NRAS Codon Research Articles

Association of various RAS codon mutations and prognostic outcomes of patients with colorectal liver metastases after hepatectomy.

The prognostic and predictive value of RAS mutations in patients with colorectal liver metastases (CRLM) who have undergone hepatectomy holds substantial importance. The present study aimed to investigate the impact of different RAS codon mutations on long-term survival in CRLM patients. A retrospective analysis was conducted on clinicopathological data from 399 CRLM patients with RAS mutations who underwent hepatectomy between January 2000 and December 2020. The RAS mutation gene status was assessed in KRAS codons (G12, G13, Q61, and A146) and NRAS codons (G12, G13, and Q61). Survival curves were generated using the Kaplan-Meier plotter and compared using the log-rank test. Univariate and multivariate analyses were performed to analyze the clinicopathological data. In the entire cohort, patients with KRAS G12 mutations exhibited the most favorable prognosis (p = 0.018). Comparatively, patients harboring KRAS Q61 mutations experienced poorer overall survival (OS) with a median of 15 months versus 33 months (p = 0.011) when compared to those with KRAS G12 mutations. Moreover, patients with NRAS Q61 mutations also showed decreased OS with a median of 26 months versus 33 months (p = 0.020) in comparison to KRAS G12 mutation patients. The results of multivariate analysis showed that both KRAS Q61 mutation (HR 2.130; 95% CI 1.088-4.168; p = 0.027) and NRAS Q61 mutation (HR 2.877; 95% CI 1.398-5.922; p = 0.004) were independent influencing factors of OS. Based on all identified risk factors, patients with RAS mutation were divided into high-risk and low-risk groups. Notably, in the high-risk group, the incorporation of postoperative chemotherapy was associated with longer OS, while it did not improve the survival of patients in the low-risk group. KRAS Q61 and NRAS Q61 mutations are promising predictors for OS in CRLM patients after hepatectomy. Postoperative chemotherapy may significantly benefit CRLM patients with RAS mutations, particularly those identified as high-risk.

NRAS mutation in melanoma: Association of codon-specific changes on patient outcomes.

e21545 Background: NRAS mutant melanoma is an aggressive subtype making up 20-30% of cutaneous melanoma cases without specific treatment options beyond immune checkpoint inhibition (ICI). Previous studies have suggested specific NRAS codon mutations are associated with unique signaling pathways. To test the hypothesis that NRAS codon mutations in cutaneous melanoma may drive a preferential response to ICI, we evaluated specific genetic subsets of NRAS mutant melanoma based on the Q61 and G12/G13 codons. Methods: Cutaneous melanoma samples (n = 4091) were analyzed by NGS at Caris Life Sciences. Samples were stratified by NRAS status and subdivided into NRASQ61, NRASG12/13, and NRAS-wildtype. PD-L1 expression was assessed using IHC (positive ≥ 1%). TMB-high was defined as ≥ 10 mutations/Mb. Transcriptomic signatures of MAPK pathway activation and T-cell signatures were calculated. Real-world overall survival (rwOS) information was obtained from insurance claims data, with Kaplan-Meier estimates calculated from time on ICI until last date of contact. Mann-Whitney U, Chi-square, and Fisher exact tests were applied as appropriate, with p-values adjusted for multiple comparisons. Results: Cutaneous melanoma samples were comprised of 22% NRASQ61 (n = 902), 3.1% NRASG12/13 (n = 125) and 74.8% NRASWT (n = 3064), with the most common NRAS mutation being Q61R. Patients harboring NRASG12/13 codon changes were associated with concurrent mutations in BRAF (21.0 vs 6.4%, p< 0.0001), NF1 (50.5% vs 8.7%, p< 0.0001), KIT (3.2% vs 0.2%, p< 0.001), ARID2 (22.1% vs 11.3%, p< 0.01), MAP2K1 (7.2% vs 2.6%, p = 0.022) compared to NRASQ61. NRASG12/13 tumors were more frequently TMB-high compared to NRASQ61 or NRASWT (83.1% vs 62.1% vs 55.1%, p = 0.0001), but no difference was observed in the prevalence of dMMR/MSI-H or PD-L1 expression. Transcriptomic analysis showed that NRASQ61 mutants had a significantly higher MPAS compared to NRASG12/13 or NRASWT cohorts (Median: 1.16 vs 0.67 vs 0.46, p = 0.0147), while T-cell-inflamed scores were significantly higher in NRASWT compared to NRASQ61 (Median: 4 vs -40, p = 0.00492) but not to NRASG12/13 (Median: 4 vs -13.5, p = 0.674). No difference in rwOS was identified in NRASG12/13 and NRASQ61 melanoma treated with ICI (HR: 0.753, [95% CI: 0.43 – 1.31], p = 0.316). Conclusions: Patients with NRAS-mutated cutaneous melanoma exhibit codon-specific ( NRASQ61 vs NRASG12/13) signaling and prevalence of immunotherapy-related biomarkers. Our findings indicate that melanoma samples with NRASG12/13 mutation tend to exhibit immune richness suggesting they could be viewed as a distinct group that might potentially benefit from immune therapies. This observation offers valuable insight into codon-specific tumor subsets, differential response to therapy, resistance mechanisms, and the potential development of combination therapies to enhance survival outcomes of patients with NRAS mutant melanoma.

Macrofollicular Architecture in Invasive Encapsulated Follicular Variant of Papillary Thyroid Carcinoma: A Pitfall in Thyroid Practice.

Predominantly macrofollicular architecture in invasive encapsulated follicular variant of papillary thyroid carcinoma (IEFVPTC-MF) is rare and often a cause of misinterpretation during pre-operative work-up and histopathology evaluation. We comprehensively evaluated the radiological, cytological, gross, microscopic, molecular and follow-up characteristics of four such cases, intending to increase its recognition and add our experience to the limited literature available. All such histopathologically-proven cases of IEFVPTC-MF were retrieved from the departmental archives. The clinical details, thyroid ultrasound, cytology and thyroid scan findings were reviewed. Allele-specific PCR for BRAF p.V600E, KRAS, NRAS, and HRAS mutations, and FISH assays for ETV6::NTRK3 fusion and RET fusions were performed. There were four cases of IEFVPTC-MF diagnosed between 2021 and 2022, involving two males and two females. The median age at presentation was 27years, and the duration of the disease was 1-10years. Thyroid ultrasound was TR1 (benign; n = 1), TR2 (not suspicious; n = 2), or TR4 (moderately suspicious; n = 1). Cytology was categorized as nondiagnostic (n = 1), benign (n = 1), andatypia of undetermined significance (n = 1). The three nodules with available cytology smears showed abundant colloid. Cells were arranged as sheets/microfollicles/clusters. Nuclei were predominantly round with minimal/focal elongation, membrane irregularity, and cellular crowding. On gross examination, cut surfaces of the tumors showed variable amounts of colloid. The tumors were solid-cystic. Histopathology revealed partially encapsulated multinodular tumors. There were prominent pseudopapillae projecting into the lumina of macrofollicles. Nuclei were predominantly round with variable nuclear atypia, including chromatin clearing and multifocal presence of nuclear grooves. Pseudoinclusions were identified in two. Molecular analysis revealed NRAS codon 61 mutation and ETV6::NTRK3 fusion in one case each. Two patients had cervical lymph node and hematogenous metastases. Post-radio-active iodine, the response was structurally incomplete (n = 2), indeterminate (n = 1) and excellent (n = 1). Macrofollicular architecture in invasive encapsulated follicular variant of papillary thyroid carcinoma is a major pitfall in thyroid oncology practice. Long-standing disease, and ultrasonographic and cytological features that overlap with benign disease, often lead to underdiagnosis during pre-operative evaluation. As patients may consequently develop distant metastases and have inadequate treatment response, there is a need for more vigilant understanding of the spectrum of macrofollicular thyroid disease for accurate diagnosis. ETV6::NTRK3 or other fusions, when found, present opportunities for targeted therapy.

KRAS/NRAS Mutations Associated with Distant Metastasis and BRAF/PIK3CA Mutations Associated with Poor Tumor Differentiation in Colorectal Cancer.

The occurrence, progression, and prognosis of colorectal cancer (CRC) are regulated by EGFR-mediated signaling pathways. However, the relationship between the core genes (KRAS/NRAS/BRAF/PIK3CA) status in the signaling pathways and clinicopathological characteristics of CRC patients in Hakka population remains controversial. Patients were genotyped for KRAS (codons 12, 13, 61, 117, and 146), NRAS (codons 12, 61, 117, and 146), BRAF (codons 600), and PIK3CA (codons 542, 545 and 1047) mutations. Clinical records were collected, and clinicopathological characteristic associations were analyzed together with mutations of studied genes. Four hundred and eight patients (256 men and 152 women) were included in the analysis. At least one mutation in the four genes was detected in 216 (52.9%) patients, while none was detected in 192 (47.1%) patients. KRAS, NRAS, BRAF, and PIK3CA mutation status were detected in 190 (46.6%), 11 (2.7%), 10 (2.5%), 34 (8.3%) samples, respectively. KRAS exon 2 had the highest proportion (62.5%). Age, tumor site, tumor size, lymphovascular invasion, and perineural invasion were not associated with gene mutations. KRAS mutations (adjusted OR 1.675, 95% CI 1.017-2.760, P=0.043) and NRAS mutations (adjusted OR 5.183, 95% CI 1.239-21.687, P=0.024) appeared more frequently in patients with distant metastasis. BRAF mutations (adjusted OR 7.224, 95% CI 1.356-38.488, P=0.021) and PIK3CA mutations (adjusted OR 3.811, 95% CI 1.268-11.455, P=0.017) associated with poorly differentiated tumor. KRAS/NRAS mutations are associated with distant metastasis and BRAF/PIK3CA mutations are associated with poor tumor differentiation in CRC. And the results provided a better understanding between clinicopathological characteristics and gene mutations in CRC patients.

Collision tumor of a papillary and follicular thyroid carcinoma: a case report

BackgroundPapillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC) are common differentiated thyroid cancers, but the detection of a collision tumor is an extremely rare event. Case presentationThe patient was a 69-year-old Japanese female with multiple cervical lymph node swellings and a thyroid tumor. Preoperative fine needle aspiration cytology of the enlarged lymph node revealed a cytological diagnosis of papillary thyroid carcinoma (PTC). A total thyroidectomy, right cervical dissection and paratracheal dissection were performed. Histopathological and immunohistochemical analyses of resected specimens revealed a collision tumor of PTC and FTC. Multiple metastases of papillary carcinoma were found in the dissected lymph nodes. In the PTC lesion, IHC for BRAF (V600E) was positive but negative for the FTC lesion. Genetic analyses further revealed a TERT promoter C228T mutation in PTC and a NRAS codon 61 mutation in FTC. The patient died of recurrent cancer 8 months after surgery.ConclusionsA case of a collision tumor of PTC and FTC is very rare, and even fewer cases have been subjected to genetic scrutiny. The present case was successfully diagnosed by pathological examination using immunohistochemical and genetic analyses. The TERT promoter mutation in the PTC lesion was consistent with the aggressive behavior of the cancer.

Abstract A005: Canine hemangiosarcoma as a model for RAS-mutated human cancers: Preliminary data

Abstract Canine cancer patients represent an important translational model for human cancer research with their spontaneous tumor pathogenesis and the many histological, genetic, molecular and clinical similarities between canine and human tumors. Canine tumors provide an opportunity to investigate shared biomarkers and their response to therapeutic interventions over an accelerated timeline compared to human cancers due to differences in expected lifespan and other factors. Dogs, like humans, have three related RAS genes, HRAS, KRAS, and NRAS, with extremely similar genetic sequence and resultantly high protein homology. These proteins have many key molecular functions including roles in cell proliferation and survival. Across multiple human cancer types, recurrent gain-of-function missense mutations in the RAS-family genes occur primarily at codons 12, 13, and 61. These mutations result in constitutively active RAS proteins. The frequencies of mutations across the three RAS genes and codons have been hypothesized to lead to distinct biological behaviors. Mutations are most common in KRAS, then NRAS followed by HRAS. We investigated RAS-family genetic mutations in a dataset of 213 canine patients with hemangiosarcoma (HSA), a common, aggressive malignancy derived from endothelial cells for which treatment with both surgery and traditional chemotherapies often fail to prolong survival beyond six months. Recent publications show that genomic profiling of canine HSA tumors can help group tumors into subtypes with potential for guiding therapy selection and improving outcomes. All 213 dogs had tumor tissue sequenced via the FidoCure® Precision Medicine Platform targeted sequencing panel and were followed until time of death or were censored to the date patients were last known to be living. All SNVs and Indels detected were missense mutations. Unlike human pan-cancer data, somatic mutations in NRAS (n=32) were most common with only 1 dog with a somatic KRAS mutation and no dogs with somatic HRAS mutations. Similar to human RAS mutations, affected codons were primarily codon 61 (n=28), codon 13 (n=5), and codon 12 (n=1). Pairwise survival comparisons between dogs with missense mutations in NRAS codon 61 showed that this mutation was associated with better prognosis (n=28, MST 251 [CI 213;327]) compared to dogs with missense mutations at other codons in the NRAS and KRAS genes (n=5, MST 66 [CI 64;NA], p=1.58E-05) and compared to dogs without missense mutations in the RAS family of genes (n=180, MST 123 [CI 100;149], p=0.036). These preliminary findings of similarities between canine and human tumors and associations between mutated codons and prognosis in our growing dataset create an important opportunity to accelerate cancer research, including clinical and biopharmaceutical studies, with great potential benefit to both dogs and humans. Citation Format: Michelle E. White, Garrett Harvey, Lucas Rodrigues, Chase Schwalbach, Dorothy Girimonte, Aubrey Miller, Abigail Hull, Lindsay Lambert, Christina Lopes, Gerald Post. Canine hemangiosarcoma as a model for RAS-mutated human cancers: Preliminary data [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A005.

ODP465 BRAF V600 and NRAS Q61 mutations in thyroid fine-needle aspiration (FNA) with indeterminate cytology in Argentina

Abstract It is known that thyroid cancer initiation and progression occurs because of gradual accumulation of genetic and epigenetic alterations at cell level. Many mutations associated with this disease have been reported to be present in genes encoding proteins in the mitogen-activated protein kinase (MAPK) signaling pathway, specifically BRAF and RAS genes, as well as in the PI3K/Akt signaling pathway, among others. In particular, molecular alterations in follicular cells in the BRAF or NRAS genes have been reported to be associated with the process of carcinogenesis. Previously we found BRAF and NRAS mutations are observed in a significant number of papillary and follicular thyroid carcinomas in our population, respectively. In this study, we tested the diagnostic performance of BRAF and NRAS mutation in thyroid carcinoma in fine-needle aspiration (FNA) specimens with indeterminate cytology. In total, residual material from 73 FNA samples was used (n= 16, Bethesda III; n=48, Bethesda IV and n=9 Bethesda V). BRAF codon 600 mutation and NRAS codon 61 mutations were investigated using qPCR and melting curve analysis was carried out. Diagnosis of malignancy was confirmed by histology on paired surgical specimen in 54 cases. Results In the cytologically indeterminate categories, 9.6% of specimens were BRAF V600 mutated: 1 of 16 were subcategorized as atypia of undetermined significance or follicular lesion of undetermined significance (6.3%); 2 of 48 as follicular neoplasm or suspicious for follicular neoplasm (4.1%); and 4 of 9 as suspicious for malignancy (44.4%). NRAS Q61 mutation was found in 4 patients (2 with Bethesda IV and 2 with cytology V). The BRAF V600 mutations were associated with carcinoma in 100% of cases (n = 7), whereas only 75% (n = 3) of the nodules with NRAS were associated with carcinoma. BRAF V600 and NRAS Q61 mutations had high specificity in predicting malignant carcinoma (96.8%); the sensitivity was 43.5% with a positive predictive value of 90.9% and a negative predictive value of 69.8%. In specific categories of indeterminate cytology, the pre-test risk of malignancy was 38%, 33% and 88% while the post molecular test risk of malignancy was 100%, 75% and 100% respectively. Conclusions BRAF V600 and NRAS Q61 molecular test in residual FNA samples improve diagnostic accuracy of the cytology analysis in our population and may help to better select patients for avoid inadequate or unnecessary surgeries. Presentation: No date and time listed

Establishment and Characterisation of Human Clear Cell Sarcoma and Malignant Melanoma Cell Lines.

Clear cell sarcoma of soft tissue (CCSST) and conventional malignant melanoma (MM) are rare and aggressive tumours with similarities in morphology and the expression of melanocytic markers. We established two CCSST cell lines (FU-CCSST-1 and FU-CCSST-2) from soft tissues of the patella and supraclavicular. A MM cell line (FU-MM-1) was established from lymph node metastases of subungual malignant melanoma. FU-CCSST-2 cells were transplantable to immunodeficient mice. Immunohistochemical studies demonstrated tumour cells were negative for cytokeratin AE1/AE3 and positive for S100 protein, HMB45, Melan-A, CD146 and SOX10 in all specimens. FU-CCSST-1 and FU-CCSST-2 harboured t(12;22)(q13;q12) translocations with expression of the EWSR1/ATF1 fusion gene. FU-MM-1 demonstrated loss of the short arm of chromosome 9 and harboured wild-type BRAF (codon 469 and 600) and NRAS (codon 12, 13 and 61). We report the establishment and characterisation of CCSST and MM cell lines that may have utility in the study of pathogenic mechanisms and development of novel therapeutic reagents.

Molecular Pathological Characteristics of Thyroid Follicular-Patterned Tumors Showing Nodule-in-Nodule Appearance with Poorly Differentiated Component.

Simple SummaryPoorly differentiated thyroid carcinoma (PDTC) is aggressive and is reportedly evoked by well-differentiated thyroid follicular-patterned tumors (TFTs). TFTs showing nodule-in-nodule (NN) appearance with PD component (PDc) but neither invasion nor metastasis are uncommon and are regarded as benign nodules despite their high-grade histological features. Our study aimed to preliminarily assess the potential role of PDc in NN in PDTC carcinogenesis using dual-color immunofluorescence of 53BP1 as a DNA damage response (DDR) molecule and the Ki-67, NRAS codon 61, and TERT-promoter (TERT-p) mutations. The prevalence of abnormal type 53BP1 expression and NRAS and TERT-p mutations in PDc was comparable to that of carcinomas. Because co-expression of 53BP1 and Ki-67 can be an indicator of altered DDR, the development of PDc in NN may be associated with DDR impairments after harboring NRAS and TERT-p mutations. Therefore, PDc in NN is potentially a precursor lesion associated with PDTC.Thyroid follicular-patterned tumors (TFTs) showing nodule-in-nodule (NN) appearance with poorly differentiated component (PDc) but neither invasion nor metastasis are diagnosed as benign nodules. Although PDc exhibits histologically aggressive features relative to the outer nodule (Out-N), its pathological significance remains unclear. TP53 binding protein-1 (53BP1) is a DNA damage response (DDR) molecule that rapidly localizes at DNA double-strand breaks. Using dual-color immunofluorescence with Ki-67, the profile of 53BP1 expression is shown to be significantly altered during diverse tumorigenesis. In this study, we aimed to elucidate the malignant potential of PDc at the molecular level. We analyzed the profile of 53BP1 expression and NRAS codon 61 and TERT-promoter (TERT-p) mutations in 16 cases of TFTs showing NN with PDc compared to 30 adenomatous goiters, 31 follicular adenomas, 15 minimally invasive follicular carcinomas (FCs), and 11 widely invasive FC cases. Our results revealed that the expression level of abnormal type 53BP1 and incidence of NRAS and TERT-p mutations in PDc were comparable to FCs, suggesting a malignant potential. Because co-expression of 53BP1 and Ki-67 can be an indicator of altered DDR, the development of PDc in NN may be associated with DDR impairments after harboring NRAS and TERT-p mutations.

The drug targets genomic alterations detected in female tumor tissue with melanoma.

e21558 Background: Due to the rapid development of next-generation sequencing (NGS) and precision medicine, molecularly driven targeted therapy, for different tumor types, plays an important role in cancer patients with drug targets. mucosal melanoma (MM) is aggressive disease with a poor prognosis. there are few studies focusing on its target drug. Methods: We enrolled female patients with melanoma (n = 24) from 2019.** to 2021.** cancer database with a gynecological tumor site. Subsequently, we chose to use WES sequencing to predict possible targets in patients. Results: In all mutations of the 24 melanoma patients, we screen the mutations about the drug targets. And the result shown that BRAF somatic mutations were identified in 6 of the 24 samples (25%), which are the highest frequency drug targets. V600E substitutions accounted for 3 (50%) of these mutations as target drug. Other activating mutations at NRAS codons 61 and 12/13 were lower frequent (Q61*, n = 3; G13D, n = 1; G12A, n = 1). TP53 and TOP2B were mutated in 4(16%) tumors were found as drug targets. Conclusions: This study aimed to analyze the target drug interaction of MM in China and lay a support for targeted therapy. NGS in MM is feasible with potential impact in half of our cohort. Earlier testing and improved trial drug from drug bank database access is necessary to increase the like hood that MM patients may benefit from drug target guided therapy.

A clinicopathologic and molecular analysis of five cases of bronchiolar adenoma with rare mutations.

Bronchiolar adenoma (BA) is a rare benign lung tumor that shows proliferation of bland bronchiolar-type epithelium containing a continuous layer of basal cells. This tumor entity has been newly added to the recent World Health Organization (WHO) classification 5th edition. This entity encompasses a spectrum of lesions: the classic ciliated muconodular papillary tumor (CMPT) and the non-classic CMPT. Although BA is reported to have driver mutations including BRAF V600E, EGFR, and KRAS, the molecular profile of BA is still incompletely understood. Five resected BAs at our institutions were analyzed. The BA lesions were subdivided into two groups: three proximal-type BAs and two distal-type BAs. NRAS codon 12/13 mutation and EML4 exon 20-ALK exon 20 fusion were found in two of the three proximal-types. BRAF V600E mutation was found in one of the two distal-types. Two cases coexisted with lung adenocarcinoma, with EGFR exon 19 deletion and KRAS mutation, respectively. No recurrence was observed at a median of 12 months (range 2-84 months) of follow-up. BA has uncommon variants of mutation seen in lung adenocarcinoma. NRAS mutation and ALK fusion partner has not been reported previously. The present cases may reinforce the distinctive biology of BA from lung adenocarcinoma.

Prognostic value of the combination of primary tumor location and RAS mutational status on patients with colorectal liver metastasis undergoing hepatectomy.

To assess prognostic influences of RAS mutational status and primary tumor site on cases with colorectal liver metastasis (CRLM) who underwent hepatectomy. Clinicopathological data of 762 patients with CRLM who underwent hepatectomy between January 2000 and November 2018 were retrospectively analyzed. The left-sided tumors (LST) included tumors located in the splenic flexure, descending colon, sigmoid colon, and rectum; while right-sided tumors (RST) included those located in the cecum, ascending colon, and transverse colon. RAS mutational status was determined using Sanger sequencing or next-generation sequencing, including KRAS (Codons 12, 13, and 61) and NRAS (Codons 12, 13, and 61), which were defined as wild-type (RASwt) and mutant-type (RASmut), respectively. Survival curves were plotted using the Kaplan-Meier plotter and compared by the log rank test. The clinicopathological data were analyzed using univariate and multivariate analyses. The 5-year overall survival (OS) in the LST group was longer than that in the RST group (OS: 47.1% vs. 31.0%, p = 0.000, respectively), and the OS in the RASwt group was longer compared with that in the RASmut group (OS: 53.6% vs. 24.0%, p = 0.000). Besides, overall survival of the patients after hepatectomy was alternative, which was stratified by primary tumor site, with the 1-, 3-, and 5-year survival rates of 93.1%, 62.1%, and 47.1% for patients with LST, and 91.1%, 42.8%, and 31.0% for patients with RST, respectively. OS and disease-free survival (DFS) were significantly different stratified by RAS mutational status, with the 1-, 3-, and 5-year rates of 96.9%, 67.9%, and 53.6% for patients with RASwt tumors, and 85.7%, 41.5%, and 24.0% for patients with RASmut tumors, respectively. The 1-, 3-, and 5-year DFS rates were 51.9%, 30.0%, and 26.7% for patients with RASwt tumors, and 35.8%, 18.2%, and 14.9% for patients with RASmut tumors, respectively. The results of multivariate analysis showed that RAS mutational status and primary tumor site were both independent influencing factors of OS. RAS mutational status and primary tumor site affect OS independently in CRLM patients undergoing hepatectomy. The worse prognosis of RST cannot be simply attributed to the imbalance of RAS mutational status in different primary tumor sites.

Carcinogen-induced DNA structural distortion differences in the RAS gene isoforms; the importance of local sequence

BackgroundLocal sequence context is known to have an impact on the mutational pattern seen in cancer. The RAS genes and a smoking carcinogen, Benzo[a]pyrene diol epoxide (BPDE), have been utilised to explore these context effects. BPDE is known to form an adduct at the guanines in a number of RAS gene sites, KRAS codons 12, 13 and 14, NRAS codon 12, and HRAS codons 12 and 14.ResultsMolecular modelling techniques, along with multivariate analysis, have been utilised to determine the sequence influenced differences between BPDE-adducted RAS gene sequences as well as the local distortion caused by the adducts.ConclusionsWe conclude that G:C > T:A mutations at KRAS codon 12 in the tumours of lung cancer patients (who smoke), proposed to be predominantly caused by BPDE, are due to the effect of the interaction methyl group at the C5 position of the thymine base in the KRAS sequence with the BPDE carcinogen investigated causing increased distortion. We further suggest methylated cytosine would have a similar effect, showing the importance of methylation in cancer development.

NGS-Based Analysis of Atypical Deep Penetrating Nevi.

Simple SummaryThe recent WHO classification of melanocytic tumors requires the implementation of combined phenotypic–genotypic diagnostics. For rare tumors, such as atypical deep penetrating nevi, there is insufficient information regarding genetic status, and it is not yet clear whether the observed unusual morphological cyto-architectures reflect a distinct genomic profile or are associated with an increased metastatic potential and aggressive clinical behavior. We report herein a comprehensive next-generation sequencing (NGS) analysis of a series of atypical DPNs, showing their mutational profile with some specific signatures for these rare and diagnostically challenging tumors.Deep penetrating nevi (DPNs) are rare melanocytic neoplasms consisting of pigmented spindled or epithelioid melanocytes with a distinctive wedge-shaped configuration showing activation of the WNT pathway, with unusual cyto-architectural features. It is unclear whether they show a distinct genomic profile associated with a diverse metastatic potential. We describe herein a cohort of 21 atypical DPNs analyzed by next-generation sequencing using the Ion AmpliSeq™ Comprehensive Cancer Panel. We found that β-catenin exon 3 was mutated in 95% and MAP kinase pathway genes in 71% of the cases. Less frequent mutations were observed in HRAS (19%) and MAP2K1 (24%). Isocitrate dehydrogenases 1 (IDH1) mutations, including R132C, V178I, and S278L, were identified in 38% of cases and co-existed with BRAF/HRAS mutations. The only case with progressive nodal disease carried alterations in the β-catenin pathway and mutations in IDH1 and NRAS (codon 61). By a comprehensive mutation analysis, we found low genetic heterogeneity and a lack of significant associations between specific gene mutations and histopathological features, despite atypical features. Whether the acquisition of an NRAS or IDH1 mutation in an atypical DPN may represent a molecular evolution implying a pathway to melanoma progression should be confirmed in a larger series.

Clinical and Functional Characterization of Atypical KRAS/NRAS Mutations in Metastatic Colorectal Cancer.

Mutations in KRAS/NRAS (RAS) predict lack of anti-EGFR efficacy in metastatic colorectal cancer (mCRC). However, it is unclear if all RAS mutations have similar impact, and atypical mutations beyond those in standard guidelines exist. We reviewed 7 tissue and 1 cell-free DNA cohorts of 9,485 patients to characterize atypical RAS variants. Using an in vitro cell-based assay (functional annotation for cancer treatment), Ba/F3 transformation, and in vivo xenograft models of transduced isogenic clones, we assessed signaling changes across mutations. KRAS exon 2, extended RAS, and atypical RAS mutations were noted in 37.8%, 9.5%, and 1.2% of patients, respectively. Among atypical variants, KRAS L19F, Q22K, and D33E occurred at prevalence ≥0.1%, whereas no NRAS codon 117/146 and only one NRAS codon 59 mutation was noted. Atypical RAS mutations had worse overall survival than RAS/BRAF wild-type mCRC (HR, 2.90; 95% confidence interval, 1.24-6.80; P = 0.014). We functionally characterized 114 variants with the FACT assay. All KRAS exon 2 and extended RAS mutations appeared activating. Of 57 atypical RAS variants characterized, 18 (31.6%) had signaling below wild-type, 23 (40.4%) had signaling between wild-type and activating control, and 16 (28.1%) were hyperactive beyond the activating control. Ba/F3 transformation (17/18 variants) and xenograft model (7/8 variants) validation was highly concordant with FACT results, and activating atypical variants were those that occurred at highest prevalence in clinical cohorts. We provide best available evidence to guide treatment when atypical RAS variants are identified. KRAS L19F, Q22K, D33E, and T50I are more prevalent than many guideline-included RAS variants and functionally relevant.

Rhinitis may be a clinical symptom of primary pulmonary lymphoepithelioma-like carcinoma, an incidental finding during the COVID-19 pandemic.

Rhinitis may be a clinical symptom of primary pulmonary lymphoepithelioma-like carcinoma, an incidental finding during the COVID-19 pandemic.

Combination of ultrasound and molecular testing in malignancy risk estimate of Bethesda category IV thyroid nodules: results from a single-institution prospective study

PurposeMalignancy prediction in indeterminate thyroid nodules is still challenging. We prospectively evaluated whether the combination of ultrasound (US) risk stratification and molecular testing improves the assessment of malignancy risk in Bethesda Category IV thyroid nodules.MethodsNinety-one consecutively diagnosed Bethesda Category IV thyroid nodules were prospectively evaluated before surgery by both ACR- and EU-TIRADS US risk-stratification systems and by a further US-guided fine-needle aspiration cytology (FNAC) for the following molecular testing: BRAFV600E, N-RAS codons 12/13, N-RAS codon 61, H-RAS codons 12/13, H-RAS codon 61, K-RAS codons 12/13, and K-RAS codon 61 point-mutations, as well as PAX8/PPARγ, RET/PC1, and RET/PTC 3 rearrangements.ResultsAt histology, 37% of nodules were malignant. No significant association was found between malignancy and either EU- or ACR-TIRADS. In total, 58 somatic mutations were identified, including 3 BRAFV600E (5%), 5 N-RAS 12/13 (9%), 13 N-RAS 61 (22%), 7 H-RAS 12/13 (12%), 11 H-RAS 61 (19%), 6 K-RAS 12/13 (10%), 8 K-RAS 61 (14%) mutations and 2 RET/PTC1 (4%), 0 RET/PTC 3 (0%), 3 PAX8/PPARγ (5%) rearrangements. At least one somatic mutation was found in 28% and 44% of benign and malignant nodules, respectively, although malignancy was not statistically associated with the outcome of the mutational test. However, the combination of ACR-, but not EU-, TIRADS with the presence of at least one somatic mutation, was significantly associated with malignant histology (P = 0.03).ConclusionUS risk stratification and FNAC molecular testing may synergistically contribute to improve malignancy risk estimate of Bethesda category IV thyroid nodules.

Papillary Thyroid Carcinoma in Patients with Acromegaly from a Single Center in China

An increased risk of thyroid cancers in patients with acromegaly has been addressed by numerous studies. However, the differences between patients with papillary thyroid cancer (PTC) with and without acromegaly remain to be clarified. We compared the clinical-pathologic data and genetic alterations of PTC between the 2 groups. Four patients with PTC and acromegaly and 32 age-matched patients with PTC without acromegaly were retrieved retrospectively from the hospital recordings. Mutational analysis was determined by direct sequencing. Insulin-like growth factor-1 receptor and insulin Rβ expression were analyzed by immunohistochemistry in acromegaly group. The prevalence of multifocality involved in bilateral lobes in the acromegaly group was significantly increased (P= 0.017). The presence of bilateral lymph node metastasis showed the increasing trend even though without a significant difference because of the limited number of PTC patients in acromegaly group (P= 0.053). There was no significant difference in other factors, such as sex, tumor size in maximum diameter, lymph node metastasis, extrathyroidal extension, and TNM stage. Two (50%) PTCs in acromegalic group and 25 (78.12%) PTCs in the nonacromegalic group were detected to harbor BRAF600E mutation, and no patient was identified to have NRAS codon 61, KRAS codon 61/12/13 mutation. Insulin-like growth factor-1 receptor and insulin Rβ immunostaining showed low positive to positive in PTC cells and negative in adjacent normal tissues in patients with acromegaly. Multifocality involved in 2 lobes is more common in patients with PTC and acromegaly, which shows more aggressive behaviors. BRAF mutation is not uncommon in patients with PTC and acromegaly.

PULSE: A randomized phase II open label study of panitumumab rechallenge versus standard therapy after progression on anti-EGFR therapy in patients with RAS wild-type metastatic colorectal cancer (mCRC).

TPS143 Background: Patients with KRAS and NRAS ( RAS) wild-type mCRC benefit from the epidermal growth factor receptor (EGFR) monoclonal antibodies (Abs) panitumumab and cetuximab, but nearly all patients experience resistance. Blood-based profiling of cell free DNA (cfDNA) can identify genomic alterations that drive acquired EGFR Ab resistance. After discontinuation of anti-EGFR Abs, acquired genomic alterations decay over time to undetectable levels. Some studies have suggested clinical benefit from EGFR Ab rechallenge, but there is limited evidence that EGFR Ab rechallenge improves survival compared to standard of care (SOC) therapies. We hypothesize that cfDNA profiling will identify patients appropriate for panitumumab rechallenge, and that these molecularly selected patients will have improved survival compared to current SOC therapies. Methods: This is a randomized phase II, open label study designed to compare the overall survival (OS) of panitumumab rechallenge versus SOC (investigator choice TAS-102 or regorafenib). Secondary objectives include comparisons of progression free survival, objective response rate, clinical benefit rate, and quality of life as measured by the linear analogue self-assessment (LASA) questionnaire. Eligible patients have radiographically measurable KRAS, NRAS, and BRAF codon 600 wild-type mCRC based on tumor tissue testing, and must have experienced progression or intolerance to treatment with a fluoropyrimidine, oxaliplatin, irinotecan, an anti-VEGF Ab, and an anti-PD-1 Ab if the tumor has mismatch repair deficiency or is MSI-H. Progression after at least 4 months treatment with an anti-EGFR Ab is required. All patients must be enrolled in the COLOMATE cfDNA screening protocol (NCT03765736) and meet molecular eligibility based on Guardant360 cfDNA profiling (absence of amplification of ERBB2, KRAS, NRAS, and MET; absence of mutations of BRAF, EGFR, ERBB2, KRAS, NRAS, and MET [mutant allele frequency > 0.5%]). Greater than 90 days must have elapsed between the most recent treatment with an anti-EGFR Ab and cfDNA profiling. Dosing for all study drugs is according to clinical SOC. 120 patients will be randomized 1:1 to panitumumab rechallenge or SOC. With 83 OS events, this study will have 80% power to detect an improvement in median OS from 6.5 to 10 months (HR=0.65; 1-sided α= 0.15). This study began enrollment in 6/2020. Recruitment is ongoing at 16 sites in the Academic and Community Cancer Research United (ACCRU) network (ACCRU-GI-1623). Clinical trial information: NCT03992456.

Mutational analysis of Ras hotspots in patients with urothelial carcinoma of the bladder.

BACKGROUNDMutational activation of Ras genes is established as a prognostic factor for the genesis of a constitutively active RAS-mitogen activated protein kinase pathway that leads to cancer. Heterogeneity among the distribution of the most frequent mutations in Ras isoforms is reported in different patient populations with urothelial carcinoma of the bladder (UCB).AIMTo determine the presence/absence of mutations in Ras isoforms in patients with UCB in order to predict disease outcome.METHODSThis study was performed to determine the mutational spectrum at the hotspot regions of H-Ras, K-Ras and N-Ras genes by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing followed by their clinical impact (if any) by examining the relationship of mutational spectrum with clinical histopathological variables in 87 UCB patients.RESULTSNone of the 87 UCB patients showed point mutations in codon 12 of H-Ras gene; codon 61 of N-Ras gene and codons 12, 13 of K-Ras gene by PCR-RFLP. Direct DNA sequencing of tumor and normal control bladder mucosal specimens followed by Blastn alignment with the reference wild-type sequences failed to identify even one nucleotide difference in the coding exons 1 and 2 of H-Ras, N-Ras and K-Ras genes in the tumor and control bladder mucosal specimens.CONCLUSIONOur findings on the lack of mutations in H-Ras, K-Ras and N-Ras genes could be explained on the basis of different etiological mechanisms involved in tumor development/progression, inherent genetic susceptibility, tissue specificity or alternative Ras dysfunction such as gene amplification and/or overexpression in a given cohort of patients.