HRAS Mutations Research Articles

Analysis of H-ras Mutations and Immunohistochemistry in Recurrence Cases of High-Grade Oral Squamous Cell Carcinoma.

This study is focused on the identification of gene mutations in H-ras which are probably associated with tumor recurrence in oral squamous cell carcinoma (OSCC) following conventional therapy. Surgically removed biopsies from OSCC patients without recurrence (n = 43) and biopsies from recurrent cases (n = 19) were analyzed. Also, gingival tissues (n = 5) from normal individuals were processed and considered as control. DNA was extracted and amplified using primers for exons 1 and 2 for the H-ras gene, and then DNA products were analyzed using Sanger's sequencing technique. Besides, H-ras expression was compared in samples by immunostaining (IHC), using anti-ras antibody. Demographic data show that smoking habit in patients and recurrent tumors was ~ 44.1 and 78%, respectively. The major site of malignancy was tongue tissue (40-60%). The rate of pathological stage III/IV were 41.8 and 100% in primary tumors and recurrence malignancy respectively. The sequencing data showed that a specific mutation in H-ras gene, Gly12Ala (G6266A) in recurrence samples and primary cases was detected in ~ 66.6% and 10% respectively. Accumulation of H-ras protein in tissues was relatively high scores (> 5) in both primary and recurrence tumors. The H-ras mutation detected was associated with increased level of H-ras protein accumulated in the malignant cells (IHC data). These data may suggest that regardless of the causes and factors involved, Gly12Ala (G6266A) is associated with recurrence in high-grade OSCC tumors.

PSAT279 Thyroid Oncocytic tissue: Presenting as metastatic thyroid carcinoma

Abstract Case A 76-year-old male with status post thyroidectomy in 2017 for compressive multi-nodular goiter. Pathology reported as benign thyroid tissue with follicular hyperplasia. In 2020, he presented with incidental finding of left neck mass on MRI. Ultrasound neck confirmed two extra-thyroidal masses left neck largest dimension of 2.9 cm. FNA reported as benign follicular cells favoring benign thyroid tissue. Surgical resection of the neck masses revealed benign thyroid tissue with focal oncocytic features and two benign lymph nodes. In 2021, he presented with left chest mass. Biopsy reported benign thyroid tissue, no evidence of papillary cancer. PET CT confirmed hypermetabolic chest mass, multiple lytic bone lesions and pulmonary nodules. Referred to Endocrinology for further evaluation, Thyroglobulin level elevated at >4500 ng/mL (Tg Ab <1.0 IU/mL). Molecular testing of chest wall mass was positive for TERT and HRAS mutations, loss of 22q and gain of 17q on microarray consistent with changes found in thyroid cancer. Surgical resection of the chest wall mass not feasible due to size and location. External beam radiation for cytoreduction of the sternal mass planned and radioactive iodine therapy if tumor RAI (radioactive iodine) avid. He is currently not a candidate for tyrosine kinase inhibitor (TKI) as he recently had acute coronary event. Zoledronic acid infusions initiated for bone involvement. Discussion Thyroid carcinomas that exhibit vascular invasion, or anaplastic dedifferentiation are readily and consistently diagnosed. However, the morphology of primary oncocytic thyroid tumors is similar to their non-oncocytic counterparts posing a huge challenge for the pathologist. This controversial and often confusing area of thyroid pathology requires careful evaluation for accurate diagnosis and management for patients with oncocytic thyroid lesions. Molecular and microarray testing should be considered when in doubt. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Increased osteoclastogenesis contributes to bone loss in the Costello syndrome Hras G12V mouse model.

RAS GTPases are ubiquitous GDP/GTP-binding proteins that function as molecular switches in cellular signalling and control numerous signalling pathways and biological processes. Pathogenic mutations in RAS genes severely affect cellular homeostasis, leading to cancer when occurring in somatic cells and developmental disorders when the germline is affected. These disorders are generally termed as RASopathies and among them Costello syndrome (CS) is a distinctive entity that is caused by specific HRAS germline mutations. The majority of these mutations affect residues 12 and 13, the same sites as somatic oncogenic HRAS mutations. The hallmarks of the disease include congenital cardiac anomalies, impaired thriving and growth, neurocognitive impairments, distinctive craniofacial anomalies, and susceptibility to cancer. Adult patients often present signs of premature aging including reduced bone mineral density and osteoporosis. Using a CS mouse model harbouring a Hras G12V germline mutation, we aimed at determining whether this model recapitulates the patients' bone phenotype and which bone cells are driving the phenotype when mutated. Our data revealed that Hras G12V mutation induces bone loss in mice at certain ages. In addition, we identified that bone loss correlated with an increased number of osteoclasts in vivo and Hras G12V mutations increased osteoclastogenesis in vitro. Last, but not least, mutant osteoclast differentiation was reduced by treatment in vitro with MEK and PI3K inhibitors, respectively. These results indicate that Hras is a novel regulator of bone homeostasis and an increased osteoclastogenesis due to Hras G12V mutation contributes to bone loss in the Costello syndrome.

166 (PB046) - Targeting oncogenic HRAS in pediatric rhabdomyosarcoma

Background: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, and RAS pathway mutations are the known driver mutations in the majority of fusion-negative (FN) RMS. Recent studies have demonstrated that HRAS mutations are enriched in infant cases of FN-RMS and can be associated with an aggressive clinical course and inferior outcomes. Using HRAS-mutant RMS cell lines and xenograft models, we have demonstrated that tipifarnib (farnesyl transferase inhibitor, FTI) decreases ERK signaling, decreases in vitro proliferation, and decreases in vivo tumor growth.

Mutational profiles of head and neck squamous cell carcinomas based upon human papillomavirus status in the Veterans Affairs National Precision Oncology Program.

Patients with advanced head and neck squamous cell carcinoma (HNSCC) associated with human papillomavirus (HPV) demonstrate favorable clinical outcomes compared to patients bearing HPV-negative HNSCC. We sought to characterize the association between HPV status and mutational profiles among patients served by the Veterans Health Administration (VHA). We performed a retrospective analysis of all Veterans with primary HNSCC tumors who underwent next-generation sequencing (NGS) through the VHA's National Precision Oncology Program between July 2016 and February 2019. HPV status was determined by clinical pathology reports of p16 immunohistochemical staining; gene variant pathogenicity was classified using OncoKB, an online precision oncology knowledge database, and mutation frequencies were compared using Fisher's exact test. A total of 79 patients met inclusion criteria, of which 48 (60.8%) had p16-positive tumors. Patients with p16-negative HNSCC were more likely to have mutations in TP53 (p < 0.0001), and a trend towards increased mutation frequency was observed within NOTCH1 (p = 0.032) and within the composite CDK/Rb pathway (p = 0.065). Mutations in KRAS, NRAS, HRAS, and FBXW7 were exclusively identified within p16-positive tumors, and a trend towards increased frequency was observed within the PI3K pathway (p = 0.051). No difference in overall mutational burden was observed between the two groups. In accordance with the previous studies, no clear molecular basis for improved prognosis among patients harboring HPV-positive disease has been elucidated. Though no targeted therapies are approved based upon HPV-status, current efforts to trial PI3K inhibitors and mTOR inhibitors across patients with HPV-positive disease bear genomic rationale based upon the current findings.

1771P Genomic characteristics and clinical outcomes of HRAS-mutated urothelial bladder cancer

HRAS gene encodes the GTPase HRAS protein, a member of the RAS superfamily, which is enriched in high-grade urothelial bladder carcinoma (UBC). We aimed to characterize genomic and clinical outcomes of patients with HRAS mutations (HRASmt) as a possible prognostic and therapeutic biomarker. A total of 3,426 UBC tissue samples underwent molecular profiling at Caris Life Sciences utilizing NGS of DNA. MAP kinase pathway activation and the likelihood of a tumor’s response to anti-PD(L)1 therapy were measured via MPAS and IFN signature, respectively. Wilcoxon, Fisher’s exact were used for statistical significance. Overall survival (OS) was calculated from the date of tissue collection or the start of treatment until the last contact from insurance claims. All comparisons were conducted between HRAS mutated tumors and the entire UBC general cohort (GC). HRASmt were detected in 107 patients with metastatic UBC (3.12%). Of those, 70% were detected in primary and 30% from metastatic sites. HRASmt were associated with lower TMB (5 vs 8 mut/Mb, q<0.01) and higher PD-L1 expression (55.3% vs 39.1%, q<0.01). HRASmt tumors harbored less TP53 (25.5% vs 60.1%, q<0.05), RB1 (5.3% vs 22.1%, q<0.05), FGFR3 (1.9% vs 13.9%, q<0.05), and KMT2D (14.0% vs 25.4%, q<0.05), but more BRAF mutations (14.0% vs 2.1%, q<0.05). The most frequent HRAS point mutation involved Q61 (47.6%). HRAS-Q61 mutant tumors showed higher MPAS scores (2.21 vs 0.6, q<0.001), enrichment in pathways (all q<0.05) such as EMT (0.86 vs 0.83), TGFb (0.91 vs 0.89), angiogenesis (0.78 vs 0.75) and notch signaling (0.82 vs 0.81). Patients with HRAS-Q61 point mutations experienced worse OS and prognosis compared to HRASwt tumors when treated with checkpoint inhibitors (HR 1.45, 95% 1.01-2.10; p <0.05). HRAS-Q61 point mutations represent a clinically relevant mutation in UBC, as evidenced by its association with worse clinical outcomes compared to HRASwt tumors. Our transcriptomic analysis suggests HRAS-Q61 mutations were associated with lower response to immunotherapy and leads to MAPK activation, as well as modulation of important TME pathways, possibly indicating a distinct biological and clinical phenotype. Our study provides rationale for therapeutic HRAS targeting in UBC.

34625 Cutaneous manifestations of a rare RASopathy: Costello syndrome

Introduction: Costello syndrome (CS) is a rare genetic condition with only approximately 350 affected individuals reported worldwide. CS manifests phenotypically in various anatomic, physiologic, and cutaneous abnormalities, from non-specific findings, such as failure to thrive, to more distinct features, like coarse facial features and papillomata of the face. CS belongs to the group of RASopathies, which have overlapping features caused by gene mutations in the RAS pathway. Presented here is a case of a young woman with previously-diagnosed CS (heterozygous germline mutation in HRAS) with characteristic phenotypic abnormalities.

Targeting HRAS in Head and Neck Cancer: Lessons From the Past and Future Promise.

HRAS mutations define a unique biologic subset of head and neck squamous cell carcinoma. Oncogenic HRAS is uniquely dependent on posttranslational farnesylation for membrane localization and activation of downstream signaling. Tipifarnib, a farnesyltransferase inhibitor, demonstrated encouraging antitumor activity for HRAS mutant head and neck squamous cell carcinoma and modest activity for HRAS mutant salivary gland cancer. New combination strategies to circumvent intrinsic and acquired resistance to TFIs are being investigated.

Clinical significance and interrelations of PD-L1 expression, Ki-67 index, and molecular alterations in sporadic medullary thyroid carcinoma from a Chinese population.

Immunotherapy shows prospects in treating advanced medullary thyroid carcinoma although controversial reports are present. Recently, histological grading has been applied to medullary thyroid carcinoma by the Ki-67 index, mitotic figures, and tumor necrosis. However, the interrelation of PD-L1 expression, the Ki-67 index, and major genetic alterations of sporadic medullary thyroid carcinoma has not been fully reported. We examined the expression of PD-L1 (SP142 and 22C3) and the Ki-67 index immunohistologically and detected the major genetic alterations by next-generation sequencing in a cohort of sporadic medullary thyroid carcinomas, studied their survival impact, and discussed their interrelation. We identified that a high Ki-67 index (> 2%) and positive RET M918T mutation were correlated with poor disease-free survival but were not correlated with PD-L1 expression. All PD-L1 positive tumors were RET M918T mutation negative, and PD-L1 expression was positively correlated with HRAS mutation. The Ki-67 index was correlated with neither PD-L1 expression nor major genetic alterations. Our results indicate that immunotherapy targeting PD-L1/PD-1 might be more effective for patients with sporadic medullary thyroid carcinoma harboring HRAS mutations.

Inflammatory linear verrucous epidermal nevus (ILVEN) encompasses a spectrum of inflammatory mosaic disorders.

Inflammatory linear verrucous epidermal nevus (ILVEN) is a rare skin disease characterized by pruritic erythematous scaly plaques distributed along the lines of Blaschko. Two cases of ILVEN with CARD14 mutations and one case with a GJA1 mutation have been previously reported. To elucidate the genetic cause of a cohort of patients diagnosed based on clinical and histopathological evaluation with ILVEN. We recruited patients diagnosed with ILVEN based on clinical and histopathological criteria. Exome sequencing of affected skin with or without blood/saliva was performed and germline and somatic pathogenic variants were identified. Five patients were enrolled. All had skin lesions from birth or early childhood. Two patients developed psoriasis vulgaris after the diagnosis of ILVEN. The first had a germline heterozygous CARD14 mutation and a post-zygotic hotspot mutation in KRT10. The histopathologic evaluation did not show epidermolytic hyperkeratosis. The second had a post-zygotic hotspot mutation in HRAS. Her ILVEN became itchy once psoriasis developed. One patient was re-diagnosed with linear porokeratosis based on a germline mutation in PMVK and a post-zygotic second-hit mutation. Two patients were re-diagnosed with congenital hemidysplasia with ichthyosiform nevus and limb defect nevus based on germline NSDHL mutations. ILVEN is a clinical descriptor for a heterogenous group of mosaic inflammatory disorders. Genetic analysis has the potential to more precisely categorize ILVEN and permits pathogenesis-directed therapies in some cases.

Targeted molecular profiling of salivary duct carcinoma with rhabdoid features highlights parallels to other apocrine and discohesive neoplasms: which phenotype should drive classification?

Salivary duct carcinoma with rhabdoid features (SDC-RF) is a recently-described salivary gland tumor that bears striking histologic similarity to lobular carcinoma of the breast. While this tumor has an apocrine phenotype that supports classification as a variant of SDC, it infrequently arises in association with conventional SDC. Furthermore, discohesive architecture can be seen in non-apocrine salivary carcinomas, raising the possibility that discohesive growth should define a separate entity. In this study, we aimed to perform comprehensive molecular profiling of SDC-RF to better understand its pathogenesis and classification. We documented the clinicopathologic features of 9 cases of SDC-RF and performed immunostains including AR, GCDFP, and e-cadherin on all cases. We also performed targeted next generation sequencing (NGS) panels on 7 cases that had sufficient tissue available. The SDC-RF represented 8 men and 1 woman with a median age of 67 years (range 63-83 years) and included 6 parotid, 2 buccal, and 1 submandibular primary. All tumors were uniformly composed of discohesive cells with abundant eosinophilic cytoplasm; signet-ring cell features were seen in 2 cases. All tumors were also positive for AR (100%) and GCDFP (100%), and 7 tumors (78%) displayed lost or abnormal e-cadherin. NGS highlighted concomitant PIK3CA and HRAS mutations in 4 tumors, with additional cases harboring TP53, PTEN, and AKT1 mutations. Furthermore, CDH1 alterations were seen in 6 cases, including a novel CDH1::CORO7 fusion. Among 5 patients with follow-up available, 3 (60%) developed local recurrence and widespread distant metastasis and died of disease at a median 20 months (range 10-48 months). Overall, our findings confirm frequent CDH1 mutations and e-cadherin inactivation in SDC-RF, similar to discohesive tumors from other sites. We also highlight an apocrine molecular profile similar to conventional SDC. However, occasional AKT1 mutation and signet-ring features suggest SDC-RF may also be related to mucinous adenocarcinoma. As more salivary tumors with discohesive growth are identified, it may become clearer whether SDC-RF should remain in the SDC family or be recognized as a separate entity.

Argonaute 2 modulates EGFR-RAS signaling to promote mutant HRAS and NRAS-driven malignancies.

Activating mutations in RAS GTPases drive nearly 30% of all human cancers. Our prior work described an essential role for Argonaute 2 (AGO2), of the RNA-induced silencing complex, in mutant KRAS-driven cancers. Here, we identified a novel endogenous interaction between AGO2 and RAS in both wild-type (WT) and mutant HRAS/NRAS cells. This interaction was regulated through EGFR-mediated phosphorylation of Y393-AGO2, and utilizing molecular dynamic simulation, we identified a conformational change in pY393-AGO2 protein structure leading to disruption of the RAS binding site. Knockdown of AGO2 led to a profound decrease in proliferation of mutant HRAS/NRAS-driven cell lines but not WT RAS cells. These cells demonstrated oncogene-induced senescence (OIS) as evidenced by β-galactosidase staining and induction of multiple downstream senescence effectors. Mechanistically, we discovered that the senescent phenotype was mediated via induction of reactive oxygen species. Intriguingly, we further identified that loss of AGO2 promoted a novel feed forward pathway leading to inhibition of the PTP1B phosphatase and activation of EGFR–MAPK signaling, consequently resulting in OIS. Taken together, our study demonstrates that the EGFR–AGO2–RAS signaling axis is essential for maintaining mutant HRAS and NRAS-driven malignancies.

Costello Syndrome

Costello syndrome is a rare RASopathy resulting from germline mutations of the protooncogene HRAS. Many of these mutations affect SHP2, SOS1, RAS, RAF and MEK proteins it was discovered by Dr. Jack Costello, a New Zealand paediatrician in 1977. Dr. White says. Costello syndrome is now known to be one of a group of related disorders, caused by abnormal functioning of the Ras‐mitogen‐activated protein kinase (RAS/MapK) pathway. Ras/MAPK pathway is an essential signaling pathway that controls cell proliferation, differentiation, survival and its dysregulation causes clinically overlapping genetic disorders, called as ‘Rasopathies’.In this pathway, Ras, a GTPase, transmits extracellular signaling from receptor tyrosine kinases to two serine/threonine kinases (Raf and MEK) and, finally, to the activation of MAPKs. Costello syndrome is a severe developmental disorder characterised by postnatal growth retardation with delayed skeletal maturation, psychomotor retardation, cutis laxa, and acanthosis nigricans. Excessive mucopolysaccharides, which accumulate in cultured fibroblasts of patients with Costello syndrome (CS). Intracellular accumulation of chondroitin non-sulphate, as a cause of functional deficiency of the 67 kDa elastin binding protein, has been described in fibroblasts of patients with Costello syndrome.This gives support to the previous hypothesis of a defect in lysosomal degradation

Abstract 5625: Molecular and immunologic characterization of HRAS mutations in a cohort of 6,329 patients with cutaneous melanoma

Abstract Background: Activation in RAS pathway has been associated with cancer development. Three RAS family members, including NRAS, KRAS and HRAS are frequently mutated across various cancer types, where NRAS mutations are present in 15-20% of melanomas. NRAS-mutant melanomas (NRASm) have been extensively characterized. However, molecular and clinical implications of HRAS mutations (HRASm) in melanoma are less well understood. Methods: A total of 6329 melanoma samples were subjected to comprehensive molecular profiling at Caris Life Sciences. Analyses included next generation sequencing of DNA (592 Gene Panel, NextSeq; whole exome sequencing, NovaSEQ), RNA (NovaSeq, whole transcriptome sequencing, WTS) and IHC. MPAS scores to evaluate MAPK pathway activation, IFN scores, QuantiSeq, neoantigen load (high, intermediate, low binding affinity: HBA, IBA and LBA) and GSEA were calculated from mRNA expression data. Wilcoxon, Fisher’s exact were used to determined statistical significance (p value without and q value with multi comparison correction; FDR for GSEA). The reference cohort was the entire melanoma cohort (MC). Results: HRASm were identified in 69 (1.09%) of melanoma samples (hotspots mutations: G13, 40%; Q61, 34%; G12, 18% and others, 9%). HRASm and NRASm had different genomic landscapes: HRASm were significantly associated with a higher mutation rate of NF1 (43.2% vs 27.7%, p&amp;lt;.05), ARID1A (17.2% vs 6.3%, p&amp;lt;.05), B2M (14.3% vs 2.4%, p&amp;lt;.05), RAF1 (12.2% vs 1.4, p&amp;lt;.0001), CTNNB1 (9.1% vs 3.3%, p&amp;lt;.05) and higher amplifications of EMSY (11.8% vs 1.8%, p&amp;lt;.01), MRE11 (4.3% vs 0.5%, p&amp;lt;.05), whereas NRASm harbored less NF1 (14.8% vs 27.7%, q&amp;lt;.0001 ), BRAF (6.9% vs 39.9%, p &amp;lt;.0001), PTEN (3.9% vs 6.9%, q &amp;lt;.05), KIT (0.8% vs 4.4%, q &amp;lt;.0001) mutations and less amplification PDGFR (0.4% vs 1.3%, p&amp;lt;.05), BRAF (0.2% vs 1.4%, p&amp;lt;.05), KIT (0.2% vs 2.1%, q&amp;lt;.05) when comparing to MC. Both HRASm and NRASm had higher MPAS scores than MC (HRASm, 0.24; NRASm, 0.11; MC, -0.41, q&amp;lt;.001). In addition, HRASm showed higher TMB (HRASm, 68.1%; NRASm, 56.9%; MC, 50.0%, q&amp;lt;.05), relatively higher IFN scores (HRASm, 0.16; NRASm, -0.23; MC, -0.24, q = .16) and higher neoantigen load (HBA: HRASm, 10.5; NRASm, 4; MC, 4, p &amp;lt; .05; IBA: HRASm, 17.5; NRASm, 8 MC, 7, p &amp;lt; .05; LBA: HRASm, 37.5; NRASm, 21; MC, 19, p =.1) when compared to NRASm and MC. Lastly, suppression of angiogenesis pathway was observed in both HRASm (NES = 1.7, FDR&amp;lt;.05) and NRASm with respect to MC (NES = 1.4, FDR&amp;lt;.25). Conclusions: The genomic landscape of HRASm are significantly different from that of NRASm, implying their distinct roles in tumorigenesis. HRASm also demonstrated higher MAPK activation, suggesting that they could potentially benefit from agents targeting on this pathway. In addition, HRASm displayed more immunogenic features, associated with down-regulation of angiogenesis pathway, revealing a potential higher susceptibility of HRASm to immunotherapy. Citation Format: Leonel F. Hernandez-Aya, Estelamari Rodriguez, Aparna Nallagangula, Jun Yin, Phillip Walker, Joanne Xiu, Justin Moser, Gino K. In, David Spetzler, Geoffery T. Gibney, Matthew Oberley, Thuy Phung, Michael Atkins, Dave S. Hoon, Wolfgang Michael Korn, Jose Lutzky, Gilberto Lopes. Molecular and immunologic characterization of HRAS mutations in a cohort of 6,329 patients with cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5625.

Abstract 3882: Translating genotype to immunophenotype in HRAS mutated head and neck squamous cell carcinoma (HNSCC) to identify effective Tipifarnib partners for optimal patient outcomes

Abstract Background: Tipifarnib is a highly selective farnesyltransferase inhibitor that has shown promising activity in HRAS mutant recurrent/metastatic HNSCC. The incidence of HRAS mutations in HNSCC is 4-10%. Since HRAS mutated HNSCC can be successfully targeted with Tipifarnib, we sought to analyze the immune and genomic landscape of these tumors to guide combinatorial strategies. Methods: We identified thirteen patients with HRAS mutant tumors. We included in the analysis 50 patients with HRAS wild-type tumors. Genomic DNA from baseline biopsies of tumors was purified and subjected to targeted sequencing (TruSightTM Oncology 500 panel). PD-L1 expression in formalin-fixed tumor samples was assessed using the PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies, Carpinteria, CA, USA) and characterized by the combined positive score (CPS). Assessment of PD-L1+ cells inside and at the periphery of the tumor was performed. HRAS mutational status was correlated with PD-L1 status. The genomic data derived from the targeted gene panel of HRAS mutant tumors were compared to those with wild-type HRAS. Results: Thirty-seven patients had both genomic and PD-L1 expression data available. The presence of HRAS mutations was associated with higher TPS PD-L1 scores (26% vs 40% vs 100% in TPS &amp;lt;1%, 1-49% and &amp;gt;50% respectively, p=0.043). Similar results were obtained for CPS (28% vs 80% in CPS &amp;lt;10% and =&amp;gt;10%, p=0.047). HRAS mutations were more commonly found with TP53 mutations (16% in WT TP53 and 55.6% in mutated TP53, p=0.073). Higher PD-L1 TPS showed increased survival without reaching significance when measuring either in the margins (64.1 vs 35.8, p=0.066) or the center of the tumor (p=0.097) due to the small sample size. Conclusions: HRAS mutant HNSCC are associated with high PD-L1 expression and co-occurrence of p53 mutations. Combinations of tipifarnib with an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA-4 antibody warrant clinical investigation in this rare subset of HNSCC. Citation Format: Amanda Psyrri, Miriam Anastasiou, Aris Spathis, Stergios Doumas, Panagiota Economopoulou, Ioannis Kotsantis, Anastasios Pantazopoulos, Anastasios Kyriazoglou, George Kavourakis, Theodoros Rampias, Periklis Foukas. Translating genotype to immunophenotype in HRAS mutated head and neck squamous cell carcinoma (HNSCC) to identify effective Tipifarnib partners for optimal patient outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3882.

Abstract 48: Molecular characteristics of HRAS mutated non-small cell lung cancer (NSCLC)

Abstract Background: Alterations in the RAS pathway have been linked to tumorigenesis, apoptosis, metabolism and angiogenesis. Mutations of KRAS in NSCLC are more frequent and better characterized, however, other family members such as HRAS remain under investigated and RAS remains a challenging therapeutic target. HRAS has been indirectly targeted with tipifarnib, a farnesyltransferase inhibitor rendering HRAS inactive in head and neck tumors. Here, we characterize the incidence, genomic landscape, and clinical context of HRAS alterations in NSCLC. Methods: A total of 29,767 NSCLC tumor samples underwent comprehensive molecular profiling at Caris Life Sciences. Analyses included next generation sequencing of DNA (592 Gene Panel, NextSeq, or whole exome sequencing, NovaSeq), RNA (NovaSeq, whole transcriptome sequencing, WTS) and immunohistochemistry. MAPK activation was assessed using the MPAS gene expression signature. Wilcoxon, Fisher’s exact, or Dunnett’s tests were used to determined statistical significance (displayed as p value without and q value with multi-comparison correction). Overall survival was calculated from date of tissue collection to last contact from insurance claims data and used for Kaplan-Meier analysis. Comparisons were conducted between HRAS mutated tumors and the entire NSCLC general cohort (GC). Results: HRAS mutations (Hm) were detected in 128 of 29767 NSCLC samples (0.4%) and were significantly enriched in older patients (median age, 71 vs. 69 years; q&amp;lt;0.01) and squamous histology (57.8% vs 21.8%, q&amp;lt;0.0001) compared to GC. Smoking status was not associated with HRAS mutational status (p=0.19). The most prevalent loci of hotspot mutations in Hm tumors were G13 (42.2%), followed by Q61 (33.6%) and G12 (21.1%). HRAS was not prognostic for overall survival (HR = 1.06, 95% CI [0.83-1.35], p=0.64), but for HRAS G13 mutant subset of NSCLC there was a trend towards worse prognosis (HR = 1.31, 95% CI [0.91-1.88], p =0.14). Hm-positive tumors harbored significantly more PIK3CA mutations (16.9% vs 5.5%, q&amp;lt;0.05) but less KRAS (3.2% vs 27.4%, q&amp;lt;0.05) and EGFR mutations (0.8% vs 11.9%, q&amp;lt;0.05) compared to GC. In addition, Hm displayed dMMR/MSI deficiency (2.6% vs 0.7%, p &amp;lt;0.05) more frequently, but had a comparable percentage of TMB-H tumors (34.2% vs 40.2%, p = 0.16) and similar median PD-L1 expression (54.7% vs 60.3%, p = 0.21) when comparing to GC. Lastly, MAPK pathway activation score was relatively higher in Hm compared to GC (-0.26 vs -0.42) with Q61 being the highest group (0.04 vs -0.42, p = 0.37). Conclusions: HRAS mutations are detectable but uncommon events in NSCLC and significantly enriched in squamous histology. HRAS mutations often occur with PIK3CA co-mutations and trended towards higher activation of MAPK pathway and MSI-H frequency. This warrants further investigation on possible clinical applications of HRAS pathway inhibitors and utility of immune checkpoint inhibitors for this subset of NSCLC. Citation Format: Asaad Trabolsi, Estelamari Rodriguez, Samuel Alexander Kareff, Michael Korn, Joanne Xiu, Stephen Liu, Philip walker, Patrick Ma, Hirva Mamdani, Jorge Nieva, Hossein Borghaei, Chadi Nabhan, Misako Nagasaka, Sonam puri, Gilberto Lopes. Molecular characteristics of HRAS mutated non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 48.

Abstract CT253: A Phase 1/2 trial to evaluate the safety and antitumor activity of tipifarnib and alpelisib for patients with HRAS-overexpressing and/or PIK3CA-mutated/amplified recurrent/metastatic head and neck squamous cell carcinoma (The KURRENT trial)

Abstract Background: Understanding connections between key cellular pathways is particularly important when selecting combinatorial cancer therapies. HRAS preferentially activates PI3K 5-fold more efficiently than KRAS, while KRAS is a more efficient activator of RAF (Yan et al. 1998). Further, mutant HRAS is insufficient for oncogenic transformation if it is unable to recruit PI3K in preclinical models (Gupta et al. 2007). Conversely, mutant PI3K requires RAS to drive tumor growth (Zhao and Vogt 2008). HRAS mutation/overexpression and PIK3CA mutations/amplifications account for up to 50% of head and neck squamous cell carcinoma (HNSCC). Recognition of these interdependencies was the basis for the evaluation of HNSCC PDX models (HRAS-mutant, HRAS wildtype-overexpressed, PIK3CA-amplified, and PIK3CA-mutant) which demonstrated additive and synergistic growth effects that confirmed the codependency of these pathways, thus providing robust rationale for investigating combined pathway inhibition in the clinic. The KURRENT trial is enrolling patients with HRAS and/or PIK3CA-dependent tumors who will receive combination treatment with tipifarnib (a potent and selective inhibitor of farnesyltransferase, a critical enzyme for HRAS activity) and alpelisib (an inhibitor of PI3K with inhibitory activity predominantly against the PI3Kα isoform). Methods: The KURRENT trial (KO-TIP-013, NCT04809233) is an ongoing multicenter, open-label, 2-cohort, phase 1/2 trial designed to evaluate the safety of the combination of tipifarnib and alpelisib, determine the recommended combination dose(s) regimen, and evaluate preliminary anti-tumor activity in patients with recurrent/metastatic (R/M) HNSCC whose tumors are dependent upon HRAS and/or PIK3CA signaling. Dependency is defined as an overexpression of the HRAS protein, and/or a PIK3CA mutation and/or amplification. The trial will enroll 40 HNSCC patients (oral cavity, oropharynx, larynx or hypopharynx, sinonasal, nasopharyngeal, or unknown primary subsites regardless of human papillomavirus status); 20 each into two biomarker defined cohorts (Cohort 1: PIK3CA; Cohort 2: HRAS). Participants must have documented treatment failure from at least one prior therapy in the R/M setting and have measurable disease by RECIST v1.1. At the starting dose level, participants will receive tipifarnib at 300 mg twice daily on days 1-7 and 15-21 and alpelisib 200 mg each morning continuously during a 28-day cycle. The trial will use an adaptive dose escalation design (based on a Bayesian logistic regression model) to characterize safety, tolerability, and clinical activity of the combination to identify the Optimal Biologically Active Dose (OBAD) while maintaining a dose limiting toxicity (DLT) rate &amp;lt;33%. No formal interim analysis is planned as the model-based dose escalation process requires decisions based on real-time evaluation of aggregate toxicity and efficacy data. All observed/available data among each cohort will be evaluated before choosing the combination dose for a subsequent cohort. The trial opened for enrollment in October 2021. Citation Format: Glenn J. Hanna, Cesar Perez Batista, Alan L. Ho, Maura L. Gillison, Douglas Adkins, Ashley Dayoub, Bridget Martell, Mollie Leoni, Stephen Dale, Kun Nie, Alex Dmitrienko. A Phase 1/2 trial to evaluate the safety and antitumor activity of tipifarnib and alpelisib for patients with HRAS-overexpressing and/or PIK3CA-mutated/amplified recurrent/metastatic head and neck squamous cell carcinoma (The KURRENT trial) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT253.

Abstract 413: WT RAS signaling is an essential therapeutic target in RAS mutated cancers

Abstract Single-agent targeting of RAS-mutated cancers using RAF/MEK/ERK or PI3K/AKT pathway inhibitors is ineffective; blocking one pathway relieves negative feedback control of RTK signaling and hyperactivates parallel effector pathways. While combined MEK and PI3K inhibition is effective in preclinical models, toxicity of this combination prevents its clinical use. Alternative strategies for treating RAS-mutated cancers are essential. Therapeutics directly targeting mutated RAS proteins have the potential to spare normal cellular function thereby lessening overall toxicity. Unfortunately, direct RAS inhibition as a monotherapy does not fully block RAS effector signaling. RAS proteins show differential activation of RAF and PI3K pathways: KRAS potently activates RAF but poorly activates PI3K, whereas HRAS potently activates PI3K but poorly activates RAF. Further, mutant RAS inhibition relieves negative feedback controls leading to rapid hyperactivation of RTK−WT RAS signaling. A more comprehensive understanding of the interplay between mutant RAS and RTK−WT RAS signaling is essential to developing rational therapeutic approaches to treat RAS-mutated cancers. We found that WT RAS enhanced mutant RAS-driven transformation by activating RAS effectors poorly engaged by mutated RAS. Further, inhibition of RAS effectors activated poorly by mutant RAS synergized with and limited resistance to mutant HRAS and KRAS inhibitors. The mutant HRAS inhibitor tipifarnib blocked PI3K signaling and synergized with MEK inhibitors in HRAS-mutated cancer cell lines; covalent KRASG12C inhibitors blocked MEK signaling and synergized with PI3K inhibitors in KRASG12C-mutated cell lines. Synergy between inhibitors of mutant RAS and RAS effectors was dependent on intact RTK/WT RAS signaling and was lost in both RASless or SOSless MEFs. Upstream of RAS, the RASGEFs SOS1 and SOS2 showed unique and overlapping functions to promote mutant RAS-driven transformation. SOS1 was critical for mutant RAS (G12V) activation and SOS1 inhibition augmented the efficacy of mutant RAS inhibitors. RTK−SOS2−PI3K signaling protected cells from anoikis and mediated mutant KRAS-driven transformation. These results should inform the design of clinical trials for patients with RAS-mutated cancers. Citation Format: Nancy E. Sealover, Robert L. Kortum. WT RAS signaling is an essential therapeutic target in RAS mutated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 413.

Multidisciplinary Management of Costello Syndrome: Current Perspectives.

Costello syndrome (CS) is a rare neurodevelopmental disorder caused by germline mutations in HRAS. It belongs among the RASopathies, a group of syndromes characterized by alterations in components of the RAS/MAPK signaling pathway and sharing overlapping phenotypes. Its typical features include a distinctive facial appearance, growth delay, intellectual disability, ectodermal, cardiac, and musculoskeletal abnormalities, and cancer predisposition. Due to the several comorbidities having a strong impact on the quality of life, a multidisciplinary team is essential in the management of such a condition from infancy to adult age, to promptly address any detected issue and to develop appropriate personalized follow-up protocols and treatment strategies. With the present paper we aim to highlight the core and ancillary medical disciplines involved in managing the health challenges characterizing CS from pediatric to adult age, according to literature and to our large clinical experience.

Identifying prognostic and predictive value of mutations associated with clinical outcomes in first line (1L) patients with advanced or metastatic non–small cell lung cancer (aNSCLC).

9047 Background: Whilst the impact of certain mutations on aNSCLC survival outcomes has been evaluated, real-world (rw) evidence on its predictive and prognostic value remains limited. We aimed to identify the predictive and prognostic value of non-driver mutations that are deemed to be clinically relevant in aNSCLC. Methods: This retrospective cohort study analysed de-identified electronic medical records from the Flatiron Clinico-Genomic (FCGD) and FoundationCORE™ databases of aNSCLC patients who have initiated 1L cancer immunotherapy (CIT, alone or in combination) or chemotherapy (chemo) under routine care between 2016 and 2021. Propensity-based multivariable models were used to determine associations between mutational status and progression-free survival (rwPFS), overall survival (rwOS) and overall response (rwR). Results: Of 10,795 patients identified, 2,999 met inclusion criteria (1,042 chemo, 1,957 CIT). Among these, 53% were men and 65% were ≥65 years old. Patients mostly had non-squamous histology (68%), a previous history of smoking (95%) and ECOG ≤1 (61%). 58% of patients were diagnosed as de novo. A total of 185 different mutations were identified in this population, and variants of the same mutation were grouped, identifying 58 mutation families. STK11, KEAP1 and CDKN2A/B mutations were significantly associated with all effectiveness outcomes (Table), indicating lower response (rwR OR&lt;1) and shorter survival (rwOS and rwPFS HR&gt;1) in patients harbouring mutated vs. wild type genes. APC and KRAS mutations were only significantly associated with lower rwR, while FGRF and HRAS mutations were related to worse rwPFS and rwOS, respectively. In contrast, a significantly higher likelihood of response or PFS was associated with ATM/R/RX and GATA3 mutations, respectively. Moreover, our results suggest that KRAS mutations potentially have predictive value for PFS in CIT-treated patients. Mutations with prognostic value on clinical outcomes (overall population). HR, hazard ratio; PFS, progression-free survival; OR, odds ratio; OS, overall survival; rw, real-world. Conclusions: STK11, KEAP1 and CDKN2A/B mutations were significantly associated with poor prognosis in all effectiveness outcomes. In addition, KRAS mutations resulted in clinically significant differences in terms of PFS. Further analyses should be conducted to confirm this clinical significance and to evaluate the relevance of these mutations in future clinical trials design.[Table: see text]