NRAS Genes Research Articles

Development of custom amplicon-based test systems for detecting KRAS, NRAS, and BRAF gene mutations in colorectal cancer using next-generation sequencing (NGS).

e15061 Background: Polymerase chain reaction (PCR)-based mutation diagnostics in colorectal cancer is widely available. However, PCR has limitations in identifying rare mutations and precise mutation coordinates. Targeted sequencing, though promising, often incurs high costs and longer research durations, demanding higher DNA sample quality. Our study demonstrates the benefits of using a customized amplicon test system for diagnosing somatic mutations in tumor tissue, including detecting rare mutations not captured by PCR, requiring less input biomaterial, and similar research costs. Methods: A targeted NGS assay system was developed using region-specific primers for KRAS (exons 2, 3, and 4), NRAS (exons 2, 3, and 4), and BRAF (exon 15). Library preparation involved a three-step PCR process: Target PCR incorporating Illumina overhang adapter sequences, Index PCR introducing multiplexing indices and sequencing adapters, and PCR cleanup using magnetic beads. Sequencing utilized MiSeq v2 reagents with paired 300‐bp reads, generating high-quality, full-length reads. Validation included re-testing known samples. Tumor cell percentages in FFPE specimens were determined, and DNA was extracted using the QIAamp DNA FFPE Tissue Kit. Data analysis utilized proprietary software. Results: Out of 1234 samples, 1133 were tested, with mutations detected in 685 samples (60.45%). KRAS mutations were found in 548 (48.36%), NRAS mutations in 40 (3.5%), and BRAF mutations in 88 (7.7%) samples. Double mutations were observed in 9 samples (0.79%). Data on rare and double mutations are presented in the table. Conclusions: The developed test system accurately identifies essential mutations in colorectal cancer, including rare and potentially clinically significant variants. The required DNA amount ranged from 10 to 30 ng in 5 μl, nearly ten times less than well-known commercial PCR test systems like COBAS (Roche) and therascreen (QIAGEN). The cost of the study is comparable to PCR with a minimum study duration of 4 days. [Table: see text]

Liquid biopsy multiomics enable clinical triage for early colorectal cancer diagnosis.

e15599 Background: Colorectal cancer (CRC) is the second most common cause of cancer death in the United State. Despite advancements in screening, diagnosis, and treatment, the disease remains a formidable public health issue. In 2023, approximately 153020 individuals were diagnosed with CRC and 52550 will die from the disease. In order to meet early diagnostic, we developed this multiomics assay to enable clinical triage for liquid biopsy screening. Methods: Xenonucleic acid (XNAs) are artificial genetic polymers that retain the Watson-Crick base-pairing capability and exhibit high chemical and biological stability. They can be employed as molecular clamps in RT-qPCR due to the stronger hybridizing/binding capability seen in XNA/DNA duplexes than DNA/DNA duplexes. We are developing an XNA-based multiplex RT-qPCR assay for APC, BRAF, CTNNB1, KRAS, NRAS, PIK3CA, SMAD4 and TP53 gene mutation panel and CRC 9 genes methylation panel combined with multiplex protein panel, called ColoscapeTM Plus. We enrolled in FIT positive subjects of both genders, aged 50-74, attending the CRC screening program, we tested 152 plasma samples, 75 health, 50 AA and 27 early CRC with this multiomics assay. Results: The assay results showed that its sensitivity was about 44% (95% CI: 0.302- 0.587) and specificity about 80% (95%CI: 0.689- 0.880) for advanced adenoma (AA). For CRC stage I and II, its sensitivity is about 63% and 78% with 80% specificity separately. Conclusions: The primary data has shown that this low cost and rapid turnaround time multiomics assay has a comparable sensitivity for early colorectal cancer diagnostic.

Results of a phase 2 study of evorpacept (E, ALX148), cetuximab (C), and pembrolizumab (P) in patients with refractory microsatellite stable metastatic colorectal cancer (MSS CRC).

3530 Background: Single-agent anti-PD-1/PD-L1 drugs are ineffective in MSS CRC. E is an engineered protein (high-affinity CD47-blocker fused to an inactive IgG Fc region), which blocks the CD47/SIRPα innate immune inhibitory phagocytosis checkpoint expressed on CRC and phagocytes, respectively. Preclinical evidence suggests ECP may be effective in MSS CRC. Methods: This phase 2, single-arm, two-stage, multicenter, investigator-initiated trial of E (15 or 10 mg/kg weekly at dose level [DL] 1 and -1, respectively), C (400 mg/m2 then 250 mg/m2 weekly), and P (200 mg every 3 weeks) in 21-day cycles enrolled patients with MSS CRC, regardless of tumor sidedness and RAS/BRAF status, refractory to ≥ 2 prior lines of therapy, including EGFR inhibitor if indicated (NCT05167409). All patients in both Stage 1 (safety run-in) and Stage 2 (expansion) received ECP until progression or unacceptable toxicity. The co-primary objectives were to determine the recommended dose (RD) of E with CP and objective response rate (ORR) by RECIST v1.1 (by one-sided exact test with α=0.05, H0 p ≤ 3% [historical controls], HA p ≥ 15%; power is 87% with N=48). Results: The safety-evaluable population included 16 patients, of whom 69% were male and median age 53 years. The primary tumor was in the right, left, and transverse colorectum in 31%, 31%, and 6% of patients, and was unknown in 31%. Mutations in KRAS, NRAS, and BRAF genes were present in 38%, 6%, and 0% of patients. The median number of prior lines of therapy was 3, including prior EGFR inhibitor in 44%. In Stage 1, 9 and 3 patients were enrolled at E DL1 and DL-1, respectively. E DL1 was established as the RD for Stage 2, in which 4 patients were enrolled. The most common any-grade treatment-emergent adverse events were headache (32%), fatigue (26%), anemia (21%), diarrhea (21%), dyspnea (16%), abdominal pain (16%), nausea/vomiting (16% each), and acneiform rash (16%). There were two on-study deaths assessed as related to all three study drugs (hemophagocytic lymphohistiocytosis and cytokine release syndrome; 1 each). While both are known associations with checkpoint inhibitors, other significant factors contributing to these event outcomes included cancer progression with high tumor burden in the former and patient decision to transition to comfort care in the latter. In the safety-evaluable population, ORR was 6.3% (1 ongoing partial response, 95% confidence interval [CI] 0.2-30.2%), disease control rate was 12.5% (95% CI 1.6-38.4%), median progression-free survival was 2.6 months (mo; 95% CI 2.1-2.9 mo), and median overall survival was 10.9 mo (95% CI 3.3 mo-infinity). Formal hypothesis testing was not performed. Conclusions: Further dose optimization of ECP is needed to establish a RD in the refractory MSS CRC population. While initial activity was seen, at the DLs evaluated, criteria for study termination were met. Clinical trial information: NCT05167409 .

Incorporating precision oncology in everyday clinical practice: First two years of comprehensive genomic profiling (CGP) testing experience in Croatia.

11151 Background: Despite increased availability of modern technologies and evolution of tailored treatment, which has already significantly changed the outcomes in some cancer types, applicability of precision medicine in everyday clinical practice is still emerging and is one of the hot topics. Croatia is among first ones in the World to have entered into force a country-level based project of precision oncology throughout the CGP analysis in everyday clinical work, fully covered by Croatian health insurance. Hence, here we would like to present our data from everyday clinical practice and analyze the penetration of CGP given the opportunity and challenges we had. Methods: The study was observational and retrospective in nature, conducted on a country level among all patients on whose tumor specimens CGP was performed between January 1, 2020 and December 31, 2021. Eligible patients for the CGP analysis were those with metastatic disease, with at least 6-12 months of life-expectancy, with ECOG performance status ≤ 2, and after the recommendation from the multidisciplinary team. The analysis was performed in accredited laboratory (Foundation Medicine Inc., Cambridge, MA, USA), through FoundationOneCDx for solid tumors, FoundationOne Liquid in case of insufficient tissue or FoundationOneHeme for sarcomas. Results: There was total of 481 patients with CGP results. Median age was 61 years (IQR 49-69) and 58% of patients were women, while 42% were men. Most commonly tested were gastrointestinal (29.1%) and reproductive tumors (28.9%) with colorectal (19.1%) and uterine cancer (11.2%) as the most prevalent. At least one clinically relevant genomic alteration was found in 48.7% of patients. The most frequent mutations were those of KRAS (27.2%), PIK3CA (12.9%), AR (10.6%), NRAS (10.4%) and PTEN (9.1%) genes. Currently clinically not relevant mutations were found in 51.8% of patients with TP53 gene mutation as the most common (42.8%). According to the Croatian cancer registry, there was around 26 000 cancer related deaths during the investigated period defining population of at least more than 10 000 potentially eligible patients for the CGP analysis. Conclusions: Our results have shown that almost 50% of tested patients could have potential treatment benefit based on the detection of clinically relevant genomic alteration. Unfortunately, taking into consideration country level insurance eligibility criteria for CGP testing and number of potentially eligible cancer patients, we could say that only less than 5% of patients with metastatic disease were tested within first two years and that penetration of CGP is rather low, resulting with a significant number of patients underserved.

Comparing the efficacy and safety of HepaSphere drug-eluting bead transarterial chemoembolization combined with hepatic arterial infusion chemotherapy versus hepatic arterial infusion chemotherapy alone in unresectable colorectal liver metastases.

3565 Background: Drug-eluting bead transarterial chemoembolization (DEB-TACE) and hepatic arterial infusion chemotherapy (HAIC) are widely employed treatments for unresectable colorectal liver metastases (CRLM). Limited evaluation exists regarding TACE plus HAIC versus HAIC alone. This study aimed to evaluate the efficacy of combining HepaSphere DEB-TACE with HAIC compared to HAIC alone in patients with unresectable CRLM. Methods: A total of 259 CRLM patients aged over 18 years, treated at Peking University Cancer Hospital between March 2015 and November 2022, were included in this retrospective study. Among them, 157 patients received HepaSphere DEB-TACE combined with HAIC (DEB-TACE-HAIC), while 102 patients received HAIC alone. Propensity score matching (PSM) was performed to avoid selection bias and enable comparative analysis of efficacy and safety between the two cohorts. The primary endpoint was progression-free survival (PFS), while secondary endpoints comprised hepatic progression-free survival (hPFS), overall survival (OS), objective response rate (ORR), disease control rate (DCR) and safety. Results: PSM resulted in 83 pairs, achieving comparable baseline between DEB-TACE-HAIC and HAIC cohorts. Of these pairs, 116 were male and 50 were female. Notably, 51.2% of patients harbored mutations in either KRAS, NRAS, or BRAF genes. Prior to enrollment, 94.6% of patients had received systemic treatments. Median hPFS, PFS and OS were all higher in the matched DEB-TACE-HAIC cohort (median hPFS: 7.6 vs. 4.1 months, P=0.023; median PFS: 5.1 vs. 3.5 months, P=0.092; median OS: 15.5 vs. 14.7 months, P=0.513). ORR was 37.3% in the DEB-TACE-HAIC group and 27.7% in the HAIC group, with DCR of 75.9% and 69.9%, respectively. There were no treatment related deaths. Serious adverse events were comparable between the two groups, except for nausea, which occurred more frequently in the HAIC group (39.8% vs. 22.9%, P=0.019). Conclusions: DEB-TACE-HAIC demonstrated superior survival compared to HAIC alone in unresectable CRLM, even in patients refractory to systemic therapy. The combination of HepaSphere DEB-TACE and HAIC appears promising as a regional treatment option with improved outcomes and a comparable safety profile.

A phase III, open-label, multicenter, randomized controlled trial of tunlametinib versus investigator-selected chemotherapy in patients with advanced NRAS-mutant melanoma who had previously received immunotherapy.

TPS9606 Background: NRAS-mutant melanoma is an aggressive subtype with worse prognosis. However, no targeted therapy has been approved to date worldwide. Tunlametinib (HL-085) has showed an encouraging efficacy (confirmed ORR:34.8%, mPFS:4.2 months) with a manageable safety profile in phase II pivotal registrational study, which was published in ASCO 2023 annal meeting (NO.:9510). Here, we present the design of phase III randomized controlled study. Methods: This is a multicenter, two-arm, open-label, randomized controlled phase III confirmatory clinical trial to evaluate the efficacy and safety of tunlametinib in comparison with the combination chemotherapy of investigator's choice in advanced NRAS-mutant melanoma patients who had previously received immunotherapy. A total of 165 subjects from about 12 sites in China will be included and randomly assigned to the corresponding treatment group in 2:1 ratio. Two stratification factors before randomization: LDH level and whether have received chemotherapy. The key inclusion criteria included: a) Patients with unresectable stage III or metastatic IV melanoma confirmed by histology or cytology; b) History of immunotherapy failure or could not tolerate immunotherapy; c) Be able to provide NRAS mutation positive test report at baseline and provide sufficient histological specimens for NRAS mutation confirmation by central laboratory. Experimental group subjects will receive continuous administration of tunlametinib 12mg BID, 28 days per cycle. Control group subjects will receive one of the following regimens which according to investigator's choice (paclitaxel plus carboplatin, or temozolomide plus cisplatin, or dacarbazine plus cisplatin) , 28 days per cycle. The two groups will receive continuous therapy until disease progression or intolerable toxicity. The primary endpoint is progression-free survival which assessed by independent radiology review committee. Secondary endpoints included overall survival, objective response rate, disease control rate, duration of response, safety, pharmacokinetics and exposure-response. Exploratory endpoints are to evaluate quality of life between two groups and to development a companion diagnostic kit for NRAS gene mutations detection. This study is currently open for enrollment. Clinical trial information: NCT06008106 .

Changes in cell morphology and function induced by the NRAS Q61R mutation in lymphatic endothelial cells.

Recently, a low-level somatic mutation in the NRAS gene (c.182 A > G, Q61R) was identified in various specimens from patients with kaposiform lymphangiomatosis. However, it is unknown how these low-frequency mutated cells can affect the characterization and surrounding environment of their lesions. To understand the pathogenesis and association of these gene abnormalities, we established NRASQ61R mutated lymphatic endothelial cells transfected with lentivirus vector and undertook morphological and functional characterization, protein expression profiling, and metabolome analysis. NRASQ61R human dermal lymphatic endothelial cells showed poor tube formation, a low proliferation rate, and high migration ability, with an increase in the ratio of mutated cells. An analysis of signaling pathways showed inactivation of the PIK3/AKT/mTOR pathway and hyperactivation of the RAS/MAPK/ERK pathway, which was improved by MAPK kinase (MEK) inhibitor treatment. This study shows the theoretical circumstances induced in vitro by NRASQ61R-mutated cells in the affected lesions of kaposiform lymphangiomatosis patients.

Incidence of common gene mutations in early-onset colorectal cancer and the association with cancer survival: a meta-analysis

Objective: The incidence of early-onset colorectal cancer (EOCRC) is increasing globally; however, the molecular characteristics and prognosis of sporadic EOCRC are unclear. In this systematic review and meta-analysis, we aimed to investigate the incidence of gene mutations and their association with cancer survival in sporadic EOCRC, focusing on six common gene mutations (TP53, BRAF, KRAS, NRAS, PTEN, and APC). Methods: Ovid Embase and Ovid Medline electronic databases were searched for studies involving patients with sporadic EOCRC (i.e., diagnosed with colorectal cancer before the age of 50 years and with no evidence of hereditary syndromes predisposing to colorectal cancer). The included articles were evaluated using quality assessment tools. Meta-analysis was performed using random-effects and fixed-effects models. Cochran's Q statistic and the I2 index were used to assess heterogeneity. The incidence of the six common gene mutations listed above in sporadic EOCRC and their association with cancer survival were evaluated. Results: (1) Incidence of specific gene mutations in sporadic EOCRC. A total of 34 articles were included in this meta-analysis. The incidence of APC gene mutation was 36% (from 13 articles, 95%CI: 19%-55%, P=0.043); of KRAS gene mutation 30% (from 26 articles, 95%CI: 24%-35%, P=0.190); of BRAF gene mutation 7% (from 18 articles, 95%CI: 5%-11%, P=0.422); of NRAS gene mutation 4% (from five articles, 95%CI: 3%-5%, P=0.586); of PTEN gene mutation 6% (from six articles, 95%CI: 4%-10%, P=0.968); and of TP53 gene mutation 59% (from 13 articles, 95%CI: 49%-68%, P=0.164). (2) Association between gene mutations and survival in sporadic EOCRC. A total of six articles were included in this meta-analysis. Compared with wild-type BRAF, mutant BRAF was significantly associated with increased overall mortality risk in patients with EOCRC (pooled HR=2.85, 95%CI: 1.45-5.60, P=0.002). Subgroup analysis showed that the incidence of BRAF gene mutation was higher in Eastern than in Western countries, whereas the incidence of TP53, KRAS, NRAS, and APC gene mutations was lower. There was no significant difference in the incidence of PTEN gene mutation between different regions. Conclusion: Compared with colorectal cancer occurring in the general population, the incidence of APC and KRAS mutations is lower in EOCRC, whereas the incidence of TP53 mutation remains consistent. BRAF mutation is associated with increased overall mortality risk in patients with EOCRC.

Local intralesional talimogene laherparepvec therapy with complete local response in oral palatine mucosal melanoma: a case report

BackgroundMucosal melanoma, an aggressive type of malignancy different from the cutaneous melanomas commonly seen in the head and neck region, represents < 1% of all malignant melanomas. The pathogenesis of mucosal melanoma is unknown. Targetable mutations commonly seen in cutaneous melanoma, such as in the BRAF and NRAS genes, have a lower incidence in mucosal melanoma. Mucosal melanoma carries a distinct mutational pattern from cutaneous melanoma. Surgery with negative margins is the first-line treatment for mucosal melanoma, and systemic therapy is not well defined. Talimogene laherparepvec, an oncolytic viral immunotherapy, is United States Food and Drug Administration approved for the treatment of advanced malignant cutaneous melanoma, with local therapeutic benefits. Mucosal melanoma was initially excluded from talimogene laherparepvec’s initial phase III clinical trial.Case presentationWe present the case of a white female patient in her 40s with past medical history of systemic lupus erythematous, scleroderma, and estrogen-receptor-positive invasive ductal breast carcinoma. Following a bilateral mastectomy, the patient was found to have BRAF-negative mucosal melanoma of her hard palate with a soft palate skip lesion. Owing to the presence of a skip mucosal lesion as well as the anticipated defect and need for free-flap reconstructive surgery, nonsurgical management was considered. The patient was referred to medical oncology, where—based on the patient’s complicated medical history and the risk of immunotherapy possibly worsening her prior autoimmune diseases—local talimogene laherparepvec injections were chosen as the primary therapy for her mucosal lesions. Though talimogene laherparepvec is approved for the treatment of cutaneous melanoma, there are limited data available on the use of talimogene laherparepvec in mucosal melanomas.ConclusionThe patient had a complete local tumor response at both the primary lesion as well as the skip lesion with the local injections. She had no side effects and maintained a high quality of life during treatment.

Exploring the Relationship Between KRAS, NRAS, and BRAF Mutations and Clinical Characteristics in Iranian Colorectal Cancer Patients.

Patients with colorectal cancer can benefit from anti-EGFR (epidermal growth factor receptor) therapy. However, this therapy is not effective for treating colorectal cancers with constitutive activating mutations in the KRAS, NRAS, and BRAF genes. Molecular analysis of tumor tissue frequently informs treatment decisions for colorectal cancer. This study aims to identify KRAS, NRAS, and BRAF mutations in Iranian patients diagnosed with colorectal cancer and to assess the prevalence of these mutations relative to the tumor differentiation stage within these populations. From April 2018 to December 2022, 2000 specimens from patients with colorectal cancer were collected. Data on sex, age, and tumor differentiation stage were recorded for all samples. For mutation detection, the KRAS and NRAS exons (2, 3, and 4) were amplified using the Diatech kit, and a specific primer was used to amplify BRAF exon 15. Pyrosequencing was then performed. Analysis of samples revealed that 1105 specimens (55.3%) contained mutations in at least one of the screened genes. Among the genes studied, the highest occurrence was the KRAS mutation at 47.4%, followed by NRAS at 5.3% and BRAF at 2.7%. Most KRAS mutations were found in exon 2 (89.7%), with the G12D mutation being the most prevalent at 32% of cases. There was a significant difference in the rate of KRAS mutations in women (52.5%) compared to men (43.5%) (P = 0.02). For NRAS, the majority mutations were observed in exon 3 (76.2%), with the Q61H mutation being the most prevalent at 28.5% of cases. There were no significant associations between the clinicopathological parameters and mutations. The study's findings indicate a rising frequency of mutations in these genes in Iran, highlighting the need to screening mutations in the main exons of all three genes for effective colorectal cancer treatment strategies.

Identification and analysis of oncogenic non-synonymous single nucleotide polymorphisms in the human NRAS gene: An exclusive in silico study

BackgroundN-ras protein is encoded by the NRAS gene and operates as GDP-GTP-controlled on/off switching. N-ras interacts with cellular signaling networks that regulate various cellular activities including cell proliferation and survival. The nonsynonymous single nucleotide polymorphism (nsSNPs)-mediated alteration can substantially disrupt the structure and activity of the corresponding protein. N-ras has been reported to be associated with numerous diseases including cancers due to the nsSNPs. A comprehensive study on the NRAS gene to unveil the potentially damaging and oncogenic nsSNPs is yet to be accomplished. Hence, this extensive in silico study is intended to identify the disease-associated, specifically oncogenic nsSNPs of the NRAS gene. ResultsOut of 140 missense variants, 7 nsSNPs (I55R, G60E, G60R, Y64D, L79F, D119G, and V152F) were identified to be damaging utilizing 10 computational tools that works based on different algorithms with high accuracy. Among those, G60E, G60R, and D119G variants were further filtered considering their location in the highly conserved region and later identified as oncogenic variants. Interestingly, G60E and G60R variants were revealed to be particularly associated with lung adenocarcinoma, rhabdomyosarcoma, and prostate adenocarcinoma. Therefore, D119G could be subjected to detailed investigation for identifying its association with specific cancer. ConclusionThis in silico study identified the deleterious and oncogenic missense variants of the human NRAS gene that could be utilized for designing further experimental investigation. The outcomes of this study would be worthwhile in future research for developing personalized medicine.

Multiple Immune Dysregulation Conditions with a Single Underlying Root Cause of NRAS Gene-Positivity: A Case Report

Hematological malignancies in particular are recognized to be linked to immunological dysregulated disorders such hematolytic anemia, immunodeficiency, and autoimmune illnesses. We describe a rare case that had multiple immune dysregulation symptoms and was subsequently found to be positive for the NRAS gene. A male, 29, complained of pleuritic chest pain. On the ECG, he showed broad PR depression and elevated PR in aVR, along with elevated troponin. He received colchicine and NSAIDs as treatment with a good response and resolution of his symptoms. After four weeks, he developed a fever which thought to be secondary to perianal abscess, and a surgical incision and drainage along with broad spectrum antibiotics was used to treat a perianal abscess. Despite receiving extensive medical and surgical care, his fever persisted. His blood film started to show circulating blast cells. Following an immediate bone marrow aspiration and biopsy, there were no notable abnormalities seen, and a 1% blast was present, which was assumed to be the result of stress or infection. Later, a weak positive +1 Coombs (DAT) test is developed and caused active hemolytic anemia required blood transfusion. Along with the anemia, a wrist expanding wound was discovered in the previous IV cannula site. Clinically wound appears as a pyoderma gangrenosum. Histopathology showed purulent dermal inflammation including many neutrophils and macrophages. This feature of micro abscess formation was consisting with the diagnosis of pyoderma gangrenosum. An intravenous steroid (1 mg/kg) was administered in response to a positive Coombs test and pyoderma gangrenosum. The patient's condition greatly improved overall. His wound began to heal as his white blood cell count dropped. After being given oral steroids with a titration-down plan, the patient was sent home. A whole genome sequence was performed on him because he had peri myocarditis, pyoderma gangrenosum, and hematolytic anemia. The results show that he is NRAS gene positive. He was attentively observed clinically with regular out-patient follow-ups as this gene is strongly associated with hematological cancers. He developed symptomatic normocytic anemia a few months later, and a bone marrow aspiration and biopsy were performed to confirm the presence of Myelodysplastic syndromes (MDS). He was transferred to another facility and underwent chemotherapy and bone marrow transplant. In otherwise healthy individuals, the idea of offering a single explanation—Occam's razor—must be considered and investigated. Finding the underlying reason could improve patient care, enable precision therapy, and alter the course of events.

Epidemiological and clinicopathological features of KRAS, NRAS, BRAF mutations and MSI in Chinese patients with stage I-III colorectal cancer.

The selection of appropriate treatment modalities based on the presence or absence of mutations in KRAS, NRAS, BRAF, and the microsatellite instability (MSI) status has become a crucial consensus in colorectal cancer (CRC) therapy. However, the distribution pattern of these genetic mutations and the prevalence of MSI status in Chinese stage I-III CRCs remain unclear. We retrospectively analyzed clinicopathological features, mutations in the KRAS, NRAS, and BRAF genes, as well as MSI status of 411 patients with stage I-III CRC who underwent surgery from June 2020 to December 2022 in the First Affiliated Hospital of Nanjing Medical University. The mutation rates of KRAS, NRAS, and BRAF were 48.9%, 2.2%, and 3.2%, respectively, and the microsatellite instability-high rate was 9.5%. KRAS mutation was independently associated with mucinous adenocarcinoma. Multivariate analysis suggested that tumor location and mucinous adenocarcinoma were independently associated with BRAF mutation. Only T stage was associated with NRAS mutations in the univariate analysis. Multivariate analysis revealed that factors such as larger tumor size, tumor location, younger age, and poor differentiation were independently associated with microsatellite instability-high status. The results illustrate the mutation frequencies of KRAS, NRAS, BRAF genes and MSI status in stage I-III CRC from the eastern region of China. These findings further validate the associations between these genes status and various clinicopathological characteristics.

A novel t(X;21)(p11.4;q22.12) translocation adds to the role of BCOR and RUNX1 in myelodysplastic syndromes and acute myeloid leukemias.

In myeloid neoplasms, both fusion genes and gene mutations are well-established events identifying clinicopathological entities. In this study, we present a thus far undescribed t(X;21)(p11.4;q22.12) in five cases with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The translocation was isolated or accompanied by additional changes. It did not generate any fusion gene or gene deregulation by aberrant juxtaposition with regulatory sequences. Molecular analysis by targeted next-generation sequencing showed that the translocation was accompanied by at least one somatic mutation in TET2, EZH2, RUNX1, ASXL1, SRSF2, ZRSR2, DNMT3A, and NRAS genes. Co-occurrence of deletion of RUNX1 in 21q22 and of BCOR in Xp11 was associated with t(X;21). BCOR haploinsufficiency corresponded to a significant hypo-expression in t(X;21) cases, compared to normal controls and to normal karyotype AML. By contrast, RUNX1 expression was not altered, suggesting a compensatory effect by the remaining allele. Whole transcriptome analysis showed that overexpression of HOXA9 differentiated t(X;21) from both controls and t(8;21)-positive AML. In conclusion, we characterized a new recurrent reciprocal t(X;21)(p11.4;q22.12) chromosome translocation in MDS and AML, generating simultaneous BCOR and RUNX1 deletions rather than a fusion gene at the genomic level.

Multimodal therapy for metastatic colorectal cancer: a case of complete clinical and radiological response of liver metastases

Background. Colorectal cancer is one of the most common cancers and the second leading cause of cancer-related deaths worldwide. Population-based studies have shown that 25–30 % of patients with colorectal cancer have synchronous liver metastases at the time of diagnosis. despite modern advances in oncology and surgery, only 25 % of patients with metastatic colorectal cancer (mCRC) are suitable for liver resection, which is the only curative treatment option for these patients. In recent years, the indications for curative treatment of mCRC have expanded. due to the introduction of new targeted drugs into clinical practice, the tumor response rate to preoperative therapy has increased, thus increasing surgical resection rate. Some patients experience a complete clinical response, which is defined as the complete disappearance of liver metastases. However, 30–70 % of patients develop recurrent metastases in the liver within the first year of follow-up, and currently, even in the presence of complete regression of metastases, it is recommended to perform resection of the initially affected hepatic segments. Case presentation. We describe a case of complete clinical and radiological response of liver metastases after chemotherapy in a patient with sigmoid colon cancer. Molecular genetic analysis revealed the wild type of the Kras, Nras and Braf genes. The patient received 3 courses of preoperative chemotherapy according to the FOLFOxIRI + Cetuximab regimen. Laparoscopic sigmoid colon resection with d3 lymph node dissection was followed by adjuvant chemotherapy with oxaliplatin and 5-fluorouracil (12 cycles). After 16 months of follow-up, no evidence of colon cancer recurrence and liver metastasis was found. Conclusion. Current targeted therapy has demonstrated efficacy in treating mCRC with synchronous liver metastases and makes it possible, in selected cases, to avoid the liver resection provided that a complete clinical and radiological response of the metastases is achieved.

Abstract 3487: Molecular dynamic simulations of RAS family protein interactions: Mutant KRAS binding with wildtype HRAS and NRAS

Abstract Introduction: Mutations in the KRAS, HRAS, and NRAS genes are frequently associated with worse patient outcomes. Previous analyses of the structure and function of the associated proteins have been explored but not conclusively. The actual binding conformations of the three isoforms, especially when mutated are essential in designing inhibitory drugs. Recent studies have identified important interactions between the three isoforms that affect the oncogenic strength of the others when they are mutated. We use a bioinformatics approach to examine the modifications of the structural properties, mechanisms, and kinetic energies of eight KRAS mutants interacting with wildtype HRAS, and again with wildtype NRAS. Methodology: AlphaFold AI predicted models of KRAS, HRAS, and NRAS were downloaded from Uniprot and relevant mutations were induced using PyMOL. Pairs of KRAS mutants and either HRAS or NRAS were submitted to ClusPro for docking. PyMOL was used to separate proteins by chain identities before being repaired in GROMACS. Molecular dynamics simulations (MDS) were performed between proteins using GROMACS, a Linux based MDS software that renders a topology regulated by Newton’s Laws. XmGrace was used to analyze structure, MMPBSA.py was used to derive energy calculations, and VMD was used to visualize the interaction mechanisms. Results: RMSF data indicated C-terminus residues in KRAS mutants and WT-HRAS and WT-NRAS are the most mobile in their interactions. Notably, when WT-HRAS interacts with G12R, G12S, G12V, and Q61H, its C-terminus decreases fluctuation. G13D had the greatest binding strength and strength fluctuation with WT-HRAS. G12V had the greatest binding strength with WT-NRAS. All other mutants had weaker binding with WT-NRAS than WT-KRAS. WT-HRAS had stronger binding at residue Val187 with WT-KRAS than all mutants did. WT-HRAS residues Cys184, Lys185, and Cys186 each had weaker binding energies with WT-KRAS than most mutants did. WT-KRAS residues Gln25 and Lys117 both exhibited stronger binding energy in interactions with WT-HRAS than most mutants did. WT-NRAS had stronger binding at residues Met182 and Met189 with WT-KRAS than all mutants did. Residue Pro185 in WT-NRAS did not have significant interaction with WT-KRAS, but had a strong interaction with all mutants. WT-NRAS residue Val187 had weaker binding energy with WT-KRAS than most mutants. WT-KRAS residues Tyr32 and Asp38 both exhibited weaker binding energy in interactions with WT-NRAS than most mutants did. Conclusion: MDS between KRAS mutants and WT-HRAS and WT-NRAS present notable structural modification and binding kinetics. In each KRAS mutation, different segments of the protein complexes are involved in the interaction, indicating unique inhibitory methods are required to approach each mutant. Citation Format: Isaac Silverman, Michael S. Gerber, Aaron Shaykevich, Yitzchak F. Stein, Alexander Siegman, Sanjay Goel, Radhashree Maitra. Molecular dynamic simulations of RAS family protein interactions: Mutant KRAS binding with wildtype HRAS and NRAS [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 3487.

Ultraviolet Radiation Biological and Medical Implications.

Ultraviolet (UV) radiation plays a crucial role in the development of melanoma and non-melanoma skin cancers. The types of UV radiation are differentiated by wavelength: UVA (315 to 400 nm), UVB (280 to 320 nm), and UVC (100 to 280 nm). UV radiation can cause direct DNA damage in the forms of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). In addition, UV radiation can also cause DNA damage indirectly through photosensitization reactions caused by reactive oxygen species (ROS), which manifest as 8-hydroxy-2'-deoxyguanine (8-OHdG). Both direct and indirect DNA damage can lead to mutations in genes that promote the development of skin cancers. The development of melanoma is largely influenced by the signaling of the melanocortin one receptor (MC1R), which plays an essential role in the synthesis of melanin in the skin. UV-induced mutations in the BRAF and NRAS genes are also significant risk factors in melanoma development. UV radiation plays a significant role in basal cell carcinoma (BCC) development by causing mutations in the Hedgehog (Hh) pathway, which dysregulates cell proliferation and survival. UV radiation can also induce the development of squamous cell carcinoma via mutations in the TP53 gene and upregulation of MMPs in the stroma layer of the skin.

Fluoropyrimidine type, patient age, tumour sidedness and mutation status as determinants of benefit in patients with metastatic colorectal cancer treated with EGFR monoclonal antibodies: individual patient data pooled analysis of randomised trials from the ARCAD database

BackgroundKRAS mutations in metastatic colorectal cancer (mCRC) are used as predictive biomarkers to select therapy with EGFR monoclonal antibodies (mAbs). Other factors may be significant determinants of benefit.MethodsIndividual patient data from randomised trials with a head-to-head comparison between EGFR mAb versus no EGFR mAb (chemotherapy alone or best supportive care) in mCRC, across all lines of therapy, were pooled. Overall survival (OS) and progression-free survival (PFS) were compared between groups. Treatment effects within the predefined KRAS biomarker subsets were estimated by adjusted hazard ratio (HRadj) and 95% confidence interval (CI). EGFR mAb efficacy was measured within the KRAS wild-type subgroup according to BRAF and NRAS mutation status. In both KRAS wild-type and mutant subgroups, additional factors that could impact EGFR mAb efficacy were explored including the type of chemotherapy, line of therapy, age, sex, tumour sidedness and site of metastasis.Results5675 patients from 8 studies were included, all with known mCRC KRAS mutation status. OS (HRadj 0.90, 95% CI 0.84–0.98, p = 0.01) and PFS benefit (HRadj 0.73, 95% CI 0.68–0.79, p < 0.001) from EGFR mAbs was observed in the KRAS wild-type group. PFS benefit was seen in patients treated with fluorouracil (HRadj 0.75, 95% CI 0.68–0.82) but not with capecitabine-containing regimens (HRadj 1.04, 95% CI 0.86–1.26) (pinteraction = 0.002). Sidedness also interacted with EGFR mAb efficacy, with survival benefit restricted to left-sided disease (pinteraction = 0.038). PFS benefits differed according to age, with benefits greater in those under 70 (pinteraction = 0.001). The survival benefit was not demonstrated in those patients with mutations found in the KRAS, NRAS or BRAF genes. The presence of liver metastases interacted with EGFR mAb efficacy in patients with KRAS mutant mCRC (pinteraction = 0.004).ConclusionThe benefit provided by EGFR mAbs in KRAS WT mCRC is associated with left-sided primary tumour location, younger patient age and absence of NRAS or BRAF mutations. Survival benefit is observed with fluorouracil but not capecitabine. Exploratory results support further research in KRAS mutant mCRC without liver metastases.

Assessment of RAS-RAF-MAPK Pathway Mutation Status in Healthy Skin, Benign Nevi, and Cutaneous Melanomas: Pilot Study Using Droplet Digital PCR.

In the present study, we employed the ddPCR and IHC techniques to assess the prevalence and roles of RAS and RAF mutations in a small batch of melanoma (n = 22), benign moles (n = 15), and normal skin samples (n = 15). Mutational screening revealed the coexistence of BRAF and NRAS mutations in melanomas and nevi and the occurrence of NRAS G12/G13 variants in healthy skin. All investigated nevi had driver mutations in the BRAF or NRAS genes and elevated p16 protein expression, indicating cell cycle arrest despite an increased mutational burden. BRAF V600 mutations were identified in 54% of melanomas, and NRAS G12/G13 mutations in 50%. The BRAF mutations were associated with the Breslow index (BI) (p = 0.029) and TIL infiltration (p = 0.027), whereas the NRAS mutations correlated with the BI (p = 0.01) and the mitotic index (p = 0.04). Here, we demonstrate that the "young" ddPCR technology is as effective as a CE-IVD marked real-time PCR method for detecting BRAF V600 hotspot mutations in tumor biopsies and recommend it for extended use in clinical settings. Moreover, ddPCR was able to detect low-frequency hotspot mutations, such as NRAS G12/G13, in our tissue specimens, which makes it a promising tool for investigating the mutational landscape of sun-damaged skin, benign nevi, and melanomas in more extensive clinical studies.

Transactivation of Met signaling by oncogenic Gnaq drives the evolution of melanoma in Hgf-Cdk4 mice

Recent pan-cancer genomic analyses have identified numerous oncogenic driver mutations that occur in a cell-type and tissue-specific distribution. For example, oncogenic mutations in Braf and Nras genes arise predominantly in melanocytic neoplasms of the epidermis, while oncogenic mutations in Gnaq/11 genes arise mostly in melanocytic lesions of the dermis or the uvea. The mechanisms promoting cell-type and tissue-specific oncogenic events currently remain poorly understood. Here, we report that Gnaq/11 hotspot mutations occur as early oncogenic drivers during the evolution of primary melanomas in Hgf-Cdk4 mice. Additional single base substitutions in the Trp53 gene and structural chromosomal aberrations favoring amplifications of the chromosomal region containing the Met receptor gene accumulate during serial tumor transplantation and in cell lines established in vitro. Mechanistically, we found that the GnaqQ209L mutation transactivates the Met receptor. Overexpression of oncogenic GnaqQ209L in the immortalized melanocyte cell line promoted in vivo growth that was enhanced by transgenic Hgf expression in the tumor microenvironment. This cross-signaling mechanism explains the selection of oncogenic Gnaq/11 in primary Hgf-Cdk4 melanomas and provides an example of how oncogenic driver mutations, intracellular signaling cascades, and microenvironmental cues cooperate to drive cancer development in a tissue-specific fashion.