NIFTPs. There was no BRAFV600E mutation in NIFTPs, whereas one of 11 invasive encapsulated FVPTCs and three of 8 infiltrative FVPTCs harbored BRAFV600E. RAS mutations were frequently detected in encapsulated FVPTCs (5 of 10 NIFTPs and 4 of 11 invasive encapsulated FVPTCs) but were only detected in one case of the infiltrative type. There were no differences in molecular or clinicopathologic profiles between non-invasive and invasive encapsulated FVPTCs, except for lymph node metastasis and the presence of BRAFV600E. NIFTP has favorable pathologic characteristics with a high frequency of RAS mutations.

Pathological and Molecular Characteristics of Colorectal Cancer with Brain Metastases.

Background: Colorectal cancers (CRC) with brain metastases (BM) are scarcely described. The main objective of this study was to determine the molecular profile of CRC with BM. Methods: We included 82 CRC patients with BM. KRAS, NRAS, BRAF and mismatch repair (MMR) status were investigated on primary tumors (n = 82) and BM (n = 38). ALK, ROS1, cMET, HER-2, PD-1, PD-L1, CD3 and CD8 status were evaluated by immunohistochemistry, and when recommended, by fluorescence in situ hybridization. Results: In primary tumors, KRAS, NRAS and BRAF mutations were observed in 56%, 6%, and 6% of cases, respectively. No ROS1, ALK and cMET rearrangement was detected. Only one tumor presented HER-2 amplification. Molecular profiles were mostly concordant between BM and paired primary tumors, except for 9% of discordances for RAS mutation. CD3, CD8, PD-1 and PD-L1 expressions presented some discordance between primary tumors and BM. In multivariate analysis, multiple BM, lung metastases and PD-L1+ tumor were predictive of poor overall survival. Conclusions: CRCs with BM are associated with high frequency of RAS mutations and significant discordance for RAS mutational status between BM and paired primary tumors. Multiple BM, lung metastases and PD-L1+ have been identified as prognostic factors and can guide therapeutic decisions for CRC patients with BM.

Abstract 3909: HDAC inhibition in combination with MEK or BCL-2 inhibition as novel therapeutic strategies in multiple myeloma

Abstract Multiple myeloma (MM) is an incurable malignancy of plasma cells. It is the second most common hematologic cancer, affecting nearly 30,000 people in the United States annually. Substantial progress has been made in the past fifteen years in the treatment of MM due to the approval of several new classes of drugs. However, patients inevitably relapse and become refractory to existing therapies. Hence, there is an immediate unmet need to develop novel therapies for MM based on a better understanding of the disease biology. Mutations in RAS have been found to occur in about 40% of newly diagnosed MM patients, with the frequency increasing to around 70% in relapsed/refractory patients. Such mutations are absent in patients with the premalignant conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). Clearly, RAS mutations contribute to both disease progression and relapse. However, targeting the MEK/ERK pathway has been unsuccessful in MM patients to date. Given the high frequency of RAS mutations in MM, we hypothesized that targeting this pathway could still be a promising strategy when combined with existing agents that have multifaceted mechanisms to promote tumor cell death, such as the recently approved histone deacetylase (HDAC) inhibitor LBH589 (panobinostat). Our results clearly demonstrate that low doses of LBH589 in combination with the MEK inhibitor AZD6244 induce BIM-dependent synergistic cell death in several MM cell lines and patient cells. Our studies also suggest that mutations in RAS/RAF could serve as a predictive biomarker for sensitivity to AZD6244/LBH589. RAS/RAF mutations appear to confer Mcl-1 dependence in MM cells, in part by driving up the phosphorylation of Mcl-1. The AZD6244/LBH589 combination is able to decrease the phosphorylation of Mcl-1 at several sites, which dissociates BIM-Mcl-1 complexes, ultimately leading to activation of the intrinsic apoptosis pathway. Additionally, we identified that wild-type RAS/RAF cells have relatively lower levels of phospho-Mcl-1, as well as higher levels of Bcl-2 and phospho-Bcl-2 when compared to mutated RAS/RAF cells. This seems to confer functional Bcl-2 dependence. Consequently, we found that wild-type RAS/RAF cells are sensitive to the BH3-mimetic ABT199 (venetoclax) when combined with LBH589. Through ongoing experiments, we hope to further confirm the mechanism of action of both these combinations, identify the particular HDAC that is required to be inhibited for the observed synergy, and validate RAS/RAF mutational status as a biomarker for predicting sensitivity to either combination. Our findings have broad therapeutic potential given the prevalence of RAS mutations in MM. Moreover, the ABT199/LBH589 combination could emerge as a targeted therapy for wild-type RAS patients, perhaps broadening the scope and capacity of Bcl-2 inhibition in MM. Citation Format: Kevin C. Miller, Jessica Haug, Teresa Kimlinger, Sanjay Kumar, Wilson Gonsalves, S. Vincent Rajkumar, Shaji K. Kumar, Vijay Ramakrishnan. HDAC inhibition in combination with MEK or BCL-2 inhibition as novel therapeutic strategies in multiple myeloma [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 3909.

A Phase II Study of Pelareorep (REOLYSIN®) in Combination with Gemcitabine for Patients with Advanced Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with 1 and 5-year survival rates of ~18% and 7% respectively. FOLFIRINOX or gemcitabine in combination with nab-paclitaxel are standard treatment options for metastatic disease. However, both regimens are more toxic than gemcitabine alone. Pelareorep (REOLYSIN®), a proprietary isolate of reovirus Type 3 Dearing, has shown antitumor activity in clinical and preclinical models. In addition to direct cytotoxic effects, pelareorep can trigger antitumor immune responses. Due to the high frequency of RAS mutations in PDAC, we hypothesized that pelareorep would promote selective reovirus replication in pancreatic tumors and enhance the anticancer activity of gemcitabine. Chemotherapy-naïve patients with advanced PDAC were eligible for the study. The primary objective was Clinical Benefit Rate (complete response (CR) + partial response (PR) + stable disease (SD) ≥ 12 weeks) and secondary objectives include overall survival (OS), toxicity, and pharmacodynamics (PD) analysis. The study enrolled 34 patients; results included one partial response, 23 stable disease, and 5 progressive disease. The median OS was 10.2 months, with a 1- and 2-year survival rate of 45% and 24%, respectively. The treatment was well tolerated with manageable nonhematological toxicities. PD analysis revealed reovirus replication within pancreatic tumor and associated apoptosis. Upregulation of immune checkpoint marker PD-L1 suggests future consideration of combining oncolytic virus therapy with anti-PD-L1 inhibitors. We conclude that pelareorep complements single agent gemcitabine in PDAC.

Mutational Characterisation and Tracking Disease Progression Using Circulating Cell-Free Tumor DNA in Multiple Myeloma Patients

Background:Multiple myeloma (MM) currently relies on bone marrow (BM) biopsy for mutational characterisation, which does not capture the putative spatial and genetic heterogeneity of this multi-focal disease. Circulating cell-free tumour DNA (ctDNA) is a powerful non-invasive biomarker, which is being utilised to monitor tumor dynamics in a number of cancers. In this study, analysis of peripheral blood plasma (PL) - derived ctDNA and contemporaneously sourced BM MM cells was undertaken to determine if ctDNA can be utilised as an adjunct to BM biopsy for mutational characterisation and non-invasive therapeutic monitoring of disease progression.Methods:Blood (30ml) from MM patients was collected into Streck Cell-Free DNA BCT tubes and DNA extracted using the QIAamp circulating nucleic acid kit (Qiagen). BM aspirates from MM patients were CD138 enriched and subject to DNA extraction (Qiagen). Paired BM MM DNA and PL samples from 54 patients (New Diagnosis (ND) = 17; Relapsed/ Refractory (RR) = 35 and monoclonal gammopathy of undetermined significance (MGUS) =2] were analysed in the high-sensitive OnTargetTM mutation detection platform (OMD, Boreal Genomics) that included 96-mutations in the KRAS, NRAS, CTNNB1, EGFR, PIK3CA, TP53, FOXL2, GNAS and BRAF genes. OMD findings were validated with ddPCR (Biorad QX200 droplet digital PCR system). Sequential ctDNA quantitation with ddPCR through longitudinal PL tracking of specific clones in two patients for the monitoring of disease was also performed.Results:OMD detected a total of 141 mutations (BM and PL=42; BM-only=63 and PL only=36) with the presence of mutations in the PL (55.3%) authenticating the existence of ctDNA harbouring mutations. The detection of PL-only mutations (25.5%) signified the existence of mutant clones predominantly or exclusively, distant to the BM biopsy site. RR patients had a higher number of PL-only mutations compared to ND (34 vs 2 mutations), with none detected in MGUS, denoting that spatial and genetic heterogeneity is more evident in patients with advanced disease.Activating RAS mutations were highly prevalent in 34/54 patients (63%) harboring at least one RAS mutation. Specifically, KRAS mutations were found in 26/54 patients (48%), with 17/35 RR (48.5%), 9/17 ND (53%) and 1/2 MGUS patients harboring at least 1 KRAS mutation, indicating that mutations in this gene occur early in MM pathogenesis. NRAS mutations were found to be present at a higher frequency in RR patients compared to ND patients (45.7% vs 35.2%). BRAF mutations were equally distributed amongst RR and ND patients, in contrast, TP53 mutations were found exclusively in RR patients. Additionally, in 2 RR patients, a total of 5 PIK3CA mutations (E542K, E545K, H1047R, C420R, E81K) were present, 4 of which were found in PL-only, denoting the presence of these mutations distant to the BM biopsy site. Two ND patients had 1 GNAS mutation each (R201H and R201C). Preliminary validation of the OMD findings was performed in 12 patients using ddPCR with 90.9% concordance between the two platforms. Furthermore, 4/13 mutations (30.7%) that were negative in OMD were detected by ddPCR, indicating a higher sensitivity for ddPCR.Sequential ddPCR of ctDNA for Patient #1 with advanced relapsed disease for previously identified TP53 R273H and KRAS G12D mutations showed that the fractional abundance of KRAS G12D rapidly increased coincident with relapse of the disease, while that of TP53 273H did not change significantly over time. For Patient #2, relapsed disease was associated with the reappearance of mutant KRAS G12V and KRAS G12D clones that had been present at diagnosis in the BM and the emergence of 2 new clones NRAS G13D and NRAS Q61K.ConclusionsThis study provides the groundwork for utilisation of PL-ctDNA as an adjunct to BM biopsy for comprehensive mutational characterisation and as a non-invasive biomarker for therapeutic monitoring in MM patients. Importantly, a higher frequency of RAS mutations and presence of mutations in PIK3CA and GNAS, which have not been previously reported in MM, provides evidence of a more complex mutational landscape in MM than previously shown with BM whole exome sequencing studies. Furthermore, sequential PL tracking of specific mutant clones in two patients enabled monitoring of tumor dynamics. In conclusion, incorporating PL ctDNA evaluation may represent a significant advance in attempts to personalize future MM treatment strategies. DisclosuresMai:Boreal Genomics: Employment. Walsh:Boreal Genomics: Employment. Broemeling:Boreal Genomics: Employment. Marziali:Boreal Genomics: Employment. Wiggin:Boreal Genomics: Employment.

The RalGEF-Ral Effector Signaling Network: The Road Less Traveled for Anti-Ras Drug Discovery.

The high frequency of RAS mutations in human cancers (33%) has stimulated intense interest in the development of anti-Ras inhibitors for cancer therapy. Currently, the major focus of these efforts is centered on inhibitors of components involved in Ras downstream effector signaling. In particular, more than 40 inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase cascade and phosphoinositide 3-kinase-AKT-mTOR effector signaling networks are currently under clinical evaluation. However, these efforts are complicated by the fact that Ras can utilize at least 9 additional functionally distinct effectors, with at least 3 additional effectors with validated roles in Ras-mediated oncogenesis. Of these, the guanine nucleotide exchange factors of the Ras-like (Ral) small GTPases (RalGEFs) have emerged as important effectors of mutant Ras in pancreatic, colon, and other cancers. In this review, we summarize the evidence for the importance of this effector pathway in cancer and discuss possible directions for therapeutic inhibition of aberrant Ral activation and signaling.

RAS Mutations Contribute to Evolution of Chronic Myelomonocytic Leukemia to the Proliferative Variant

The biological and clinical heterogeneity of chronic myelomonocytic leukemia features renders its classification difficult. Moreover, because of the limited knowledge of the mechanisms involved in malignant evolution, chronic myelomonocytic leukemia remains a diagnostic and therapeutic challenge and a poor prognosis disease. We aimed to verify the biological and clinical significance of the discrimination, based on the leukocyte count, between myelodysplastic chronic myelomonocytic leukemia (MD-CMML) and myeloproliferative chronic myelomonocytic leukemia (MP-CMML). Peripheral blood samples from 22 patients classified as MD-CMML and 18 as MP-CMML were collected at different time points during disease course, and patients' clinical characteristics were examined. RAS mutational screening was done by sequencing and, for each substitution identified, a highly selective allele-specific PCR was set up to screen all specimens. MP-CMML patients showed a significantly poorer survival (P = 0.003) and a higher frequency of RAS mutations (P = 0.033) by sequencing compared with MD-CMML. Overall, five MD-CMML patients progressed to myeloproliferative disease: in two, allele-specific PCR unveiled low levels of the RAS mutations predominating in the myeloproliferative phase at the time of myelodysplastic disease, documenting for the first time the expansion of a RAS mutated clone in concomitance with chronic myelomonocytic leukemia evolution. Moreover, one of the progressed patients harbored the FLT3-ITD and two MP-CMML patients presented with the JAK2 V617F substitution. All these lesions were mutually exclusive. Our results strongly suggest RAS mutations to function as a secondary event that contributes to development of the chronic myelomonocytic leukemia variant with the poorer prognosis (MP-CMML) and therefore advise their detection to be implemented in chronic myelomonocytic leukemia diagnostics and monitoring.

Thematic review series: Lipid Posttranslational Modifications. Farnesyl transferase inhibitors

Some proteins undergo posttranslational modification by the addition of an isoprenyl lipid (farnesyl- or geranylgeranyl-isoprenoid) to a cysteine residue proximal to the C terminus. Protein isoprenylation promotes membrane association and contributes to protein-protein interactions. Farnesylated proteins include small GTPases, tyrosine phosphatases, nuclear lamina, cochaperones, and centromere-associated proteins. Prenylation is required for the transforming activity of Ras. Because of the high frequency of Ras mutations in cancer, farnesyl transferase inhibitors (FTIs) were investigated as a means to antagonize Ras function. Evaluation of FTIs led to the finding that both K- and N-Ras are alternatively modified by geranylgeranyl prenyltransferase-1 in FTI-treated cells. Geranylgeranylated forms of Ras retain the ability to associate with the plasma membrane and activate substrates. Despite this, FTIs are effective at inhibiting the growth of human tumor cells in vitro, suggesting that activity is dependent on blocking the farnesylation of other proteins. FTIs also inhibit the in vivo growth of human tumor xenografts and sensitize these models to chemotherapeutics, most notably taxanes. Several FTIs have entered clinical trials for various cancer indications. In some clinical settings, primarily hematologic malignancies, FTIs have displayed evidence of single-agent activity. Clinical studies in progress are exploring the antitumor activity of FTIs as single agents and in combination. This review will summarize the basic biology of FTIs, their antitumor activity in preclinical models, and the current status of clinical studies with these agents.

Molecular Epidemiological Study in Pediatric Acute Myeloid Leukemia.

It is hypothesized that AML arises from two cooperative types of mutations: type I mutations mainly induce proliferation and type II mutations involved in the maturation arrest. AML is a rare disease in children and few molecular data are available on pediatric AML. We therefore studied N-RAS, K-RAS, FLT3-ITD, FLT3 , C-KIT mutations (type I), and CEBPA mutations (type II) as well as FLT3, EVI-1 and WT1 gene expression in 77 de novo AML.Patients and methods: All the patients (aged 1 month-17 years, median age: 6.9 years, male/female ratio 1.26) were treated for de novo AML between 1995 and 2003 in two French institutions and prospectively enrolled in LAM91, LAM01 and APLs French collaborative protocols. According to the FAB classification the repartition was: M0:6.5%, M1: 5.2%, M2: 22%, M3: 13%, M4 :14.3%, M5 :30%, M7: 6.5% and unclassified :2.5%. Cytogenetics features according to the MRC classification were favorable, intermediate or poor in 25% (t(8;21) n=5; t(15,17) n=8, inv(16) n=5), 65% (normal n=20 and 11q23 abnormalities n=15) and 10% (−7, n=4) respectively. With a median follow-up of 26 months (range 2–98 months), Complete Remission was obtained in 92% (71/77) of patients, OS was 71% and EFS 61%. CEBPA, N-RAS, K-RAS, C-KIT and FLT3 mutations detection was performed by direct sequencing. FLT3, EVI-1 and WT1 transcripts were quantified by RQ-PCR.Results: (1) Frequency of N-RAS and K-RAS mutations were 11% (8/75) and 16% (12/75) respectively. RAS-mutated patients belonged to favorable (30%), intermediate (60%) and poor (10%) cytogenetic subgroups. In univariate analysis only N-RAS mutations is associated with adverse outcome (OS 37% vs 79%, p<0.05). (2) CEBPA mutations were found in 8% (6/75), mostly belonged to the intermediate cytogenetic risk subgroup (66%). (3) C-KIT mutations were observed in 4% (4/75) always associated with fusion CBFb/MYH11 transcript and excellent outcome. (4) FLT3-ITD and FLT3 Asp835 mutations were obtained in 12% (9/74) and 4% (3/74) of patients respectively. Cytogenetic subgroups were favorable (33%) and intermediate (67%). (5) At diagnosis FLT3 overexpression was detected in 34% (24/70) and 11q23 abnormalities were associated in 7/24 patients. (6) EVI-1 overexpression was found in 21% (16/76), belonged to intermediate (85%) and poor (15%) cytogenetic subgroups with a significant frequency in monosomy 7 (p<0.025). The EVI-1 expression was specifically expressed (p<0.001) in M5 and M7-FAB subtypes. (7) WT1 overexpression was detected in 81% (62/76).Conclusion: In total, 48% of de novo AML in children had a mutation in N-RAS, K-RAS, FLT3-ITD, FLT3 Asp835, C-KIT or CEBPA with a high frequency of RAS mutations (27%) compared to adult AML and a significantly bad survival. Additional gene expression quantification of EVI-1, FLT3 and WT1 allows MDR detection in 95% of patients.

The Role of Phosphatidylinositol 3-Kinase, Rho Family GTPases, and STAT3 in Ros-induced Cell Transformation

Using loss-of-function mutants of Ros and inducible epidermal growth factor receptor-Ros chimeras we investigated the role of various signaling pathways in Ros-induced cell transformation. Inhibition of the mitogen-activated protein kinase (MAPK) pathway with the MEK (MAP/extracellular signal-regulated kinase kinase) inhibitor PD98059 had little effect on the Ros-induced monolayer and anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells even though more than 70% of the MAPK was inhibited. In contrast, inhibiting the phosphatidylinositol 3-kinase (PI3K) pathway with the drug LY294002, a dominant negative mutant of PI3K, Deltap85, or the phosphatidylinositol phosphatase PTEN (phosphatase and tensin homologue deleted in chromosome ten) resulted in a dramatic reduction of v-Ros- and epidermal growth factor receptor-Ros-promoted anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells, respectively. Parallel and downstream components of PI3K signaling such as the Rho family GTPases (Rac, Rho, Cdc42) and the survival factor Akt were all shown to contribute to Ros-induced anchorage-independent growth, although Rac appeared to be less important for Ros-induced colony formation in NIH3T3 cells. Furthermore, the transformation-attenuated v-Ros mutants F419 and DI could be complemented by constitutively active mutants of PI3K and Akt. Finally, we found that overexpressing a constitutively active mutant of STAT3 (STAT3C) conferred a resistance to the inhibition of Ros-induced anchorage-independent growth by LY294002, suggesting a possible overlap of functions between PI3K and STAT3 signaling in mediating Ros-induced anchorage-independent growth.

Characteristic association between K- ras gene mutation with loss of heterozygosity in X-ray-induced thymic lymphomas of the B6C3F1 mouse

Purpose : To elucidate the characteristics of radiation carcinogenesis, the spectra of K- and N- ras oncogene mutations, loss of heterozygosity (LOH) and their association in X-ray-induced thymic lymphomas (TL) were determined by comparing with those of N -ethyl- N -nitrosourea (ENU)-induced and spontaneously occurring TL. Materials and methods : TL that arose in untreated, X-ray-irradiated and ENU-treated B6C3F1 mice were examined both for K- and N- ras mutations by PCR-SSCP and DNA sequencing and for LOH by PCR with polymorphic microsatellite markers. Results : (1) ras gene mutations were found in a proportion of TL from X-ray-exposed (~20%) and ENU-treated (30-40%) mice while no ras gene mutations were found in spontaneous TL. N- ras mutations were rare. (2) The spectrum of ras gene mutations was diverse and seemed to differ little between X-ray-induced and ENU-induced TL, even though there was a higher frequency of ras mutations in ENU-induced TL that clustered to K- ras codon 12. (3) The X-ray-induced TL showing K- ras mutation were associated with LOH on chromosome 6, while those showing no K- ras mutation were associated with high frequency of LOH on chromosomes 4, 11 and 12. Conclusion : These results demonstrate that, in the B6C3F1 mouse TL, X-ray-induced lymphomagenesis showed both the co-expression, yet low occurrence of allelic imbalance on chromosome 6 and K- ras mutation, and exclusive expression of frequent allelic imbalance on chromosomes 4, 11 and 12 and K- ras mutation.

Induction of cell death by stimulation of protein kinase C in human epithelial cells expressing a mutant ras oncogene: a potential therapeutic target.

Ras oncogene activation is a key genetic event in several types of human cancer, making its signal pathways an ideal target for novel therapies. We previously showed that expression of mutant ras sensitizes human thyroid epithelial cells to induction of cell death by treatment with phorbol 12-myristate 13-acetate (PMA) and other phorbol esters. We have now investigated further the nature and mechanism of this cell death using both primary and cell line models. The cytotoxic effect of PMA could be blocked by bisindolylmaleimide (GF 109203X), a well-characterized inhibitor of c and n protein kinase C (PKC) isoforms, and by prior down-regulation of PKC, indicating that it is mediated by acute stimulation, rather than down-regulation. Western analysis identified two candidate isoforms--alpha and epsilon--both of which showed PMA-induced subcellular translocation, either or both of which may be necessary for PMA-induced cell death. Immunofluorescence showed that PMA induced a rapid nuclear translocation of p42 MAP kinase of similar magnitude in the presence or absence of mutant ras expression. Cell death exhibited the microscopic features (chromatin condensation, TdT labelling) and DNA fragmentation typical of apoptosis but after a surprising lag (4 days). Taken together with recent models of ras-modulated apoptosis, our data suggest that activation of the MAPK pathway by PMA tips the balance of pro- and anti-apoptotic signals generated by ras in favour of apoptosis. The high frequency of ras mutations in some cancers, such as cancer of the pancreas, which are refractory to conventional chemotherapy, together with the potential for stimulating PKC by cell-permeant pharmacological agents, makes this an attractive therapeutic approach.

Both K-ras and H-ras protooncogene mutations are associated with Harderian gland tumorigenesis in B6C3F1 mice exposed to isoprene for 26 weeks.

Isoprene is the 2-methyl analog of 1,3-butadiene, a genotoxic and carcinogenic compound in rats and mice. Male B6C3F1 mice were exposed to 0, 2200 or 7000 ppm isoprene by inhalation (6 h/day; 5 days/week) for 26 weeks. Following a 26-week recovery period, an increased incidence of Harderian gland (HG) neoplasms was observed at both concentrations. The present study was designed to characterize genetic alterations in the K-ras and H-ras protooncogenes in HG neoplasms. Mutations in K-ras and H-ras were identified by single-strand conformational analysis and direct sequencing of polymerase chain reaction (PCR) amplified DNA, isolated from paraffin-embedded sections of HG neoplasms. A higher frequency of ras mutations, in particular K-ras mutations, was detected in isoprene-induced neoplasms than in 1,3-butadiene-induced or control HG neoplasms. All of the isoprene-induced HG neoplasms exhibited activated K-ras (60%) or H-ras (40%) mutations. In contrast, ras mutations were detected in 69% of HG neoplasms from 1,3-butadiene exposed mice (14% K-ras and 55% H-ras) and in 56% of HG neoplasms obtained from control B6C3F1 mice (8% K-ras and 48% H-ras). The predominant mutations in isoprene-induced HG neoplasms, but not in previously or newly analysed 1,3-butadiene-induced HG neoplasms, consisted of A-->T transversions (CAA-->CTA) at K-ras codon 61 (15/30) and C-->A transversions (CAA-->AAA) at H-ras codon 61 (8/30). Two-thirds of the K-ras CTA mutations were detected in HG neoplasms from the 2200 ppm exposure group while one-third was present in the 7000 ppm group. Isoprene-induced HG neoplasms with K-ras or H-ras mutations had an elevated proliferating cell nuclear antigen (PCNA) index, compared to spontaneous HG neoplasms without ras mutations. The high frequency and specificity of the ras mutation profile suggest that ras protooncogene activation contributes to isoprene-induced HG tumorigenesis.