Presence Of RAS Mutations Research Articles

Disparities in prophylactic tetracycline use among patients with metastatic colorectal cancer undergoing treatment with EGFR inhibitors.

109 Background: Epidermal growth factor receptor (EGFR) inhibitors have been shown to improve survival in metastatic colorectal cancer and are considered a standard of care option for patients with RAS/RAF wild-type disease. However, anti-EGFR agents are associated with an acneiform rash that can lead to decreased quality of life and early discontinuation of treatment. Prophylactic doxycycline has been shown in a randomized clinical trial to decrease any skin toxicity ≥ grade 2 by 50%. In this study we sought to determine the rate of prophylactic tetracycline use in metastatic colorectal cancer patients receiving anti-EGFR therapy in the United States. Methods: The Flatiron Health Electronic Health Record-derived, de-identified database was used to analyze the records of patients who were at least 18 years old, diagnosed with metastatic colorectal cancer, and received treatment with an anti-EGFR agent (cetuximab or panitumumab). Patients were stratified by receipt of prophylactic tetracycline versus not. This was defined by an order for a tetracycline 28 days before to 1 day after initiation of an anti-EGFR agent. Demographic and clinical characteristics were compared via multivariable logistic regression. The change in percentage of patients who received a prophylactic tetracycline over time was assessed and was compared to the rate of anti-emetic prescription which served as a control to ensure drug administration data was accurately captured. Results: The records of 6,713 patients who met inclusion criteria and received an anti-EGFR agent between 2013 and 2023 were included in our analysis. A logistic regression model assessing the association between patient characteristics and the receipt of a prophylactic tetracycline suggested that older age, race (black/African American or Other), presence of RAS mutation, and higher line of therapy were all significantly associated with not receiving prophylactic tetracyclines. Other patient characteristics including gender, ECOG status, mismatch repair status, and RAF mutation status did not appear to impact the likelihood of receiving a prophylactic tetracycline. The rate of prophylactic tetracycline use increased from 10.9% in 2013 to 22.3% in 2023 while the rate of prophylactic anti-emetic remained stable at around 82%. Conclusions: This real-world analysis suggests the use of preemptive treatment for skin toxicity in patients with metastatic colorectal cancer who are treated with an anti-EGFR in US is suboptimal. Disparities in cancer care are also evidenced by lower use of tetracyclines in elderly and African Americans. Randomized controlled trials support the use of prophylactic tetracycline in these patients; thus, further research into the reason for this practice gap is necessary. Educational efforts should be undertaken to improve tetracycline prescribing rates in this population.

Decoding RAS mutations in thyroid cancer: A meta-analysis unveils specific links to distant metastasis and increased mortality.

RAS mutations are common in thyroid cancer, but their impact on clinical outcomes remains controversial. This study aimed to evaluate the prevalence of RAS mutations in thyroid cancer and their association with various clinical and pathological features. We conducted a systematic review and meta-analysis of studies reporting on RAS mutations in thyroid cancer. Both one-arm and pairwise meta-analyses were performed to compare outcomes between RAS-mutated (RAS+) and wild-type (RAS-) thyroid cancers. Our analysis included 2552 thyroid cancer patients from 17 studies. The overall prevalence of RAS mutations was 35.4% (95% CI: 22.7%-50.7%). NRAS mutations were most common (69.47%, 95% CI: 66.15%-72.66%), followed by HRAS (25.83%, 95% CI: 22.77%-29.14%) and KRAS (6.92%, 95% CI: 5.27%-9.04%). No statistically significant differences were found between RAS+ and RAS- cases in rates of T1/2 tumors, lymph node metastasis, extrathyroidal extension, or recurrence. The risk of distant metastasis was significantly higher in RAS+ cases (15%, 95% CI: 6%-34%) compared to RAS- cases (4%, 95% CI: 1%-12%), with a relative risk of 3.23 (95% CI: 1.49-7.02). Notably, RAS+ cases showed a significantly higher mortality rate (8%, 95% CI: 3%-18%) compared to RAS- cases (2%, 95% CI: 1%-5%), with a relative risk of 4.36 (95% CI: 1.23-15.50, p=0.03). While RAS mutations are prevalent in thyroid cancer, they do not significantly impact most clinical and pathological features. However, the presence of RAS mutations is associated with a significantly higher risk of distant metastasis and mortality, suggesting their potential role as a prognostic marker in thyroid cancer. These findings underscore the importance of RAS mutation testing in risk stratification and treatment planning for thyroid cancer patients.

Significance of apparent diffusion coefficient in diagnosis of rectal carcinoma.

The apparent diffusion coefficient (ADC) is a quantitative parameter that facilitates the detection and reliable differentiation of rectal cancer. MR differentiation between rectal carcinoma, post-radiation proctitis, and normal rectal wall with the ADC values and their comparison depending onthe level of tumor markers and pathohistological characteristics of rectal carcinoma. The retrospective study performed at the Oncology Institute of Vojvodina included 300 patients, 100 each with rectal cancer, post-radiation proctitis, and normal rectum. Mean ADC values were obtained by measuring the region of interest (ROI) of the rectal wall. Rectal cancer showed lower ADC values (0.665 ± 0.086 x 10-3mm2/s) compared to both post-radiation proctitis (1.648 ± 0.268 x 10-3mm2/s) and normal rectum (1.180 ± 0.110 x 10-3mm2/s) (p<0.001). No significant differences in ADC values were observed between different grades of rectal cancer (p=0.874; p>0.05), depending on the presence of metastases in the lymph nodes (p=0.357; p>0.05), different TN stage (p=0.196; p>0.05), local spread of the tumor (p=0.312; p>0.05), the presence of RAS mutation (p=0.829; p>0.05) and the value of tumor markers (p=0.923; p>0.05). ADC values below 1.013 x 10-3mm2/s with 100% sensitivity and 96% specificity indicate the presence of rectal cancer in relation to normal wall, with a positive predictive value of 96.1% and a negative of 100%. ADC values below 1.255 x 10-3mm2/s with 100% sensitivity and 95% specificity indicate rectal cancer in relation to post-radiation proctitis. ADC values above 1.339 x 10-3mm2/s with 87% sensitivity and 89% specificity indicate post-radiation proctitis in relation to normal wall. The ADC is a useful marker in differentiating between rectal cancer, post-radiation proctitis, and normal rectal wall with high sensitivity and specificity, but it cannot be used to distinguish the histological grades of rectal cancer, nor other pathohistological parameters.

Comprehensive multigene profiling impact on clinical decisions in patients with advanced cancers: A multicenter, retrospective analysis.

e15163 Background: Comprehensive molecular profiling (CMP) via next generation sequencing (NGS) is currently applied in clinical practice and has enabled the detection of biomarkers of response to targeted treatment. Given the lack of the data about optimal range of analyzed alterations, the clinical usefulness of CMP has not been established. To address the challenges in incorporating genomics in treatment of oncology patients, we conducted a retrospective study. Methods: Preliminary analysis of NGS reports and clinical data of patients with advanced solid tumors was performed. CMP was conducted in 261 cancer patients (non-small cell lung cancer - 30,6%, colorectal cancer - 24,8%, pancreatic cancer - 7,4%, breast cancer- 6,2%; 59,3% were female, median number of prior systemic treatment lines was 2 (0-12)). In the present study, tumor molecular profile analysis was performed using 160 - 400 gene NGS panels, containing the majority of clinically significant genes for cancer treatment selection. We assessed the percentage of patients who were recommended to receive molecularly-matched therapy (MMT) after CMP, immunotherapy (IT) distribution of recommended therapies according to ESCAT tiers, median progression-free survival (PFS) – ratio ( = PFS after CMP / PFS before CMP, PFS2/PFS1), median PFS2. Results: MMT after CMP was recommended for 55 (21,1%) patients. ESCAT tiers could be assessed in 48 MMT patients (87%). For 31% of patients, administered therapy was considered as ESCAT I tier recommendation (data on mPFS2/1, mPFS2 not available). ESCAT tiers II, III, IV and IT administration were considered in 31, 8, 8 and 22% of patients respectively (detailed data is presented in Table 1). Most commonly altered genes were TP53 (53%), RAS (29%), APC (18%), CDKN2A/B (16%), PIK3CA(14%), NF1 (10%), BRCA1/2 (9%). Positive predictive factors for MMT after CMP were MSI-high status (HR 0,13, 95% CI 0,03 - 0,67), presence of EGFR alteration (HR 0,32, 95% CI 0,12 - 0,86). Presence of RAS mutation was considered a negative predictive factor (HR 2,78, 1,07 - 7,26). Conclusions: Available NGS panels offer a wide range of analyzed alterations that are not feasible in clinical practice. Most of the alterations that can cause impact on treatment plan can be detected with other methods than NGS. Further studies are needed to determine optimal range of alterations depending on tumor site. [Table: see text]

Real-world data of early-onset colorectal cancer (EOCRC) in private health system in Brazil.

e18830 Background: Incidence of EOCRC has been sharply rising in the past decades in the US and present distinct clinical features when compared to late-onset CRC (LOCRC). Data from other countries are scarce. We compared clinical characteristics and treatment outcomes between patients ≤45y (EOCRC) and ≥60y (LOCRC). Methods: Retrospective study evaluating 510 patients with metastatic CRC treated at 5 Oncoclinicas centers in Brazil from January 2011 to December 2019. Demographic and clinical data were retrieved from electronic medical records. The median OS was calculated by Kaplan-Meier method and prognostic factors were evaluated via multivariate Cox Regression. Results: After excluding patients with ages between 46 and 59, 326 patients were identified. EOCRC was represented by 72 (22%) patients (median age 39) and LOCRC by 254 patients (median age 69). Median follow-up was 39.5m, with 231 deaths (71%). Median OS was 33.2m and 40.9m (p = 0.971), respectively. Likewise, stage IV at presentation was similar between the groups (68% vs 58%, p = 0.215), as well as the rate of dMMR status (4% vs 6%, p = 0.655). Clinical and treatment characteristics that were statistically different between the groups are described in Table 1. In multivariate analysis, presence of RAS mutations (HR:1.83), and undergoing triplet chemotherapy (HR:3.24), primary tumor resection (HR:0.40), and metastasectomy (HR:0.45) were the variables with prognostic effect on OS. Conclusions: In this real-world analysis of mCRC, EOCRC patients presented similar clinical presentation and OS, but they were more aggressively treated compared to the older patients. There is no evidence that more intensive treatments translate into superior clinical outcomes in young patients. Real-world data have been progressively adopted in clinical practice and should be considered before clinical decision making in order to offer treatments with minimal toxicity burden to the patients. Future studies with larger population are needed and may bring additional data. [Table: see text]

Evaluation of Overall Survival Rate of Patients with Metastatic Colorectal Cancer Depending on Choice of Treatment, Location of Primary Focus and RAS Genes Mutation Status

INTRODUCTION: Morbidity with colorectal cancer (CRC) in the Yaroslavl region (YR) accounts for 13.4% of all cases identified in 2021, and ranks third after skin cancer and lung cancer. In the mortality structure, CRC makes 14.2% and is the second leading cause of death. Locally advanced and metastatic forms of the tumor process are identified in more than half the patients.&#x0D; AIM: To evaluate the overall survival rate of patients with metastatic CRC (mCRC) in the territory of the YR depending on the volume of surgical treatment, chemo- and targeted treatment regimens and the presence of RAS genes mutations.&#x0D; MATERIALS AND METHODS: On the base of the Yaroslavl Regional Clinical Oncology Hospital, the data of 291 patients with mCRC who underwent treatment in the period from 2015 to 2022, were analyzed. The mean age of patients was 63.0 8.6 years. There were 52% of men (n = 151), and 48% of women (n = 140). The patients were divided to two groups depending on the status of RAS genes mutations: group I (n = 145) patients with the identified mutation; group II (n = 146) patients with wild-type mutation. The patients were additionally divided to three subgroups depending on the type of treatment: subgroup A (58.1%; n = 169) removal of the primary focus (PF) in combination with antitumor chemotherapy (CT); subgroup B (31.6%; n = 92) CT without surgical treatment; subgroup C (10.3%; n = 30) removal of the PF and resection of liver metastases in combination with CT.&#x0D; RESULTS: The overall survival rate (OSR) depending on the type of treatment was in subgroup A 21.0 (95% confidence interval (CI) 18.6-23.3) months; in subgroup B 11.2 (95% CI 9.912.5) months; in subgroup C 21.0 (95% confidence interval (CI) 18.623.3) months. OSR with RAS mutation: in subgroup 1A 17.7 (95% CI 13.821.7) months; in subgroup IB 11.1 (95% CI 8.313.2) months; in subgroup IC 13.2 (95% CI 4.0722.7) months. OSR with wild-type mutation: subgroup IIA: Cetuximab 33.6 (95% CI 26.740.4) months, Panitumumab 23.8 (95% CI 19.727.9) months (p = 0.01); subgroup IIB: Cetuximab 22.3 (95% CI 17.027.5) months, Panitumumab 15.2 (95% CI 10.719.6) months (p = 0.012); subgroup IIC: Cetuximab 27.5 (95% CI 17.837.1) months, Panitumumab 38.8 (95% CI 13.963.6) months (p = 0.013).&#x0D; CONCLUSIONS: In patients with mutation in RAS genes, the best OSR values were noted in case of surgical removal of the PF in combination with palliative drug therapy. In patients with wild-type mutation of RAS genes the best OSR parameters were recorded in surgical removal of the PF and of metastases in the liver in combination with palliative polyCT and Panitumumab. The lowest OSR was found in the subgroup of patients with use of CT without surgical treatment in the presence of RAS mutation. High OSR parameters were found with mutation in G13codon, and with use of surgical treatment with mutation in A146 codon.

Retrospective evaluation of platelet indices and RAS mutations in patients with colon cancer

Aims: Colon cancers are one of the most common cancer types in the world and cause significant mortality. The presence of RAS mutation is also associated with poor prognosis in colon cancers and plays an important role in the choice of treatment. Platelet indices, which have recently been evaluated with an easy-to-reach method, have been discussed as an inflammatory marker in many studies. In this study, it was aimed to investigate the relationship between platelet distribution width (PDW), colon cancer stage and the presence of RAS mutation. Methods: File records of 132 patients who were followed up with the diagnosis of colon cancer in Kırıkkale University Faculty of Medicine, Department of Internal Medicine and Medical Oncology Department between January 1, 2015 and January 1, 2021 were retrospectively analyzed. Patients with RAS mutations and who did not receive chemotherapy were included in the study. Patients with and without RAS mutation were divided into two groups and their platelet index (especially PDW) values were compared. PDW values were compared according to the stages of the patients. The correlation between stage and PDW was examined. Results: Of the 132 patients included in the study, 82 (62.1%) were male and 50 (37.9%) were female. Participants; 12 (9.1%) stage 1, 23 (17.4%) stage 2, 29 (22%) stage 3, 68 (51.5%) stage 4 colon cancer patients. There were no RAS mutations in 73 (55.3%)patients, and 59 (44.7%) patients had RAS mutations. There was no significant difference in PDW between the two groups with negative and positive RAS mutations (p=0.826). According to the stages; PDW values were significantly different between the four stages (X2=9.878, p=0.020). Conclusion: In the study, PDW increased as the stage increased, but there was no significant relationship between RAS mutation and PDW. This was attributed to the fact that as the stage increases, the level of inflammation may be associated with an increase. Larger studies on this subject are needed.

Immunotherapy in RAS mutant mCRC: Could CTLA-4 blockade increase the efficacy of anti PD-1 agents? Preliminary clinical results of the NERDY study.

218 Background: Immune checkpoint inhibitors (ICI) showed high efficacy in both first and subsequent lines in metastatic colorectal cancer with mismatch repair deficiency (dMMR-mCRC); however, they still fail in a minority of patients (pts). In non-preplanned analyses from previous studies, RAS mutations ( RASm) have been related to limited activity of ICI monotherapy (ICIm) as compared to ICI doublets (ICId) in dMMR-mCRC. Emerging data suggest different immunological features in presence of RASm, resulting in lower immunogenicity. Methods: NERDY is a retrospective, monocentric study designed to investigate the effect of ICIm and ICId on dMMR-mCRC basing on RASm. Pts with dMMR-mCRC treated with ICIm/ICId at our Institute were included. Clinical-pathological features for each patient were collected. On primary tumor specimens, proinflammatory pathways and CMS subgroup will be assessed by Nanostring and RNA-Seq respectively. The study is exploratory and no formal hypothesis has been postulated. Both PFS and OS were calculated with Kaplan-Meier. Cox proportional hazard model was adopted in the interaction tests. Primary objective was to assess OS and PFS according to RAS in dMMR-mCRC pts treated with ICIm or ICId. Secondary objectives were to describe the inflammatory infiltrate and TMB in dMMR-mCRC according to RAS status. Results: From June 2015 to January 2022, a total of 126 consecutive dMMR-mCRC pts treated with ICI were included, 33 RASm/93 RASwt. RASm pts were more frequently males (p=0.015) and younger (median age 47 vs 65). An imbalance was observed in sidedness (more right-CRC in RASwt than in RASm as BRAF effect, p=0.001) and timing of ICI (administered in later lines in RASm, p=0.013). No differences in ECOG-PS, histotype, disease burden, stage at diagnosis, treatment with ICIm vs ICId and best response. At a median follow up of 53.5 months (95%CI 34.7-56.9), a trend toward longer PFS in pts treated with ICId over ICIm was found in the overall population (HR 0.62; 95%CI 0.36-1.05; p=0.055), being significantly longer in RASm-only pts (HR 0.41; 95%CI 0.13-1.28; p=0.047) but not in RASwt-only pts (HR 0.64; 95%CI 0.34-1.20; p=0.139). No difference was observed in OS between ICId and ICIm in the overall population (HR 0.64; 95%CI 0.36-1.16; p=0.121), in RASwt (HR 0.59; 95%CI 0.29-1.20; p=0.132) nor in RASm pts (HR 0.59; 95%CI 0.19-1.78; p=0.275). Interaction test for RAS and ICI treatment type was not significant for PFS (HR 0.63; 95%CI 0.21-1.94; p=0.423) nor for OS (HR=1.00; 95%CI 0.29-3.41; p=0.999). Conclusions: Preliminary clinical results of the NERDY study suggest enhanced activity of ICId compared to ICIm in pts with RASm dMMR-mCRC. Further data are expected from pending translational analyses and a pre-planned adjunctive cohort from two other Italian centers will be used for external validation.

Prognostic value of gender and primary tumor location in metastatic colon cancer.

Sex might influence prognosis in patients affected by colorectal cancer. We retrospectively studied a cohort of patients affected by metastatic colon cancer (mCC) stratified by sex and primary tumor location. RAS mutational status was also included in the analysis. Overall, 616 patients met the eligibility criteria, 261 women and 355 men. Neither gender, nor RAS mutational status influenced overall survival (OS) in the entire population. As expected, patients with right-sided colon cancer (RCC) had a significant shorter OS compared to those with left-sided colon cancer (LCC) (21.3 vs 33.1 months, p= 0.002). When the analysis was performed stratifying for gender, RCC retained worse prognosis among men (OS 20.5 vs 33.9 months, p= 0.008), but not among women (p= 0.132). Similarly, the presence of RAS mutations had no prognostic effect in women, but was significantly associate with shorter survival in men (OS 29.5 vs 33.7 months, p= 0.046). In addition, when comparing clinical outcome of women or men according to sidedness and RAS mutational status, RCC was associated with dismal prognosis only in men with RAS mutated tumor (OS 17.2 vs 32.3 months, p= 0.008). Our study highlights the importance of gender in the outcome of patients with mCC.

Molecular Genetic Investigation of Digital Melanoma in Dogs.

Canine digital melanoma, in contrast to canine oral melanoma, is still largely unexplored at the molecular genetic level. The aim of this study was to detect mutant genes in digital melanoma. Paraffin-embedded samples from 86 canine digital melanomas were examined for the BRAF V595E variant by digital droplet PCR (ddPCR), and for exon 11 mutations in c-kit. Furthermore, exons 2 and 3 of KRAS and NRAS were analysed by Sanger sequencing. Copy number variations (CNV) of KITLG in genomic DNA were analysed from nine dogs. The BRAF V595E variant was absent and in c-kit, a single nucleotide polymorphism was found in 16/70 tumours (23%). The number of copies of KITLG varied between 4 and 6. KRAS exon 2 codons 12 and 13 were mutated in 22/86 (25.6%) of the melanomas examined. Other mutually exclusive RAS mutations were found within the hotspot loci, i.e., KRAS exon 3 codon 61: 2/55 (3.6%); NRAS exon 2 codons 12 and 13: 2/83 (2.4%); and NRAS exon 3 codon 61: 9/86 (10.5%). However, no correlation could be established between histological malignancy criteria, survival times and the presence of RAS mutations. In summary, canine digital melanoma differs from molecular genetic data of canine oral melanoma and human melanoma, especially regarding the proportion of RAS mutations.

Abstract 2233: Landscape of driver mutations in MAPK/PI3K/AKT signaling pathways reveals insights into therapeutic targeting strategies

Abstract Alterations in the signaling cascade of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) pathways are common mechanisms of cancer initiation and progression. RAS proteins (KRAS/HRAS/NRAS), a crucial component of this cascade, are often found to be activated in cancer cells. Recent advancements in RAS targeting offer new treatment strategies for patients, however studies suggest that the clinical responses may be tissue-dependent. Thus, understanding the landscape of RAS alterations and the concomitant mutations observed in their effector pathways may be crucial in developing appropriate disease-specific therapeutic strategies for effectively addressing this biology. In this study, we utilized comprehensive genomic profiling data from 282,792 cancer patients across 77 tumor types, tested in the course of routine clinical care (FoundationOne) to assess the landscape of gene alterations within this signaling cascade. RAS alterations were identified in 27% of the cohort. Mutations in PI3K and PTEN were also frequent (&amp;gt;10%); alterations in FGFR1-4, EGFR, RAF (ARAF/BRAF/RAF1) and ERBB2 were each identified in over 5% of samples. A closer inspection of non-small cell lung carcinomas (NSCLC, n=55,893), colorectal carcinomas (CRC, n=35,104) and pancreatic cancers (PC, n=17,447), revealed distinct mutational spectrums. RAS alterations, particularly KRAS, exhibited varying prevalence: PC (85%), CRC (54%) and NSCLC (32%). In NSCLC, 35% of the KRAS alterations were identified to be G12C. In contrast, the predominant KRAS alterations in CRC and PC were G12D (29% CRC, 41% PC) and G12V (20% CRC, 30% PC). Further, only CRCs exhibited a high rate of KRAS G13D (16% of all KRAS alterations); KRAS G12R was found exclusively to PC (15%) and KRAS amplifications were most frequently identified in NSCLC (11%). Distinct patterns were also observed in genes upstream and downstream of the RAS proteins. Alterations in ALK, EGFR, STK11 and MET were significantly more prevalent in NSCLC (P &amp;lt; 10−5); whereas gene alterations in PI3K, RAF, MTOR and ERBB2 were more frequently identified in CRC (P &amp;lt; 10−5). In contrast, PC showed a predominant presence of RAS mutations, with limited mutations in other genes of the signaling cascade. Of note, PI3K alterations were identified at a much lower prevalence in PC (4%) compared to NSCLC (11%) and CRC (19%). Our findings suggest a tissue-dependent spectrum of driver mutations exist across the MAPK/PI3K/AKT signaling pathways. The observed mutational profiles may explain some of the efficacy differences observed by targeting RAS across these diseases and have implications on acquired resistance mechanisms in a tissue-specific manner. Additional insights may be gleaned by investigating immunotherapy related biomarkers with these alterations to suggest combination strategies for effectively targeting the RAS signaling cascade. Citation Format: Smruthy Sivakumar, Ethan S. Sokol, Garrett M. Frampton, Priti S. Hegde, David Fabrizio. Landscape of driver mutations in MAPK/PI3K/AKT signaling pathways reveals insights into therapeutic targeting strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2233.

NeoRAS wild-type in metastatic colorectal cancer: Myth or truth?—Case series and review of the literature

Upfront KRAS and NRAS gene testing (‘RAS’) is the standard of care for metastatic colorectal cancer (mCRC), to guide first-line treatment. The presence of RAS mutation (MT) is a negative predictor for the efficacy of anti-EGFR antibodies and the use of cetuximab and panitumumab is restricted to RAS wild-type (WT) mCRC. Conversion from RAS WT to RAS MT mCRC after treatment with anti-EGFR antibodies is a known and well-described acquired resistance mechanism. The by far less frequent ‘NeoRAS wild-type’ phenomenon (reversion from RAS MT to RAS WT) has recently drawn attention. The proposed effect of chemotherapy on RAS status in mCRC patients is not fully understood. Because of the intriguing biological consequence of a RAS MT to RAS WT reversion, subsequent treatment of NeoRAS WT patients with anti-EGFR antibodies is increasingly being discussed. Here, we report three clinical cases of NeoRAS WT mCRC patients, which received standard-of-care regimens for RAS MT mCRC. Anti-EGFR antibodies were used in two out of three patients after progression of the disease. One of the patients had a long-term response. In line with our observations, NeoRAS WT phenomenon occurs in clinical practice. Retesting of RAS status during treatment should be discussed in patients with unusual long-term clinical courses of RAS MT mCRC to optimise treatment strategy and to evaluate the use of anti-EGFR antibodies.

Evidence of Cooperation between Hippo Pathway and RAS Mutation in Thyroid Carcinomas.

Simple SummaryRAS mutations have been reported in a wide range of thyroid cancer histological types, from benign to aggressive phenotypes. The presence of RAS mutations in benign lesions suggests that the mutation alone is unlikely to lead to a malignant transformation per se, and thus, additional aberrations are necessary for this transformation. In this study, we initially screened a cohort of 120 thyroid carcinomas with a panel of known driver mutations and identified 11 RAS-mutated samples. An RNA-Seq analysis of those 11 RAS-positive samples identified that the Hippo pathway was both mutated and differentially expressed in the RAS-positive tumors. The gene expression analysis of 60 RAS-positive The Cancer Genome Atlas (TCGA) papillary thyroid carcinomas (PTC) samples supported our findings. Thyroid cancer incidences have been steadily increasing worldwide and are projected to become the fourth leading cancer diagnosis by 2030. Improved diagnosis and prognosis predictions for this type of cancer depend on understanding its genetic bases and disease biology. RAS mutations have been found in a wide range of thyroid tumors, from benign to aggressive thyroid carcinomas. Based on that and in vivo studies, it has been suggested that RAS cooperates with other driver mutations to induce tumorigenesis. This study aims to identify genetic alterations or pathways that cooperate with the RAS mutation in the pathogenesis of thyroid cancer. From a cohort of 120 thyroid carcinomas, 11 RAS-mutated samples were identified. The samples were subjected to RNA-Sequencing analyses. The mutation analysis in our eleven RAS-positive cases uncovered that four genes that belong to the Hippo pathway were mutated. The gene expression analysis revealed that this pathway was dysregulated in the RAS-positive samples. We additionally explored the mutational status and expression profiling of 60 RAS-positive papillary thyroid carcinomas (PTC) from The Cancer Genome Atlas (TCGA) cohort. Altogether, the mutational landscape and pathway enrichment analysis (gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genome (KEGG)) detected the Hippo pathway as dysregulated in RAS-positive thyroid carcinomas. Finally, we suggest a crosstalk between the Hippo and other signaling pathways, such as Wnt and BMP.

Double Genetic Hits and Subclonal Mosaicism in the Ras Signaling Pathway in Myeloid Neoplasia

The high prevalence of oncogenic mutations in the RAS family genes including their down or upstream partners (e.g., NRAS, KRAS, PTPN11, RIT1α) has strong implication in abnormal Ras signaling in the pathogenesis of myeloid neoplasia (MN). For many years RAS mutations have been considered non-targetable. However, recently multiple targeted agents have been introduced as possible future therapies. While common in JMML, RAS mutations are rarely seen in adult MN as the ancestral event. Instead they serve as subclonal hits. To that end, the clinical impact of RAS mutations has been controversial: whereas some studies did not find prognostic impact, others demonstrated that these mutations may confer a poor prognosis. Nevertheless, molecular analysis of core binding factor AML patients showed the coexistence of RAS clones harboring independent hits targeting the same pathway. We stipulate that occurrence of RAS mutations is not random and reasoned that studying cases with multiple RAS mutant subclones may reveal distinct molecular or etio-pathologic background attracting the emergence of RAS mutations as a way to augment MYC activity as a ubiquitous pathway of clonal progression. In a review of a molecular collection of data of a cohort of MN patients (n=1876) including MDS (n=692), MDS/MPN (n=282), AML (pAML, 710; sAML, 192) we found RAS mutations in 21% (403/1876) of the patients. Among them we encountered a subgroup of patients (9%; 38/403) harboring multiple RAS mutations. These multiple hits affected PTPN11, NRAS, KRAS and RIT1-α. Most of these multiple RAS mutants represented a subclonal mosaicism rather than biallelic subclones as indicated by single cell DNA sequencing. Three patients were assessed serially (2 patients/ 2 time points and 1 patient/ 5 time points). In one case NRAS (VAF-24%) and PTPN11 (VAF-28%) were acquired at the time of AML evolution. In an MDS/MPN-U, NRAS/KRAS were both detected at diagnosis with a VAF of 1% and 12% each. Both clones increased over 2 years' time span with NRAS ramping to 43% and reaching KRAS (50%). In the 3rd case, samples were collected over a period of 5 years with NRAS and PTPN11 being detected only at the later time point (VAF of 5% and 15%, respectively). In contrast to competing RAS subclones in adults, our historical cohort of JMML showed that at least one RAS mutation was found in 89% (82/92) of the patients as ancestral events and in mutually exclusive manner while biallelic RAS mutations occurred in 15% (12/82) of the cohort. In adult MN, the most common subclonal mosaicism was encountered in NRAS/KRAS (63%; 24/38), while less fraction of patients had competing subclonal mutations in NRAS (26%; 10/38) or in KRAS (8%; 3/38). Most of these mutations were found in canonical sites (NRAS: G12D/S/A/C, G13D/R/V, Q61A/H/R; KRAS: G12D/A/R, G13D/R/V, A146P/T). Patients carrying RAS mutations had significantly higher WBCs (20.5 vs 7.2, P&amp;lt;.001) consistent with the role of RAS in proliferation, possibly via augmentation of MYC. Isolated chromosome 7 abnormalities were more common among RAS mutant carriers who otherwise showed less complex karyotypes. RAS mutations were mostly enriched in MDS/MPN (26%) while absent in sAML suggesting the impact of these lesions on OS but not on PFS. Analysis of clonal architecture showed that accumulation of RAS subclones was most commonly related to the presence TET2 (17% vs. 8%) and EZH2 (6% vs. 1%) compared to WT. At the time of AML evolution, the presence of RAS mutation did not correlate with OS (18 vs. 13 mo., P=0.07). However, median OS was shorter in MDS and MDS/MPN carrying multiple RAS mutations compared to WT (10 vs. 30 mo., P=0.005) again supporting the contention that these clones may be markers of non-progression-related mortality. In sum, our study supports the notion that ancestral RAS hits are common in childhood MN with a hereditary component whereby the progression is related to the acquisition of secondary hits in biallelic configuration. In contrast in adult and elderly MN, specific genetic background predisposes to RAS mutant mosaicism such as ancestral TET2 and EZH2 lesions and MPN features reflecting the direction of selection pressure towards accumulation of multiple RAS pathway hits. Lack of the gene/dose effect on progression indicates, that in contrast to the compound heterozygous RAS childhood diseases, RAS mutant mosaicism reflects branching rather than linear evolution mode. Disclosures Sekeres: Pfizer: Consultancy; Takeda/Millenium: Consultancy; BMS: Consultancy. Carraway:Abbvie: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Takeda: Other: Independent Advisory Committe (IRC); Stemline: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; BMS: Consultancy, Other: Research support, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents &amp; Royalties: University of Illinois at Chicago. Patel:Alexion: Other: educational speaker. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Abstract 800: Exploiting the proteasome to overcome RAS and WT1 mutation mediated resistance to FLT-3 inhibition in acute myeloid leukemia

Abstract FLT3 mutations (mFLT3) in acute myeloid leukemia (AML) carry a poor prognosis.The mandatory co-occurrence of other driver mutations with mFLT3 leads to suboptimal clinical responses with single agent FLT3 inhibitors (FLT3i). Pre-existing or emergent RAS mutations are one mechanism of primary and secondary resistance to single agent FLT3i. Therefore development of combinatorial strategies to overcome resistance conferring co-mutations is critical. Herein we report a novel in-vitro combination of the FLT3/SYK inhibitor (FSI) TAK-659 with Proteasome inhibitor (PI) ixazomib, to co-operatively inhibit proliferation of mFLT3 and RAS or WT1 co-mutated AML blasts, which are otherwise less sensitive to Flt3i and FSI. Peripheral blood or bone marrow derived cells from patients with AML were isolated and treated in vitro with TAK-659 and Ixazomib alone or in combination. Cytosolic/nuclear protein lysates were obtained at 12 hours of drug/control treatment and western blotting performed for proteins of interest. Tritiated thymidine proliferation assays were also performed with titrated doses of each agent alone, and combination of fixed dose of one agent with titration of the second. Patient samples were annotated for mutations by Next Generation Sequencing. Similar experiments were carried out on cell lines MV411 (Flt3ITD/MLL), THP-1 and OCI-AML3 (both NRAS). Proliferation assays on a series of primary AML samples revealed an inherent dichotomy in response patterns. mFLT-3 samples with co-mutations in DNMT3A, NPM1 or tMLL were very sensitive to TAK-659 (IC50 &amp;lt;75 nM) but much less sensitive to Ixazomib alone (IC50&amp;gt;100nM). The co-occurrence of RAS or WT1 mutations predicted for resistance to TAK-659 but interestingly sensitized cells to PI alone and in combination. In fact, presence of RAS mutations irrespective of FLT3 status predicted for sensitivity to PI (IC50&amp;lt;50nM). We also observed sensitivity to Ixazomib in THP-1 and OCI-AML3, while MV4-11 was sensitive to TAK-659 but relatively resistant to Ixazomib alone. Based on genome-wide CRISPR screen, overactivity of RAC-PAK signaling mediated by PREX1 is essential to RAS mutated AML (T Wang etal. Cell 168:890, 2017). Similarly, WT1 mutation in AML is known to ablate expression of a major nuclear chaperone for beta-catenin, TBL1X, and RAC has been found to be a substitute (Y Wang et al. Mol Cell. 57:662, 2015). We therefore hypothesized that PI decreased nuclear availability of RAC1 along with its chaperone partner beta-catenin - a determinant of leukemic stem cell activity, also regulated by RAC/PAK and the proteasomal system. Indeed, in primary RAS or WT1 mutated AML samples, we observed PI treatment led to significant reduction in nuclear RAC1 levels and decreased nuclear beta-catenin levels, specifically PAK phosphorylated p675 active beta-catenin. These findings suggest PI effects RAC1, PAK and beta-catenin simultaneously. Similar results were also noted in RAS mutant cell lines. siRNA knockdown experiments also linked p62 SQSTM1 in a pathway to RAS/RAC/PAK and beta-catenin. In summary, we delineate a RAS/RAC/PAK/Beta-catenin pathway to be linked to critical events in RAS or WT1 mutated AML progression and resistance. FLT3 mutated AML with these co-mutations are insensitive to single-agent activity of FLT3i or FSI alone and the addition of PI is critical to overcome them. Citation Format: Sravanti Rangaraju, Santosh Kumar Pasupuleti, Silvia Marino, Daniela Nicoleta Petrusca, Larry D. Cripe, Bhaskar Ramdas, Lakshmi Reddy Palam, Katie J. Sargent, Hamid Sayar, Heiko Konig, Reuben Kapur, David G. Roodman, Scott H. Boswell. Exploiting the proteasome to overcome RAS and WT1 mutation mediated resistance to FLT-3 inhibition in acute myeloid leukemia [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 800.

Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids

The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.

RAS Mutations Are Independently Associated with Decreased Overall Survival and Event-Free Survival in Patients with AML Receiving Induction Chemotherapy

Cai: Imago Biosciences, Inc.: Consultancy. Viny:Hematology News: Membership on an entity's Board of Directors or advisory committees; Mission Bio: Other: Sponsored travel. Goldberg:American Society of Clinical Oncology: Research Funding; Abbvie: Research Funding; ADC Therapeutics: Research Funding; American Society of Hematology: Research Funding; DAVA Oncology: Honoraria; Pfizer: Research Funding; Arog Pharmaceuticals: Research Funding; Abbvie: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Celgene: Consultancy. Tallman:BioLineRx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biosight: Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Consultancy; Cellerant: Research Funding; ADC Therapeutics: Research Funding; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KAHR: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Stein:Astellas Pharma US, Inc: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Membership on an entity's Board of Directors or advisory committees; Bioline: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees.

Combination of KIR2DS4 and Fc\u03b3RIIa polymorphisms predicts the response to cetuximab in KRAS mutant metastatic colorectal cancer

Cetuximab is a standard-of-care treatment for RAS wild-type metastatic colorectal cancer (mCRC) but not for those harbor a KRAS mutation since MAPK pathway is constitutively activated. Nevertheless, cetuximab also exerts its effect by its immunomodulatory activity despite the presence of RAS mutation. The aim of this study was to determine the impact of polymorphism FcγRIIIa V158F and killer immunoglobulin-like receptor (KIR) genes on the outcome of mCRC patients with KRAS mutations treated with cetuximab. This multicenter Phase II clinical trial included 70 mCRC patients with KRAS mutated. We found KIR2DS4 gene was significantly associated with OS (HR 2.27; 95% CI, 1.08–4.77; P = 0.03). In non-functional receptor homozygotes the median OS was 2.6 months longer than in carriers of one copy of full receptor. Multivariate analysis confirmed KIR2DS4 as a favorable prognostic marker for OS (HR 6.71) in mCRC patients with KRAS mutation treated with cetuximab. These data support the potential therapeutic of cetuximab in KRAS mutated mCRC carrying non-functional receptor KIR2DS4 since these patients significantly prolong their OS even after heavily treatment. KIR2DS4 typing could be used as predictive marker for identifying RAS mutated patients that could benefit from combination approaches of anti-EGFR monoclonal antibodies and other immunotherapies to overcome the resistance mediated by mutation in RAS.

A Novel Prognostic Model Including Cytogenetic and Molecular Data in Patients with Primary and Post-Essential Thrombocythemia (ET)/Polycythemia Vera (PV) Myelofibrosis (MF)

Introduction: MF is a Philadelphia-negative myeloproliferative neoplasm (Ph-negative MPN) with an heterogeneous outcome. In 2009, Cervantes et al. published the International Prognostic Score System (IPSS) to better determine outcomes in this disease. In the last decade, several recurrently mutated genes have been described in MF, some of them associated with prognostic impact in survival. We propose a novel prognostic score that incorporates molecular and cytogenetic data in patients with MF.Methods: We analyzed clinical, cytogenetic and molecular data from 623 patients with a diagnosis of primary MF (N=445), post-PV MF (N=109) and post-ET MF (N=69). Data was extracted from medical records at time of sample collection for analysis. Mutation data was obtained by next-generation sequencing analysis, performed with either paired tumor-normal whole exome sequencing (N=46) or selected gene panel for genes associated with myeloid malignancies (N=577). The following 16 genes were analyzed in all 623 patients and were considered as the common denominator for analysis: ASXL1, CALR, DNMT3A, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NRAS, RUNX1, TET2, TP53, WT1. RAS mutations were considered as oncogenic mutations in NRAS and/or KRAS. Molecular high risk (MHR) mutations were considered as mutations in any one of the 4 genes: ASXL1, EZH2, IDH1, IDH2 (SRSF2 mutations were not included since they were not evaluated in all cases). Cytogenetic data was stratified into 4 risk categories (based on Tam et al, Blood 2009): (1) Diploid; (2) Del(13q)/Del(20q)/Trisomy 9; (3) Abnormalities of chromosomes 5, 7, 17 and complex karyotype; (4) Other abnormalities. To develop the model, the data was split into a training dataset (N=434) and a test dataset (N=189). Variables initially included in the initial training model were those with a p-value<0.05 in the univariate analysis; subsequently, this initial model was internally validated with a bootstrap procedure for stepwise backwards selection and determination of variables to be included the final model. Score points were assigned based on the value of the hazard ratio (HR). The final model was externally validated in the test dataset. Akaike information criterion (AIC) was calculated on the final model and on the Dynamic IPSS (DIPSS) model on the full dataset, and the best model was considered the one with the lowest AIC value and the highest AIC weight.Results: In the training cohort, after a median follow-up of 30.8 months, there were 176 deaths (40.5%). The initial variables included in the multivariate Cox model were: age (>65 years), hemoglobin (<10 g/dL), white blood cell count (WBC; >25x109/L), peripheral blood blasts (>1%), presence of constitutional symptoms, sex (male vs female), platelet count (<100x109/L), type of JAK-STAT driver mutation (CALR vs JAK2 vs MPL vs Triple-negative), karyotype risk category, presence of MHR mutations and presence of RAS mutations. After bootstrapping, variables sex, peripheral blood blasts and WBC were excluded, and the final model with associated HRs is presented on table 1. Since most HRs were near 1, 1-point was given to each variable, with the exception of Triple-negative molecular status and RAS mutations (2 points each). We grouped patients based on similarity of HR into three risk categories: low (0-2 points; N=243), intermediate (3-4 points, N=141) and high (5+ points, N=50). These 3 groups were associated with significantly different survival outcomes (Figure 1): 3-years OS was 71% (95% CI 64-78%), 38% (95% CI 29-50%) and 18% (95% CI 9-37%) for low, intermediate and high risk groups (P=1.33e-13). Validating the model on the test dataset revealed significantly differences in OS among the 3 risk categories (Figure 2): 74% (95% CI 63-86%), 50% (95% CI 36-68%) and 16% (95% CI 6-39%) (P=1.42e-11). Analyzing the full dataset, the novel score model had a lower AIC than the DIPSS model (2713 vs 2722) and a higher AIC weight (0.99 vs. 0.01).Conclusion: Incorporating molecular data, including MHR mutations, karyotype and RAS mutations leads to the development of an improved prognostic model. Further validation of this model in other cohorts is necessary. The impact of novel therapies should be evaluated among the different risk categories. [Display omitted] DisclosuresRampal:Stemline: Research Funding; Celgene: Honoraria; Constellation: Research Funding; Incyte: Honoraria, Research Funding; Jazz: Consultancy, Honoraria. Verstovsek:Celgene: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy.

The mutational analysis in the diagnostic work-up of thyroid nodules: the real impact in a center with large experience in thyroid cytopathology

Fine-needle aspiration (FNA) cytology is a mainstay in the evaluation of thyroid nodules, but fails to reach reliable results in 25-30% of cases. The role of molecular markers in helping clinical decisions has been investigated for the last years, but their clinical usefulness is still unsettled. Mutation analysis of BRAF, RAS genes and TERT promoter was performed in a series of 617 consecutive cytological specimens undergoing FNA. The 617 nodules had the following cytological diagnosis: non diagnostic 22 (3.6%), benign 425 (68.9%), indeterminate 114 (18.5%), suspicious 11 (1.8%) and malignant 45 (7.3%). BRAF mutations were found in 31 cases (5.0%), all but two in suspicious and malignant nodules. RAS mutations were detected in 47 samples (7.6%): 25 benign (5.9%) and 19 indeterminate nodules (16.7%). TERT promoter mutation alone was detected in three samples. Histological outcome was available for 167 nodules, 81 of which proved malignant: all the 48 with suspicious or malignant cytology; 25 out of 56 (44.6%) with indeterminate and 8 out of 57 (14%) with benign cytology. BRAF mutations were associated with worse tumors pathological features. The presence of RAS mutations was indicative of follicular-patterned malignancies in 5 out of 8 benign nodules and 9 out of 11 indeterminate nodules. Our study established mutational rates for BRAF and RAS genes in a large series of FNA specimens. BRAF mutations were confirmed as highly specific but not able to improve cytological diagnosis, while RAS testing proved effective in assessing malignancy in nodules with indeterminate and benign cytology.