Ras Gene Mutations Research Articles

The prominent pervasive oncogenic role and tissue specific permissiveness of RAS gene mutations

In cancer research, RAS biology has been focused on only a handful of tumor types. While RAS genes have long been suspected as common contributors to a wide spectrum of cancer types, robust evidence is required to firmly establish their critical oncogenic significance. We present a data mining study using DepMap genome-wide CRISPR screening data, which provide substantial evidence to support the prominent pervasive oncogenic role and tissue-specific permissiveness of RAS gene mutations. Differential analysis of CRISPR effect scores identifies K- or N-RAS genes as the most differential gene in contrasts of (K-, N-, combined) RAS mutant versus wild-type cell lines across multiple tissue types. The distinguished tissue-specific pattern of KRAS vs. NRAS as top differential genes in subsets of tissue types and evidence from genome data supported the idea of KRAS- and NRAS-engaged tissue types. To our knowledge, this is the first report of prominent pervasive oncogenic role of RAS mutations revealed by gene dependency data that is beyond the current understanding of the oncogenic role of RAS genes and their well-known involved tissue types. Our findings strongly support RAS mutations as primary oncogenic drivers beyond traditionally recognized cancer types and offer insights into their tissue-specific permissiveness.

Multi-gene mutations in ultrasound-guided fine-needle aspiration specimens of thyroid micronodules and diagnostic value in thyroid microcarcinomas using next-generation sequencing

Objective: To explore the detection of BRAF, RAS, TERT promoter, and TP53 gene mutations in solitary thyroid micronodule (TMN) specimens obtained by ultrasound-guided fine-needle aspiration (US-FNA) using next-generation sequencing (NGS) technology and assess its diagnostic value in thyroid microcarcinomas (TMC). Methods: On-site recruitment of 428 patients with single suspicious TMC who underwent thyroid ultrasound examination, US-FNA, and NGS from September 2018 to July 2021 at Beijing Tongren Hospital affiliated to Capital Medical University. A total of 147 patients were finally included. NGS was used to detect mutations in the BRAF, RAS, TERT promoter, and TP53 genes in the US-FNA specimens. Comparisons were made between patients with and without gene mutations in terms of age, gender, and the maximum diameter of nodules. The diagnostic efficiency of BRAF mutation for TMC was calculated using the receiver operating characteristic (ROC) curve, with postoperative pathology as the gold standard. Results: The age [M (Q1, Q3)] of the 147 patients was 43.0 (32.0, 51.0) years, and 37 were male (25.2%). Among the 147 US-FNA specimens, 97 (66.0%) were detected with BRAF gene mutations, all of which were p.V600E point mutation; 6 (4.1%) were detected with RAS gene mutation, and no TERT promoter or TP53 gene mutations were detected. Postoperative pathology confirmed that 136 cases were TMC, all of which were papillary thyroid microcarcinomas (PTMC); 11 cases (7.5%)were benign. Among 136 TMC samples, BRAF gene mutations were detected in 97 cases (71.3%). There were no statistically significant differences in age, gender, and maximum nodule diameter between patients with and without BRAF gene mutations (all P>0.05). The sensitivity and specificity of BRAF gene mutation in diagnosing TMC were 71.3% and 100.0%, respectively, with an area under the ROC curve (AUC) (95%CI) of 0.857 (0.789-0.925). For nodules classified as Bethesda Ⅲ-Ⅴ, the sensitivity and specificity were 63.0% and 100.0%, respectively, with an AUC (95%CI) of 0.815 (0.680-0.950). Conclusions: NGS technology can successfully detect multiple gene mutations in US-FNA specimens from TMN patients, especially BRAF gene mutation, and BRAF gene mutation has certain value in diagnosing TMC.

8787 A Case of Indolent Medullary Thyroid Cancer with Sporadic HRAS Mutation and Prolonged Stability

Abstract Disclosure: M. Lozano: None. A. Pinero-Pilona: None. Background: Medullary thyroid cancer (MTC) is a rare neuroendocrine tumor that is derived from calcitonin-producing C-cells that accounts for the smallest portion (<5%) of thyroid cancer. Most of these cases are attributed to mutations in the RET oncogene. Of the remainder, approximately 70% of MTC cases without a RET mutation have been attributed to RAS gene mutations. We report a case of a patient who presented with a thyroid nodule present and stable for years and was found to have MTC with a HRAS mutation. Clinical Case: A 73-year-old Caucasian male with a history of metastatic squamous cell carcinoma (SCC) of the neck, orthostatic hypotension, pacemaker implantation, and coronary artery disease with prior stenting presented to the clinic for evaluation after an abnormal thyroid biopsy. His SCC was diagnosed 16 years ago and was treated with surgery and cetuximab. It was a poorly differentiated squamous cell carcinoma with 2 of 7 lymph nodes positive for malignancy. Fifteen years ago, as part of the evaluation for his SCC, the patient had CAT and PET scans that showed a small nodule with increased uptake in the right lower neck in the thyroid bed (2.7 SUVs) measuring about 1.4 cm in diameter. No further interventions were done back then, and the patient was unaware of the mass until his records were reviewed. The patient was more recently noted to have the incidental thyroid nodule 2 months prior to clinic evaluation after routine carotid ultrasound. The gland was described to be homogeneous; in the mid to inferior portion of the right lobe, there was a 1.2 cm TR5 nodule of which biopsy was recommended. Fine needle aspiration biopsy showed a follicular lesion of unknown significance Bethesda category 3 using AFIRMA GCS RNA molecular analysis. Subsequently, gene sequencing classification was performed and abnormal: results were positive for HRAS mutation. Sonography in the office showed a right-sided, slightly hypoechoic, taller-than-wide vascular nodule with well-preserved borders and intermediate risk of malignancy. No abnormal lymphadenopathy was observed regionally in the central or lateral neck. The patient then underwent total thyroidectomy with modified right level 3 dissection. The right mid-superior nodule was consistent with MTC without tumor necrosis and without lymphatic, perineural, or extrathyroidal extension, but there was angioinvasion of at least 3 vessels. Preoperative calcitonin was elevated at 71 pg/mL and the level was undetectable (<2 pg/mL) 8 weeks post operatively (n<=10 pg/mL). The patient was deemed cured based on these biochemical studies and is recovering uneventfully on supplemental levothyroxine. Conclusion: Given the predominant association of MTC with RET, it is worth noting this case of a sporadic HRAS mutation in a patient with prior SCC. This case illustrates an indolent MTC without aggressive behavior that stayed stable in size and without metastasis for 15 years. Presentation: 6/1/2024

Locally advanced rectal cancer in a young adult affected with dyskeratosis congenita (Zinsser–Cole–Engman syndrome): a case report

BackgroundDyskeratosis congenita (DKC), also known as Zinsser–Cole–Engman syndrome, is a progressive genetic disease with a triad of reticulate skin pigmentation, nail dystrophy, and leukoplakia. Approximately 8–10% of patients with DKC develop malignancies, and cases of colorectal cancer with DKC in young people have been reported previously.Case presentationA 25-year-old man with DKC since approximately 10 years of age developed fever and lower abdominal discomfort. Diagnostic imaging revealed locally advanced rectal cancer with lymph node metastasis, direct invasion of the prostate, and pelvic abscess due to tumor microperforation (cT4bN2M0 cStage IIIC). Biopsy showed well to moderately differentiated ductal adenocarcinoma. Genetic testing was negative for RAS and BRAF gene mutations, and microsatellite instability (MSI) testing was also negative. After sigmoid colostomy, the patient was treated with total neoadjuvant therapy (TNT) with systemic chemotherapy (six courses of FOLFOX + panitumumab) followed by chemoradiation therapy (50.4 Gy with capecitabine). After TNT, the primary tumor and metastatic lymph nodes shrank. According to the findings of colonoscopy and magnetic resonance image (MRI), we diagnosed near complete response (near-CR) and decided to follow the patient without surgery by every 3 months re-evaluation. However, 5 months after TNT, tumor regrowth was detected on colonoscopy and imaging, and the patient underwent total pelvic exenteration. He developed paralytic ileus as a postoperative complication, and was discharged on the 38th postoperative day. Pathological examination revealed a residual tumor with invasion of the periprostatic tissue. There was no metastasis in the pararectal and lateral pelvic lymph nodes, but one extramural non-contiguous cancerous extension (tumor deposit) was observed (ypT4bN1cM0 ypStage IIIC). The patient has been free of recurrence for one year after surgery.ConclusionsDKC often develops into various tumors in the digestive system at an early age; therefore, appropriate surveillance may be required. In addition, considering that cancers in patients with DKC occur at a young age, fertility preservation and survivorship are also important, and adequate explanations and care should be provided to patients before and after treatment.

Survival Analysis, Clinical Characteristics, and Predictors of Cerebral Metastases in Patients with Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer globally and a leading cause of cancer-related deaths. While liver metastasis is common, brain metastasis (BM) is rare, occurring in 0.1% to 14% of cases. Risk factors for BM include lung metastasis at diagnosis, rectal cancer, and mutations in RAS and KRAS genes. Due to its rarity, guidelines for BM screening and treatment are limited. The aim of this study is to identify the clinical characteristics and predictors of BM at the time of the initial diagnosis of CRC. We evaluated patients ≥18 years old with metastatic colorectal cancer and brain metastases at diagnosis from the SEER database (2010-2021). A retrospective cohort study was conducted to analyze overall survival and predictive factors for brain metastasis, utilizing multivariate logistic regression, Kaplan-Meier survival analysis, and the Cox proportional hazards models, with p-values < 0.05 considered significant. Out of 24,703 patients with metastatic colorectal cancer (mCRC), 228 (0.92%) had brain metastasis (BM) at diagnosis. BM was more prevalent in average-onset mCRC (≥50 years) compared to early-onset (<50 years) (1% vs. 0.55%, p = 0.004). Certain factors, such as older age and adenocarcinoma subtype, were associated with BM. Additionally, Asians/Pacific-Islanders (HR 1.83 CI: 1.01-3-33, p = 0.045) and American Indians/Alaska Natives (HR 4.79 CI 1.15-19.97, p = 0.032) had higher mortality rates, while surgical treatment and chemotherapy were linked to decreased mortality. Patients with BM had significantly worse overall survival (6 months vs. 21 months, p < 0.001). BM in mCRC is uncommon, but it is associated with significantly worse outcomes, including markedly reduced overall survival. Our study highlights several critical factors associated with the presence of BM, such as older age and specific racial/ethnic groups, which may inform risk stratification and early-detection strategies. Our findings emphasize the need for heightened awareness and screening for BM in high-risk mCRC patients, as well as the inclusion of these patients in clinical trials to explore tailored therapeutic approaches aimed at improving survival and quality of life.

The pathological significance of HRAS gene mutation in colorectal cancer patients in Erbil province

Background and objective: RAS gene mutations, including Harvey RAS, are a biomarker in studying many types of cancer, including colorectal cancer. Studying the molecular and mutations of the HRAS gene help in understanding the nature of the tumor and helps to formulate a suitable plan for prevention and treatment. The aim of the present study is to identify HRAS mutations among a sample of colorectal cancer. Methods: The sample for the present study involved 20 Formalin fixed embedded tissue of colorectal carcinoma and 10 adjacent normal tissues were obtained from Rizgary Teaching Hospital in Erbil. Results: The sample of the present study involved 20 patients (60% males and 40% females) with CRC. The mean age was 54.20 years, with an SD ±19.80. The study found a single HRAS mutation in the sample study. The size of the tumor mutated by HRAS is significantly different than other tumors (P = 0.009). Conclusion: there was an HRAS mutation in a sample of colorectal cancers and the size of the tumor was different than other tumors in the study sample. Further studies needed about the mutation of the HRAS gene.

Efficacy and safety of trifluridine/tipiracil plus bevacizumab across different subgroups of patients with refractory colorectal cancer: a meta-analysis.

Metastatic colorectal cancer (mCRC) patients who are refractory to initial treatment lines exhibit a challenging clinical scenario characterised by a poor prognosis and constrained therapeutic options. This systematic review and meta-analysis assess the integration of bevacizumab into trifluridine-tipiracil (TFD/TPI) therapy for mCRC, examining its benefits across patient subgroups and evaluating safety relative to TFD/TPI monotherapy. Following preferred reporting items for systematic reviews and meta-analysis statements, we conducted a thorough literature search from 15 October to 11 November 2023, covering MEDLINE, Embase and the Cochrane database. Data extraction and quality assessment followed Cochrane guidelines, and hazard or odds ratios with 95% confidence intervals (CI) were pooled (p < 0.05 significance threshold). The study protocol is registered in PROSPERO (CRD42023484695). Analysing 770 database results, we included two randomised controlled trials and five observational studies covering over 4,000 patients. Combined therapy exhibited significant improvements in overall survival (OS) hazard ratios (HR 0.60; 95% CI 0.49-0.72; p < 0.01) and progression-free survival (HR 0.48; 95% CI 0.40-0.59; p < 0.01). Subgroups, including prior bevacizumab exposure (HR 0.70; 95% CI 0.64-0.77; p < 0.01) and mutated RAS gene (HR 0.64; 95% CI 0.53-0.77; p < 0.01), demonstrated improvements in OSwith bevacizumab. This meta-analysis underscores the heightened efficacy of TFD/TPI combined with bevacizumab for refractory mCRC compared to TFD/TPI monotherapy across diverse subgroups. Combined therapy has increased grade ≥3 neutropenia and hypertension, while monotherapy is associated with fatigue and anemia.

Predicting prognosis in colorectal cancer patients with curative resection using albumin, lymphocyte count and RAS mutations

Colorectal cancer (CRC) poses a significant global health challenge, demanding reliable prognostic tools to guide treatment decisions. This study introduces a novel prognostic scoring system, the albumin-total lymphocyte count-RAS index (ALRI), integrating serum albumin, lymphocyte count, and RAS gene mutations. A cohort of 445 stage I–III CRC patients undergoing curative resection was analyzed, revealing ALRI's association with clinicopathological factors, including age, tumor location, and invasion depth. The ALRI demonstrated superior prognostic value, with a cutoff value of 2 distinguishing high and low-risk groups. The high-ALRI group exhibited elevated rates of recurrence. Univariate and multivariate analyses identified ALRI as an independent predictor for both 5 year recurrence-free survival (RFS) and overall survival (OS). Kaplan–Meier curves illustrated significant differences in RFS and OS between high and low-ALRI groups, emphasizing ALRI's potential as a prognostic marker. Importantly, ALRI outperformed existing nutritional indices, such as controlling nutritional status and neutrophil-to-lymphocyte ratio, in predicting overall survival. The study underscores the comprehensive insight provided by ALRI, combining inflammatory, nutritional, and genetic information for robust prognostication in CRC patients. This user-friendly tool demonstrates promise for preoperative prognosis and personalized treatment strategies, emphasizing the crucial role of inflammation and nutrition in CRC outcomes.

MEK inhibitors and DA-Raf, a dominant-negative antagonist of the Ras-ERK pathway, prevent the migration and invasion of KRAS-mutant cancer cells.

The Ras-induced ERK pathway (Raf-MEK-ERK signaling cascade) regulates a variety of cellular responses including cell proliferation, survival, and migration. Activating mutations in RAS genes, particularly in the KRAS gene, constitutively activate the ERK pathway, resulting in tumorigenesis, cancer cell invasion, and metastasis. DA-Raf1 (DA-Raf) is a splicing isoform of A-Raf and contains the Ras-binding domain but lacks the kinase domain. Consequently, DA-Raf antagonizes the Ras-ERK pathway in a dominant-negative manner and can serve as a tumor suppressor that targets mutant Ras protein-induced tumorigenesis. We show here that MEK inhibitors and DA-Raf interfere with the in vitro collective cell migration and invasion of human KRAS-mutant carcinoma cell lines, the lung adenocarcinoma A549, colorectal carcinoma HCT116, and pancreatic carcinoma MIA PaCa-2 cells. DA-Raf expression was silenced in these cancer cell lines. All these cell lines had high collective migration abilities and invasion properties in Matrigel, compared with nontumor cells. Their migration and invasion abilities were impaired by suppressing the ERK pathway with the MEK inhibitors U0126 and trametinib, an approved anticancer drug. Expression of DA-Raf in MIA PaCa-2 cells reduced the ERK activity and hindered the migration and invasion abilities. Therefore, DA-Raf may function as an invasion suppressor protein in the KRAS-mutant cancer cells by blocking the Ras-ERK pathway when DA-Raf expression is induced in invasive cancer cells.

Updated survival results of BBCAPX-II: Sintilimab combined with bevacizumab and CapeOx as first-line treatment in patients with RAS-mutant, microsatellite stable, unresectable metastatic colorectal cancer.

3563 Background: Microsatellite stable (MSS) and RAS-mutant metastatic colorectal cancer (mCRC) patients are characterized by an immunosuppressive microenvironment and a low response rate to immunotherapy. Sintilimab plus bevacizumab and CapeOX (BBCAPX) has proved its efficacy and safety in above unresectable mCRC patients (Ying Yuan et al. ASCO 2023 and Xuefeng Fang et al. ASCO 2022). Here, we report updated survival results of this single arm, open-label, phase 2 trial. Methods: Eligible patients were aged 18 to 75 years with histologically confirmed unresectable metastatic colorectal adenocarcinoma by multidisciplinary team, and had RAS gene mutation and confirmed MSS status. All patients received treatment with sintilimab (200 mg, day 1) plus bevacizumab (7.5mg/kg, day 1) and oxaliplatin (135 mg/m2, day 1) and capecitabine (1g/m2, bid, day 1-14) of each 21-day cycle. The primary endpoint included objective response rate (ORR) and adverse events. Secondary endpoint was disease control rate (DCR) and progression-free survival (PFS). Results: From April 2021 to December 2021, 25 patients were enrolled. The ORR was 84% and the DCR was 100%. Six (24%) patients underwent surgical treatment and unexpectedly achieved no evidence of disease status. At the data cut-off day (January 26, 2024), the median PFS in the full analysis set was 17.9 months (95% CI, 8.84-27), and the median PFS in the per-protocol set was 9.79 months (95% CI, 6.44-24.3). Median PFS was 23.7 months (95% CI, 4.8-NA) in the patients with only liver metastasis and 11.5 months (95% CI, 4.83-24.3) in the patients with other metastasis. The PFS rate at 6 months was 84% (95% CI, 70.8-99.7). The PFS rate at 12 months was 56% (95% CI, 39.6-79.3). The most frequent grade 3 or 4 TRAEs were neutropenia (3/25) and increased alanine transaminase (2/25). No grade 5 adverse events occurred during the study. Conclusions: The combination of sintilimab plus bevacizumab and CapeOx showed promising antitumor activity and manageable toxicity in RAS-mutant, MSS, unresectable mCRC. Patients with liver metastases presented better prognosis compared other metastases. Furthermore, we are currently launching a phase III, randomized, open-label, multicentric clinical trial (NCT05171660) to further analyze the effects, safety, and prognostic biomarkers of this regimen. Clinical trial information: NCT04194359 . [Table: see text]

Abstract 3320: In vivo and in vitro experience with novel direct Pan-RAS inhibitors

Abstract RAS oncogenes are frequently activated by mutations in human cancer, where they act as driving elements of the disease. Mutation rates can range from ~25% of Non Small Cell Lung cancers to over 90% of pancreatic cancers. RAS proteins can also be hyper-activated to drive cancer at a significant frequency in the absence of RAS gene mutations when their protein regulatory systems are damaged. Thus, RAS may be the most frequently activated oncoprotein in cancer. We have developed a series of novel, direct, RAS inhibitors with a unique binding site on RAS. The binding of the agents alters the structure of the RAS effector loop and blocks the association with RAS downstream effectors. We used in silico library screening followed by Medicinal Chemistry optimization to develop the compounds. We have validated the agents using Microscale Thermophoresis and NMR to quantify binding to recombinant RAS protein. We have used mutant and wild type RAS driven tumor cell lines in 3D growth and protein-based RAS signaling assays to quantify and characterize the action of the family of inhibitors. We have used xenograft assays to demonstrate compound anti-tumor activity in RAS driven cell lines and pdx systems. We have identified compounds that can bind mutant and wild type K, H, N and M-RAS. We can suppress the association of mutant and wild type RAS protein with its effector RAF-1 in treated cells. The compounds can suppress 3D growth of mutant or wild type RAS driven tumor cell lines and repress tumor development in vivo. The compound family bind to a different site than either clinical agent AMG-510 (K-RAS G12C specific) or MRTX-1133 (K-RAS G12D specific). They exhibit distinct RAS signal inhibition patterns compared to these agents. They can also co-operate with the clinical agents and reduce drug resistance effects. Citation Format: Tariq Arshad, Howard Donninger, Goeff Clarke, Joe Burlison, Rob Monsen, Mike Sabo. In vivo and in vitro experience with novel direct Pan-RAS inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3320.

Abnormalities in the KRAS Gene and Treatment Options for NSCLC Patients with the G12C Mutation in This Gene-A Literature Review and Single-Center Experience.

Mutations in the KRAS gene are among the most common mutations observed in cancer cells, but they have only recently become an achievable goal for targeted therapies. Two KRAS inhibitors, sotorasib and adagrasib, have recently been approved for the treatment of patients with advanced non-small cell lung cancer with the KRAS G12C mutation, while studies on their efficacy are still ongoing. In this work, we comprehensively analyzed RAS gene mutations' molecular background, mutation testing, KRAS inhibitors' effectiveness with an emphasis on non-small cell lung cancer, the impact of KRAS mutations on immunotherapy outcomes, and drug resistance problems. We also summarized ongoing trials and analyzed emerging perspectives on targeting KRAS in cancer patients.

The Abundance of KRAS and RAS Gene Mutations in Cancer.

Mutant forms of the RAS genes KRAS, NRAS, and HRAS are important and common drivers of cancer. Recently, two independent teams that integrated cancer genomics with cancer epidemiology estimated that approximately 15-20% of all human cancers harbor a mutation in one of these three RAS genes. These groups also estimate KRAS mutations occur in 11-14% of all human cancers. Although these estimates are lower than many commonly encountered values, these estimates continue to rank KRAS and the ensemble of RAS oncogenes among the most common genetic drivers of cancer across all forms of malignancy.

Antigen presentation deficiency, mesenchymal differentiation, and resistance to immunotherapy in the murine syngeneic CT2A tumor model.

The GL261 and CT2A syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM) but demonstrate distinct differences in their responses to immunotherapy. To decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their characteristics using whole exome and transcriptome sequencing, and protein analysis through immunohistochemistry, Western blot, flow cytometry, immunopeptidomics, and phosphopeptidomics. The transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of some huGBMs, despite neither line sharing the essential genetic or histologic features of huGBM. Both models exhibited striking hypermutation, with clonal hotspot mutations in RAS genes (Kras p.G12C in GL261-luc2 and Nras p.Q61L in CT2A-luc). CT2A-luc distinctly displayed mesenchymal differentiation, upregulated angiogenesis, and multiple defects in antigen presentation machinery (e.g. Tap1 p.Y488C and Psmb8 p.A275P mutations) and interferon response pathways (e.g. copy number losses of loci including IFN genes and reduced phosphorylation of JAK/STAT pathway members). The defect in MHC class I expression could be overcome in CT2A-luc by interferon-γ treatment, which may underlie the modest efficacy of some immunotherapy combinations. Additionally, CT2A-luc demonstrated substantial baseline secretion of the CCL-2, CCL-5, and CCL-22 chemokines, which play important roles as myeloid chemoattractants. Although the clinical contexts that can be modeled by GL261 and CT2A for huGBM are limited, CT2A may be an informative model of immunotherapy resistance due to its deficits in antigen presentation machinery and interferon response pathways.

Tumor Grade and Molecular Characteristics Associated with Survival in Sporadic Medullary Thyroid Carcinoma.

Background: The International Medullary Thyroid Carcinoma Grading System (IMTCGS) divides medullary thyroid carcinoma (MTC) into two categories, high- and low-grade tumors, which has a profound impact on patient outcomes. The aim of this study was to explore the association between IMTCGS grading, clinical data, and molecular status in sporadic MTC. Methods: A retrospective cohort study was performed on consecutive sporadic MTCs from patients undergoing initial surgery between January 2000 and January 2022 at the Padua Endocrine Surgery Unit. Clinical, pathological, and follow-up data were collected, tumors were graded, and somatic mutations of RET and RAS genes were analyzed. Patient outcomes were based on Ct levels and MTC-related deaths. Survival analyses were carried out employing the Kaplan-Meier method and the log-rank test. A Cox proportional hazard regression model was employed for multivariable survival analysis with the following covariates: somatic RET mutation, MTC stage at diagnosis, sex, age at diagnosis, and IMTCGS grade. Results: We included 141 consecutive sporadic MTCs. The median follow-up was 80.0 months (interquartile ranges: 41.5-122.5 months). Seventeen patients (12.1%) died from disease-related causes. 107/141 (76.9%) were classified as low-grade tumors, 32/141 (23.1%) as high-grade. Patients carrying a RET mutation had more aggressive features and shorter disease-specific survival (DSS) (p = 0.001) and were more frequently classified high-grade than low-grade MTC (p < 0.001). At multivariable survival analysis, only IMTCGS grading was independently associated with DSS (hazard ratio 8.8 [confidence interval: 2.7-28.3], p = 0.005). RET mutations, in particular RET-M918T, were more frequent in high-grade than in low-grade MTC (68.8% vs. 29.4% mutated in RET, 46.9% vs. 12.7% mutated in RET-M918T; p < 0.001). None of the high-grade tumors was mutated in the RAS gene, but the mutation was present in 11.8% of low-grade tumors. Conclusions: IMTCGS grading was associated with DSS independently of other clinical, pathological, and molecular factors. Moreover, MTC grading was associated with RET and RAS patterns, which explains, at least in part, the molecular basis of the aggressive behavior of high-grade MTC.

Genetic and Functional Characterization of NUP98-Rearranged Leukemia

NUP98 rearrangements ( NUP98r) are recurrent alterations in pediatric acute myeloid leukemia (AML) and are associated with dismal outcomes (PMID:21813447). These structural variantsresult in fusion oncoproteins comprised of the N-terminus of NUP98 and the C-terminus of fusion partners. Among more than 30 fusion partners discovered so far, the associations between NUP98::NSD1 with myeloblastic AML and NUP98::KDM5A with acute megakaryocytic/erythroid leukemia (AMKL/AEL) have been appreciated, which often harbor distinct cooperating mutations (e.g., NUP98:: NSD1 with FLT3-ITD and WT1 mutations). In contrast, other fusions, such as NUP98:: RAP1GDS1 or NUP98:: HOXA9 can be found also in myelodysplastic syndrome (MDS) or T-cell acute lymphoblastic leukemia (T-ALL) (PMID:32766874). However, how these fusions and cooperating mutations contribute to the specific phenotypes of NUP98r leukemias is poorly understood. To investigate the genetic background of NUP98r leukemia, we collected RNA-seq, WGS, and WES data for 91 pediatric cases from St. Jude (n=71),AEIOP (n=3), and the TARGET study (PMID:29227476, n=17; Panel.A), which included AML (n=82), MDS (n=4), and T-ALL (n=5). NUP98::NSD1 (n=47, 51.6%) and NUP98::KDM5A (n=26, 28.6%) were the most common fusions, in line with previous observations. NUP98::RAP1GDS1 (n=6, 6.6%), an understudied NUP98 fusion, was the third most common fusion in this study. We also confirmed frequent mutations of WT1 and FLT3-ITD in NUP98::NSD1 AML and entire or partial chromosome 13 loss involving RB1 locus in NUP98::KDM5A AMKL, while we observed frequent mutations in RAS or epigenetics-related genes, CCND2/3, and RUNX1 preferentially in cases without alterations of WT1, FLT3 or RB1 loss. NUP98:: RAP1GDS1 and NOTCH1 mutations were strongly enriched in T-ALL. These findings suggest that fusions and cooperating alterations both contribute to the disease phenotypes. To study how NUP98 fusion oncoproteins (FOs) drive leukemogenesis and specific phenotypes, we applied Cleavage Under Targets and Release Using Nuclease (CUT&amp;RUN) to determine NUP98 FO binding for a primary sample (myeloblastic with NUP98::NSD1), CHRF-288-11 cell line (megakaryocytic with NUP98::KDM5A), and HA-tagged CD34 NUP98::KDM5A model (Panel.B-top). CUT&amp;RUN using antibodies against the N-terminus NUP98 and histone modifications (H3K4me3, H3K27ac) revealed that these models shared NUP98 FO binding sites at the HOXA/B and RUNX1 loci, together with active histone marks. In contrast, binding patterns at differentiation-related gene loci were heterogenous. NUP98::NSD1 uniquely binds to CEBPA, which encodes a master regulator of myeloid differentiation, contrary to NUP98::KDM5A binding to GFI1B encoding regulator of erythromegakaryocytic differentiation . These data indicate that NUP98 FOs directly and uniquely regulate differentiation-related genes. We also performed CUT&amp;RUN in AML cells with RUNX1::RUNX1T1 or UBTF tandem duplications, in which the N-terminus NUP98 antibody did not show any detectable peaks, confirming the specificity of the N-terminus NUP98 signals in NUP98r AML models. Experiments using a HA antibody in HA-tagged NUP98::KDM5A CD34 models confirmed that fusion oncoproteins are binding to these target regions. However, the C-terminus NUP98 antibody also detected similar signals as the N-NUP98 antibody, suggesting that wild-type NUP98 is also recruited to NUP98 FO target genes, possibly by a heteromer formation with NUP98 FOs and other NPC components. To understand the possible association between somatic mutations and the genomic binding of NUP98 FOs, we next investigated chromatin immunoprecipitation sequencing (ChIP-seq) data from the ENCODE study (PMID:22955991) for RB1, WT1, and STAT5 (downstream FLT3-ITD). Binding peaks of these proteins partly coincided with NUP98 peaks on representative target genes (Panel.B-bottom), suggesting that frequent mutations in NUP98r leukemia converge on the genomic interaction of NUP98 FOs. In conclusion, genomic characterization of NUP98r leukemia showed the association among fusion partners, cooperating mutations, and disease phenotypes. Epigenetic profiling revealed that NUP98 FOs directly regulate target genes in a fusion and differentiation-specific manner and provide a basis for further study to investigate how NUP98 FOs and cooperating partners drive leukemogenesis and disease phenotypes.

Monomorphic Epitheliotropic Intestinal T-Cell Lymphoma:Pathological Phenotype and Genetic Mutation Analysis of 12 Patients

Introduction: Monomorphic Epitheliotropic Intestinal T-Cell Lymphoma (MEITL) is a primary intestinal lymphoma. Previously, it was referred to as Enteropathy Associated T-cell Lymphoma (EATL) type II. However, in the 2016 revision of the WHO classification, MEITL was recognized as a separate entity independent of EATL. EATL is mainly observed in people of Nordic descent and is closely associated with celiac disease, whereas MEITL is more commonly found in Asians and Hispanics without a history of celiac disease. MEITL is an extremely rare condition, accounting for approximately 5% of all Peripheral T-cell Lymphomas (PTCLs). It predominantly affects the small intestine, especially the jejunum. The tumor cells often display an activated cytotoxic T-cell phenotype and express CD8 and CD56. Due to its rarity, there is limited understanding of this disease. Between 2017 and 2022, our center diagnosed only 14 cases of MEITL. We analyzed the clinical characteristics and pathological phenotypes of these patients and further investigated the status of gene somatic mutations using a panel-based genomic exome analysis. Results: Clinical features: From the medical records system, a total of 622 gastrointestinal live tissue biopsy samples were searched from October 2017 to September 2022. Among them, 67 cases (10.8%) were diagnosed as PTCLs, with only 15 cases (2.4%) being identified as MEITL, accounting for 22.3% of all gastrointestinal PTCLs. Out of the 15 MEITL cases, 9 were male, and 6 were female, with a median age of 61 years(figure). Treatment and Efficacy: Seven patients underwent elective surgery due to the involvement of malignant tumors in the gastrointestinal tract, while six patients received emergency surgery for intestinal perforation. Three patients did not undergo chemotherapy, and the remaining 12 patients received a median of 5 (range 1-8) cycles of chemotherapy using the CHOP/CHOP-like regimen. For elderly and frail patients, the dosage was reduced by 20% to 50%. After treatment, one patient achieved complete remission (CR/CRu), three patients had a partial response (PR), six patients had stable disease (SD), and two patients had uncontrolled disease (PD). Pathological Characteristics: The diagnosis of MEITL was confirmed by two hematopathologists. The immunohistochemical staining results are shown in Figure. The results revealed that all patients expressed CD3, CD8, and CD56. Only four cases expressed CD4, including three cases with partial expression. TIA1 was expressed in 11 patients, and granzyme B was expressed in 11 patients, with eight cases showing co-expression of TIA1 and granzyme B. None of the patients expressed CD20, while one case showed weak expression of CD30. Ki67 expression was observed in all cases, with seven cases having Ki67 expression above 80% and all others showing expression above 50%. Gene Mutations: We conducted the Next-Generation Sequencing in 12 patients. The genes include STAT5B (9 cases), SETD2 (7 cases), NRAS (3 cases), KRAS (4 cases), JAK3 (4 cases), BRAF (1 case), BCOR (1 case), CREBBP (4 cases), EZH2 (1 case), EP300 (1 case), JAK1 (1 case), TP53 (3 cases), ATM (1 case), SOCS1 (1 case), CHD2 (1 case), and JAK2 (1 case). All patients showed abnormalities in two or more genes. Survival: Median follow-up time was 4.45 months, and among 15 patients, 14 experienced disease progression. The median progression-free survival (PFS) was 2.9 months (95% CI: 1.7 months to 4.2 months). Up to the follow-up cutoff, 11 patients had died, and the median overall survival (OS) was 4.8 months (95% CI: 3.2 months to 6.4 months). Patients who underwent early elective surgery showed a significant improvement in OS (P=0.012), with a median OS of 18.0 months. Based on gene mutation analysis, mutations in STAT5B and RAS (NRAS or KRAS) (P=0.038, 0.007) were associated with poorer OS, whereas abnormalities in genes like SETD2 did not affect patient OS. Conclusion: Our results show that MEITL is extremely rare, accounting for only 2.4% of all lymphomas involving the intestines. Patients with MEITL have a poor prognosis, with a median OS of less than six months. However, early elective surgery can significantly improve patient survival. MEITL is characterized by a cytotoxic T-cell phenotype, with the majority expressing TIA1 and granzyme B. Commonly occurring gene mutations are found in STAT5B, SETD2, and NRAS or KRAS. Mutations in STAT5B and RAS genes (NRAS or KRAS) often indicate a shorter OS.

Mutations of RAS genes identified in acute myeloid leukemia affect glycerophospholipid metabolism pathway.

Acute myeloid leukemia (AML) is a malignant disease originating from myeloid hematopoietic stem cells. Recent studies have shown that certain gene mutations promote tumor cell survival and affect the prognosis of patients by affecting metabolic mechanisms in tumor cells. RAS gene mutations are prevalent in AML, and the RAS signaling pathway is closely related to many metabolic pathways. However, the effects of different RAS gene mutations on AML cell metabolism are unclear. The main purpose of this study was to explore the effect of RAS gene mutation on the metabolic pathway of tumor cells. In this study, we first used a retrovirus carrying a mutant gene to prepare Ba/F3 cell lines with RAS gene mutations, and then compared full-transcriptome data of Ba/F3 cells before and after RAS gene mutation and found that differentially expressed genes after NRASQ61K and KRASG12V mutation. We found a total of 1899 differentially expressed genes after NRASQ61K and KRASG12V mutation. 1089 of these genes were involved in metabolic processes, of which 167 genes were enriched in metabolism-related pathways. In metabolism-related pathways, differential genes were associated with the lipid metabolism pathway. Moreover, by comparing groups, we found that the expression of the DGKzeta and PLA2G4A genes in the glycerophospholipid metabolism pathway was significantly upregulated. In conclusion, our study revealed that RAS gene mutation is closely related to the glycerophospholipid metabolism pathway in Ba/F3 cells, which may contribute to new precision therapy strategies and the development and application of new therapeutic drugs for AML.

A Contrast-Enhanced CT-Based Deep Learning System for Preoperative Prediction of Colorectal Cancer Staging and RAS Mutation.

This study aimed to build a deep learning system using enhanced computed tomography (CT) portal-phase images for predicting colorectal cancer patients' preoperative staging and RAS gene mutation status. The contrast-enhanced CT image dataset comprises the CT portal-phase images from a retrospective cohort of 231 colorectal cancer patients. The deep learning system was developed via migration learning for colorectal cancer detection, staging, and RAS gene mutation status prediction. This study used pre-trained Yolov7, vision transformer (VIT), swin transformer (SWT), EfficientNetV2, and ConvNeXt. 4620, and contrast-enhanced CT images and annotated tumor bounding boxes were included in the tumor identification and staging dataset. A total of 19,700 contrast-enhanced CT images comprise the RAS gene mutation status prediction dataset. In the validation cohort, the Yolov7-based detection model detected and staged tumors with a mean accuracy precision (IoU = 0.5) (mAP_0.5) of 0.98. The area under the receiver operating characteristic curve (AUC) in the test set and validation set for the VIT-based prediction model in predicting the mutation status of the RAS genes was 0.9591 and 0.9554, respectively. The detection network and prediction network of the deep learning system demonstrated great performance in explaining contrast-enhanced CT images. In this study, a deep learning system was created based on the foundation of contrast-enhanced CT portal-phase imaging to preoperatively predict the stage and RAS mutation status of colorectal cancer patients. This system will help clinicians choose the best treatment option to increase colorectal cancer patients' chances of survival and quality of life.

P04.01.B MEK INHIBITORS IN PEDIATRIC RECURRENT SYMPTOMATIC UNRESECTABLE LOW-GRADE GLIOMAS HARBORING MAPK ALTERATION: A SINGLE CENTER EXPERIENCE

Abstract BACKGROUND Treatment of inoperable, recurrent, progressive pediatric low-grade gliomas (pLGGs) remains controversial. Recent studies focused on aberrant activation of the mitogen-activated protein kinase (MAPK) pathway, due to gain of function mutations in RAS or BRAF genes or loss or inactivation of NF1. Selumetinib and trametinib, orally bioavailable and selective MEK1/MEK2 inhibitors (MEKi), act on MAPK pathway. In April 2020 the US Food and Drug Administration and in June 2021 the European Medicines Agency approved selumetinib for inoperable plexiform neurofibromas in Neurofibromatosis type 1 (NF1). We evaluated MEKi efficacy and safety in pLLGs harboring MAPK alteration. MATERIAL AND METHODS From July 2021 to February 2023 eight patients (5 females, 3 males, median age 8 years, range 4-18) were admitted to our hospital for symptomatic, recurrent, inoperable pLGGs (location: 3 hypothalamic-optic chiasm region, 2 posterior fossa, 1 temporo-mesial, 1 quadrigeminal plate, 1 diffuse leptomeningeal; MAPK alteration: 3 NF1 inactivation, 4 KIAA1549/BRAF gene fusion, 1 RNF130/BRAF fusion). MEKi were administered (6 selumetinib, 2 trametinib) after disease progression for failure of previous treatments (6 partial resection, 7 multiple chemotherapies, 2 radiotherapy). MRI assessment was performed every 4 months and radiological response was defined in accordance with RAPNO criteria. RESULTS At first evaluation, 3/8 patients (37,5%) showed a partial radiological response (-64,96% mean volume reduction, range 52,61%-77,19%) and 4/8 (50%) stable disease (0-41.67% volume decrease). Disease progression was observed in a patient heavily pretreated for acute leukemia. One patient with leptomeningeal spreading showed progressive disease at second evaluation. Median time to progression has not yet been reached. Six of 8 patients experienced at least one toxicity. The most frequent toxicities reported were mucocutaneus disorders (rash, mucositis, eczema). Three patients presented creatine phosphokinase (CPK) increase (one grade 2 with rapid normalization after therapy discontinuation). All patients on trametinib had to reduce treatment dose (for skin toxicity or increasing blood pressure). CONCLUSION MEKi, selumetinib and trametinib, represent promising strategies in children with recurrent/progressive LGGs harboring MAPK alterations. In our small series, the treatment was effective and well tolerable in patients with localized disease and without other risk factors. The most common toxicities were CPK elevation and rash. Further studies are needed to identify patients who may benefit most from MEKi treatment and to establish whether such treatments are superior to chemotherapy in front line setting for MAPK altered pLGGs.