HRAS Research Articles

Phosphocholine inhibits proliferation and reduces stemness of endometrial cancer cells by downregulating mTOR-c-Myc signaling

BackgroundEndometrial cancer (EC) represents a serious health concern among women globally. Excessive activation of the protooncogene c-Myc (c-Myc) is associated with the proliferation and stemness of EC cells. Phosphocholine (PC), which is synthesized by choline kinase alpha (CHKA) catalysis, is upregulated in EC tumor tissues. The present study aimed to investigate the effect of PC accumulation on EC cells and clarify the relationship between PC accumulation and c-Myc activity in EC.MethodsThe c-Myc and CHKA expression in EC tumor tissues were examined using immunohistochemistry. Cell Counting Kit-8 assay, colony formation assay, flow cytometry, western blotting, BrdU staining, and tumorsphere formation assay were used to assess the effect of PC accumulation on EC cells. The mechanism by which PC accumulation inhibits c-Myc was evaluated using RNA-sequencing. Patient-derived organoid (PDO) models were utilised to explore the preclinical efficacy of PC against EC cells.ResultsPC accumulation suppressed EC cell proliferation and stemness by inhibiting the activation of the mammalian target of rapamycin (mTOR)-c-Myc signaling. PC accumulation promoted excessive reactive oxygen species production, which reduced the expression of GTPase HRAS. This, in turn, inhibited the mTOR-c-Myc axis and induced EC cell apoptosis. Finally, PC impeded proliferation and downregulated the expression of the mTOR–MYC signaling in EC PDO models.ConclusionsPC accumulation impairs the proliferation ability and stem cell characteristics of EC cells by inhibiting the activated mTOR-c-Myc axis, potentially offering a promising strategy to enhance the efficacy of EC clinical therapy through the promotion of PC accumulation in tumor cells.

Salirasib Inhibits the Expression of Genes Involved in Fibrosis in Fibroblasts of Systemic Sclerosis Patients.

Fibrosis is a principal sign of systemic sclerosis (SSc) which can affect several organs including the lung, heart, and dermis. Dermal fibroblasts of SSc patients are characterized by persistent and activated Ras and ERK1/2 signaling which stimulates extreme collagen and extracellular matrix synthesis. Salirasib is a Ras inhibitor that competitively prevents the adherence of GTP-bound Ras to the plasma membrane, that inhibits Ras signaling. This study intended to clarify whether salirasib can influence fibrotic mediators in SSc fibroblasts. Dermal fibroblasts from 10 SSc patients were treated with salirasib in the presence of TGF-β1, and mRNA levels of H-Ras and genes related to fibrosis, such as COL1A1, COL1A2, CTGF, TGF-β1, fibronectin, ACTA2, and MMP1 was measured by real-time PCR. The α-SMA protein expression was analyzed by immunofluorescence staining. In dermal fibroblasts of SSc patients, salirasib treatment, markedly downregulated the H-Ras gene expression. In addition, the protein expression of α-SMA and gene expression of ACTA2 were inhibited upon salirasib treatment. Salirasib also significantly reduced the expression of COL1A1, and COL1A2 genes and augmented the gene expression of MMP1. The mRNA levels of other genes related to fibrosis such as FN1, CTGF, and TGF-β1 were significantly decreased upon salirasib treatment. Considering salirasib significantly reduced the expression of genes related to the fibrosis process and α-SMA gene and protein expression, and given significant upregulation of MMP1 by salirasib, it can be considered as a new curative strategy for fibrotic diseases like SSc.

Identification and Characterization of Novel Small-Molecule Enhancers of the CUL3LZTR1 E3 Ligase KRAS Complex.

The RAS family of GTPases is among the most frequently mutated proteins in human cancer, creating a high clinical demand for therapies that counteract their signaling activity. An important layer of regulation that could be therapeutically exploited is the proteostatic regulation of the main RAS GTPases KRAS, NRAS, and HRAS, as well as the closely related members, MRAS and RIT1, by the leucine zipper-like transcriptional regulator 1 cullin 3 RING E3 ubiquitin ligase complex (CUL3LZTR1). Genetic inactivation of LZTR1, as observed in different cancer entities and Noonan syndrome leads to enhanced RAS GTPase abundance and altered MAPK pathway activation state. Novel therapeutic approaches to interfere with hyperactive RAS signaling, thereby complementing existing treatments, are highly sought after. Motivated by the growing arsenal of molecular glue degraders, we report the identification of novel chemical fragments that enhance the protein-protein interaction (PPI) of the KRAS-LZTR1 complex. We established a split-luciferase-based reporter assay that monitors the RAS GTPase-LZTR1 interaction in a scalable format, capable of capturing chemical, as well as mutational perturbations. Using this screening system, in combination with a small fragment library, we identified two fragments, C53 and Z86, that enhance the interaction of the KRAS-LZTR1 complex in a dose-dependent manner. Further orthogonal validation experiments using proximity biotinylation (BioID), thermal shift assays, and NMR spectroscopy demonstrated fragment-dependent enhanced recruitment of endogenous LZTR1 and physical engagement of KRAS. The two fragments, which potentiate the KRAS-LZTR1 interaction, serve as starting points for fragment-based drug discovery. Additionally, the assay we introduced is amenable to high-throughput screening to further explore the pharmacological modulation of the CUL3LZTR1-RAS GTPase complex.

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

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

Dysplastic Mitral Valve in Costello Syndrome

Costello syndrome is an autosomal dominant condition caused by variants in the HRAS gene. Cardiac presentation includes valvular disease (usually valvar pulmonary stenosis), arrhythmias, and hypertrophic cardiomyopathy. To our knowledge, this is the first such report of dysplastic mitral valve associated with Costello syndrome.

The Surgical Management of Severe Scoliosis in Immature Patient with a Very Rare Disease Costello Syndrome-Clinical Example and Brief Literature Review.

Costello syndrome (CS) is a rare genetic syndrome in which, due to the occurrence of a mutation in the HRAS gene on chromosome 11 that causes the manifestation, a set of features such as a characteristic appearance, many congenital defects, intellectual disability and a genetic predisposition to cancer, friendly personality, and others can be identified. CS is very rare, with an incidence of ~1/300,000, but it belongs to one of the largest groups of congenital syndromes, called RASopathies, occurring with an incidence of 1/1000 people. Scoliosis and kyphosis, as well as other spinal defects, are common, in 63% and 58% of patients, respectively, and a study conducted among adult patients showed the presence of scoliosis in 75% of patients; there may be excessive lordosis of the lumbar section and inverted curvatures of the spine (lordosis in the thoracic section and kyphosis in the lumbar section). The aim of our study is to present a case report of treatment of severe scoliosis of 130 degrees in a 14-year-old patient with Costello syndrome, with coexisting Chiari II syndrome and syrinx in the absence of skeletal maturity. This patient underwent foramen magnum decompression 3 months before planned surgical correction for severe scoliosis. The patient was qualified for surgical treatment using magnetically controlled growing rods (MCGR). After spine surgery using MCGR, we gradually performed MCGR distraction over the next 2 years; we performed the final surgery, conversion to posterior spinal fusion (PSF) with simultaneous multi-level Ponte osteotomy, which gave a very good and satisfactory surgical result. In the perioperative period, two serious complications occurred: pneumothorax caused by central catheter and gastrointestinal bleeding due to previously undiagnosed gastrointestinal varices. This case shows that the treatment of severe and neglected scoliosis is complicated and requires special preparation and a surgical plan with other cooperating specialists. The scoliosis was corrected from 130 degrees to approximately 48 degrees, sagittal balance was significantly improved, and the surgical outcome was very pleasing, significantly improving quality of life and function for the patient.

MULTIFOCAL ATRIAL TACHYCARDIA AND HYPERTROPHIC CARDIOMYOPATHY IN THREE PATIENTS WITH COSTELLO SYNDROME

Abstract Introduction Costello syndrome is a rare disease, caused by mutation of the HRAS gene, associated with intellectual disability, growth retardation, facial dimorphisms. Cardiac abnormalities, mainly cardiac hypertrophy and multifocal atrial tachycardia, are present in 1/3 of patients. Clinical Cases From 2019 to 2023, 3 newborns with Costello syndrome came under our observation. M.M. showed respiratory distress, hypotonia and dysmorphic notes; genetic analysis highlighted the pathogenic variant c.64C>A (p.Gln22Lys). On the echocardiogram, septal hypertrophic cardiomyopathy was found with left outflow obstruction and mitral insufficiency. Therapy with propranolol was undertaken. Holter ECG never revealed arrhythmias. At the last check–up, SIV hypertrophy, continent mitral valve, free left ventricular outflow. The second patient, O.L., arrived at 10 days of age following a run of supraventricular tachycardia. He had bilateral clubfoot and facial dimorphisms. The echocardiogram was normal. On Holter ECG atrial tachycardia interrupted by rare sinus beats. Therapy with flecainide was undertaken, without benefit. Subsequently, was undertaken amiodarone, then associated with digitalis. She was discharged with satisfactory control of the average ventricular rate, but later due to the appearance of chaotic atrial arrhythmia, propranolol was added; on the Holter ECG in triple therapy, periods of sinus rhythm alternating with short episodes of atrial tachycardia were observed. Genetic analysis revealed the pathogenic variant c.34G>A, p.(Gly12Ser) of the HRAS gene. The third patient, F.A., presented atrial tachycardia. Flecainide therapy had transient benefit. Subsequently propranolol was added, without benefit. Therapy with amiodarone was initiated. Transesophageal electrophysiological study demonstrated multifocal atrial tachycardia, unresponsive to overdrive pacing. Partial control of the average ventricular rate was achieved in triple therapy. On the echocardiogram, mild concentric hypertrophy and dysplastic pulmonary valve. Genetic testing detected the c.35G>C, p(Gly12Ala) variant of the HRAS gene. Conclusions Costello syndrome requires cardiac monitoring for the possible presence of multifocal atrial tachycardia and hypertrophic cardiomyopathy. Arrhythmia, which can also occur independently of hypertrophy, can be self–limited in the first months of life with aggressive pharmacological treatment but can persist or worsen in ¼ of patients.

H-ras-targeted genetic therapy remarkably surpassed docetaxel treatment in inhibiting chemically induced hepatic tumors in rats

AimsHepatocellular carcinoma (HCC) is still a leading cause of cancer-related death worldwide. But its chemotherapeutic options are far from expectation. We here compared H-ras targeted genetic therapy to a commercial docetaxel formulation (DXT) in inhibiting HCC in rats. Main methodsAfter the physicochemical characterization of phosphorothioate-antisense oligomer (PS-ASO) against H-ras mutated gene, the PS-ASO-mediated in vitro hemolysis, in vivo hepatic uptake, its pharmacokinetic profile, tissue distribution in some highly perfused organs, its effect in normal rats, antineoplastic efficacy in carcinogen-induced HCC in rats were evaluated and compared against DXT treatment. Mutated H-ras expression by in situ hybridization, hep-par-I, CK-7, CD-15, p53 expression patterns by immunohistochemical methods, scanning electron microscopic evaluation of hepatic architecture, various hepatic marker enzyme levels and caspase-3/9 apoptotic enzyme activities were also carried out in the experimental rats. Key findingsPS-ASO showed low in vitro hemolysis (<3 %), and had a sustained PS-ASO blood residence time in vivo compared to DTX, with a time-dependent hepatic uptake. It showed no toxic manifestations in normal rats. PS-ASO distribution was although initially less in the lung than liver and kidney, but at 8 h it accumulated more in lung than kidney. Antineoplastic potential of PS-ASO (treated for 6 weeks) excelled in inhibiting chemically induced tumorigenesis compared to DTX in rats, by inhibiting H-ras gene expression, some immonohistochemical modulations, and inducing caspase-3/9–mediated apoptosis. It prevented HCC-mediated lung metastatic tumor in the experimental rats. SignificancePS-ASO genetic therapy showed potential to inhibit HCC far more effectively than DXT in rats.

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.

Computational Exploration of Single-Nucleotide Polymorphisms in the Human hRAS Gene: Implications and Insights.

Background A group of genes called oncogenes includes the Harvey rat sarcoma virus (hRAS) gene. Along with hRAS, Kirsten rat sarcoma viral oncogene homolog (kRAS) and neuroblastoma RAS viral oncogene homolog(nRAS) genes belong to the Rat sarcoma (Ras) family of oncogenes. These three genes result in Rho guanosine triphosphate hydrolases (GTPases) as their protein product. Instructions for producing the protein hRAS, which is mainly involved in controlling cell division, are provided by the hRAS gene. The hRAS protein transfers signals from outside through a process called signal transduction.Because the hRAS protein is a GTPase, it changes the chemical guanosine-5'-triphosphate (GTP) into guanosine diphosphate (GDP). GTP and GDP molecules operate as switches to turn on and off the hRAS. This study aimed to anticipate the structure and stability of the protein resulting from missense single-nucleotide polymorphisms (SNPs) in the human hRAS genes. Methodology To investigate the possible negative effects associated with these SNPs, bioinformatic analysis is typically essential. The following tools were employed for forecasting harmful SNPs: Scale-Invariant Feature Transform (SIFT), Protein Analysis Through Evolutionary Relationships (PANTHER), non-synonymous SNP by Protein Variation Effect Analyzer (PROVEAN), and non-synonymous SNP by Single Nucleotide Polymorphism Annotation Platform (SNAP). Results The present study identified a total of 11 SNPs using the SIFT approach, which were shown to have functional significance. Only two of these 11 SNPs were determined to be tolerable, whereas nine were shown to be detrimental. Among the 11 SNPs analyzed, seven (Q61H, Q99H, K117R, A121D, A146V, R169W, R169Q) were classified as possibly damaging,and four (G13V, Q22K, Q61K, Q13V) were categorized as probably benign according to the predictions made by PANTHER tools. Therefore, the seven SNPs were identified as high-risk SNPs. Conclusions Given that SNPs have the potential to be candidates for cellular alterations brought on by mutations that are associated with cancer, this study provides vital information about how SNPs might be utilized as a diagnostic marker for cancer.

Abstract B089: Oncogenic signaling and responses to treatment in RAS mutant cancers are mutation-specific

Abstract The KRAS, NRAS, and HRAS genes are frequently mutated in cancer, often at hotspots altering codons 12, 13, and 61. Yet how different RAS isoforms and mutations precisely regulate oncogenic signaling is still largely unknown. For example, the role of mutant RAS in driving overactivation of ERK signaling in cancer has long been elucidated, but its involvement in the oncogenic activation of PI3K is still controversial, and different studies have reported conflicting findings regarding the requirement of RAS for physiological and oncogenic PI3K signaling. One of the reasons for this lack of understanding has been the inability to directly target RAS, which has historically been circumvented through genomic perturbation and murine models. However, these approaches are limited because it is not possible to distinguish between the direct effects of RAS on PI3K and compensatory events that occur as an adaptation to the oncogene. With the discovery of RAS inhibitors, we can now directly interrogate the biological effects of RAS isoforms and hotspot mutations in cancer cells with improved time resolution. Here, we use RM-042, a first-in-class broad-spectrum RAS·GTP inhibitor (RASMULTI(ON)), to characterize the features and therapeutic vulnerabilities of mutant RAS signaling in human cancers. Through a detailed biochemical analysis of the signaling response to RAS and MEK inhibition, we reveal that RAS-mediated oncogenic signaling is largely independent of the mutant RAS isoform, rather depending on the mutant codon. Specifically, RASQ61X is exquisitely sensitive to MAPK pathway inhibition, confers near-complete independence of upstream regulation, and is impaired in its signaling to PI3K/Akt/mTOR. Conversely, RASG12X enables oncogenic signaling to both MAPK and PI3K/Akt/mTOR by amplifying upstream input from receptor tyrosine kinases (RTKs). Simultaneous RAS and RTK inhibition selectively impairs the growth of RASG12X-mutant tumors, irrespective of RAS isoform and tumor type, and we investigate the molecular mechanisms behind this synergistic effect. Additionally, we have generated a genetically modified mouse model (GEMM) harboring liver tumors induced by RASG12D or RASQ61R. In cell lines derived from these respective models, RASG12D but not RASQ61R activates PI3K upon growth-factor stimulation. Furthermore, in this system, RASQ61R is less oncogenic than RASG12D, an effect that can be rescued by the concurrent deletion of PTEN. These findings provide mechanistic insights into how mutant RAS interacts with upstream and downstream partners to maximize oncogenic signaling. Furthermore, our results shed light on the selective pressure driving the emergence of RASQ61X mutations as a mechanism of resistance against RTK inhibition: we show that colorectal cancer cells that acquire RASQ61X mutations to overcome EGFR inhibition become more independent of upstream signaling than those that acquire RASG12X. Citation Format: Michelangelo Marasco, Dinesh Kumar, Riccardo Mezzadra, Wei Luan, Ilinca Aricescu, Rona Yaeger, Vladimir Markov, Yu Zhu, Elisa de Stanchina, Scott Lowe, Sandra Misale, Neal Rosen. Oncogenic signaling and responses to treatment in RAS mutant cancers are mutation-specific [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B089.

O06 Clinical phenotype of the Klippel-Trenaunay syndrome with mosaic PIK3R1 mutations

Abstract Segmental overgrowth syndromes with cutaneous vascular malformations (Klippel-Trenaunay syndrome (KTS), CLOVES syndrome) are mainly due to mosaic PIK3CA mutations, but the GNAQ, GNA11, HRAS, KRAS, MAP2K1, AKT1, AKT3, and PIK3R1 genes may also be involved. We sought to determine the frequency and phenotype of patients with PIK3R1-related segmental overgrowth. Patients were ascertained nationwide in the French MUSTARD cohort of skin mosaic phenotypes. We searched for PIK3R1 mutations by next-generation sequencing on affected tissue in 297 patients with a clinical presentation suggestive of PIK3CA related overgrowth (PROS), but without PIK3CA variants. We retrospectively studied the clinical phenotype of PIK3R1-positive patients. We identified three missense variants and six in-phase indels of PIK3R1 in 15 patients (5.0%), exclusively in affected tissue. Variant allele fraction was 2.0% to 12.0%. We found extensive pale or dark capillary malformations in 14 patients, segmental overgrowth in 11, venous truncal anomalies (varicose veins or persistent lateral marginal vein) in 10, lymphatic malformation in six and a café-­au-lait macule or naevus spilus in three, thus defining phacomatosis pigmento-vascularis (PPV). One patient had limb hypoplasia. None had MCAP (Megalencephaly-Capillary malformation). PIKR1 mutations account for a minority of patients with KTS/CLOVES (5%). They are about 20 times less common than PIK3CA mutations. Truncal venous malformations, limb hypoplasia and PPV (spilorosea or melanorosea type) may be more common in PIK3R1-related KTS. No patient had severe or life-threatening involvement. Whether Sirolimus or Alpelisib are treatment options remains to be determined.

НОВООБРАЗОВАНИЯ У ДЕТЕЙ С СИНДРОМОМ КОСТЕЛЛО: ОПЫТ РОССИЙСКОЙ ДЕТСКОЙ КЛИНИЧЕСКОЙ БОЛЬНИЦЫ

Costello Syndrome is an extremely rare genetic disorder caused by a mutation in the HRAS gene that encodes for a highly active protein that in its turn affects the cell growth and division. Patients have a unique phenotype and multiple developmental abnormalities that suggest the anomaly from the first days of life. The important feature of the disease is a predisposition to the development of neoplasms, both benign and malignant. This fact must obligatory be taken into consideration by physicians who are treating children with Costello Syndrome. Authors present the three clinical cases of children with Costello Syndrome coupled with neoplasms who underwent treatment at the Department of Surgical Treatment Methods of Oncology with the Russian Children's Clinical Hospital with the Pirogov Russian National Research Medical University of the Ministry of Healthcare of Russia (Moscow, Russia). The Authors’ experience in treating these patients, including a risk-adapted approach to therapy and early removal of detected formations, can be considered successful as yet. The descriptions of these clinical cases are of scientific and practical interest not only for pediatric oncologists but also for pediatricians, neurologists, cardiologists and other specialists due to the rarity of Costello Syndrome as they contribute to the formation of oncological vigilance.

Comprehensive analysis of miRNA-mRNA regulatory pairs associated with colorectal cancer and the role in tumor immunity

BackgroundMicroRNA (miRNA) which can act as post-transcriptional regulators of mRNAs via base-pairing with complementary sequences within mRNAs is involved in processes of the complex interaction between immune system and tumors.In this research, we elucidated the profiles of miRNAs and target mRNAs expression and their associations with the phenotypic hallmarks of colorectal cancers (CRC) by integrating transcriptomic, immunophenotype, methylation, mutation and survival data.ResultsWe conducted the analysis of differential miRNA/mRNA expression profile by GEO, TCGA and GTEx databases and the correlation between miRNA and targeted mRNA by miRTarBase and TarBase. Then we detected using qRT-PCR and validated the diagnostic value of miRNA-mRNA regulator pairs by the ROC, calibration curve and DCA. Phenotypic hallmarks of regulatory pairs including tumor-infiltrating lymphocytes, tumor microenvironment, tumor mutation burden, global methylation and gene mutation were also described. The expression levels of miRNAs and target mRNAs were detected in 80 paired colon tissue samples. Ultimately, we picked up two pivotal regulatory pairs (miR-139-5p/ STC1 and miR-20a-5p/ FGL2) and verified the diagnostic value of the complex model which is the combination of 4 signatures above-mentioned in 3 testing GEO datasets and an external validation cohort.ConclusionsWe found that 2 miRNAs by targeting 2 metastasis-related mRNAs were correlated with tumor-infiltrating macrophages, HRAS, and BRAF gene mutation status. Our results established the diagnostic model containing 2 miRNAs and their respective targeted mRNAs to distinguish CRCs and normal controls and displayed their complex roles in CRC pathogenesis especially tumor immunity.

Whole exome sequencing uncovers HRAS mutations as potential mediators of resistance to metronomic chemotherapy

ObjectivesThe aim of this pilot study is to identify the genetic factors that contribute to the response of metronomic chemotherapy in head and neck squamous cell carcinoma (HNSCC) patients using whole-exome sequencing (WES). This study would facilitate the identification of predictive biomarkers, which would enable personalized treatment strategies and improve treatment outcomes for patients with HNSCC. Materials and MethodsWe have selected patients with recurrent head and neck cancer who underwent metronomic chemotherapy. Sequential tumor biopsies were collected from the patients at different stages of treatment to capture the genomic alterations and tumor evolution during metronomic chemotherapy and sequenced using WES. ResultsWe identified several known HNSCC hallmark genes reported in COSMIC, including KMT2B, NOTCH1, FAT1, TP53, HRAS, CASP8, and CDKN2A. Copy number alteration analysis revealed amplifications and deletions in several oncogenic and tumor suppressor genes. COSMIC Mutational Signature 15 associated with defective DNA mismatch repair was enriched in 73% of HNSCC samples. Further, the comparison of genomic alterations between responders and non-responders identified HRAS gene uniquely mutated in non-responders that could potentially contribute to resistance against metronomic chemotherapy. DiscussionOur findings corroborate the molecular heterogeneity of recurrent HNSCC tumors and establish an association between HRAS mutations and resistance to metronomic chemotherapy, suggesting HRAS as a potential therapeutic target. Combining HRAS inhibitors with metronomic regimens could improve treatment sensitivity in HRAS-mutated HNSCC patients. Further studies are needed to fully elucidate the genomic mechanisms underlying the response to metronomic chemotherapy.

Sialadenoma papilliferum-like intraductal papillary tumour: an emerging entity with intercalated duct differentiation showing MAPK pathway activation in both ductal and myoepithelial cells

Sialadenoma papilliferum-like intraductal papillary tumour (SP-IPT) is a recently described salivary gland tumour that shows identical morphology to sialadenoma papilliferum (SP) except for the lack of an exophytic papillary component. However, the immunohistochemical phenotypes and molecular profiles of SP-IPT remain unclear. This study aims to report new cases of SP-IPT and to determine its cellular differentiation and molecular basis. After histopathological review, four cases of SP-IPT were retrieved. Immunohistochemical staining was performed to analyse the expression patterns of cytokeratin 7 (CK7), p63, smooth muscle actin (SMA), vimentin, S100, mammaglobin, androgen receptor, SOX10, BRAF V600E-mutated protein, and phosphorylated ERK. Sanger sequencing was performed to determine the mutation status of the BRAF, KRAS, NRAS, and HRAS genes. All four cases affected the posterior mandible with a mean age of 62 years and a male-to-female ratio of 3:1. Histologically, all cases consisted of multiple tubular and cystic structures with varying sizes and shapes. The tubulocystic components were lined by a double or few-layered epithelium frequently showing a micropapillary pattern. The outer layer consisted of a rim of myoepithelial cells, which were CK7+/p63+/SMA+/vimentin+/S100+/SOX10+. The inner ductal cells were CK7+/S100+/SOX10+, consistent with intercalated duct differentiation. All cases harboured BRAF V600E mutations, but no other mutations were detected. The BRAF V600E-mutated protein and phosphorylated ERK were expressed in both ductal and myoepithelial cells. These findings demonstrate the immunohistochemical and molecular similarities between SP-IPT and SP and the role and extent of MAPK pathway activation in the pathogenesis of SP-IPT.

OR32-06 Disease-related Outcomes In Patients With Thyroid Cancer On Long Term Follow-up: Analysis Of 1487 Patients

Abstract Disclosure: W.R. Doerfler: None. E. Morariu: None. S.E. Carty: None. M. Nikiforova: Other; Self; Thyroseq. Y.E. Nikiforov: Other; Self; Thyroseq. L. Yip: None. Introduction: Molecular genotype of differentiated thyroid cancer (DTC) has been shown to be associated with histology and disease-related outcomes including local recurrence and distant metastatic disease. In an updated analysis with long follow-up of a large cohort of thyroid cancer patients with prospective 7-gene molecular testing results, we aim to identify the risk of long-term recurrence and associated factors to guide optimal surveillance strategies. Methods: We reviewed a consecutive series of 1487 patients with DTC who had index thyroidectomy performed from 2007-2013 and routine molecular testing to detect alterations in 7 genes: BRAF, NRAS, HRAS, and KRAS genes, and RET/PTC1, RET/PTC3, and PAX8/PPARG fusions. We assessed for disease recurrence at latest follow-up which was defined as date of thyroglobulin measurement or neck ultrasound. Recurrence (occurring &amp;gt; 6 months after initial treatment) was defined per 2015 ATA guidelines as disease confirmed on histology, a rise in unstimulated thyroglobulin to &amp;gt;1 ng/mL without clear structural recurrence, or structural recurrence on imaging without confirmed biopsy. Results:98% of patients were diagnosed with papillary thyroid carcinoma (PTC). BRAFV600E was the most common mutation identified (43%). At least 5 years of follow-up occurred in 966 (64.9%) patients. Median follow up was 7.3 years ± 4.1 years. Of 1390 patients for whom recurrence could be assessed, 93 (6.4%) recurred including 55 (59%) recurrences &amp;lt; 2 years after surgery, 25 (27%) between 2-5 years, and 13 (14%) who were diagnosed &amp;gt;5 years. Of those who recurred between 2 to 5 years, 16 had BRAFV600E, 1 had NRAS, and 1 had RET/PTC1 cancer. Of those who recurred &amp;gt;5 years, 7 had BRAFV600E-positive DTC, 5 were 7-gene negative, and 1 had a complex BRAF mutation. Of the late recurrences (diagnosed &amp;gt;5 years), 9 were structural including 6 locoregional which were biopsy-confirmed, and 4 were biochemical. Metachronous distant metastatic disease occurred in 1 of 13 with late recurrence and was a patient with pulmonary metastasis whose tumor was positive for complex BRAF mutation. Disease-free survival at 5 years for patients with RAS, PAX8PPARG, and BRAFK601E positive cancer (99-100%)was higher than for patients with cancers positive for RET/PTC (87%) or BRAFV600E (88.7%)(p&amp;lt;0.001), and remained higher at 10 years (99-100% vs 87%, p&amp;lt;0.001). Conclusion: In this large cohort of consecutive DTC patients with prospective 7-gene molecular testing and long-term follow-up, 5- and 10-year DFS was higher in patients with RAS-like TC compared to those with BRAF-like TC. ∼60% of recurrences occurred in the first 2 years, while most recurrences &amp;gt;5 years were locoregional or biochemical and only one patient developed distant metastatic disease. Further analysis with additional comprehensive genetic variables is ongoing to better identify patients who are at risk for long-term recurrence. Presentation: Sunday, June 18, 2023

36P Comparison of pelitinib, tepotinib or docetaxel efficacy according to the copy number or gene alteration status of EGFR, MET, HRAS, KRAS and NRAS genes

36P Comparison of pelitinib, tepotinib or docetaxel efficacy according to the copy number or gene alteration status of EGFR, MET, HRAS, KRAS and NRAS genes

Different Mutational Landscapes in Human Papillomavirus–Induced and Human Papillomavirus–Independent Invasive Penile Squamous Cell Cancers

Penile squamous cell carcinomas (SCC) are rare cancers that arise after transforming human papillomavirus (HPV) infections or independent of HPV in the background of chronic dermatoses. Limited knowledge about genetic alterations driving penile carcinogenesis comes from studies of mainly small cohorts of typically mixed etiology. In this comparative genetic study of HPV-induced and HPV-independent invasive penile SCC of 156 patients from a single institution in a low-incidence country, hotspots of 50 cancer-relevant genes were analyzed with targeted next-generation sequencing. Seventy-nine of 156 SCC were classified as HPV induced, and 77 of 156 SCC arose independent of HPV. Only 28 (35%) of 79 HPV-induced penile SCC, but 69 (90%) of 77 HPV-independent SCC carried somatic gene mutations. PIK3CA, FGFR3, and FBXW7 mutations occurred in both groups in similar numbers as seen in other human cancers. In contrast, mutations in TP53 (44/77; 57%), CDKN2A (35/77; 45%), and HRAS (13/77; 17%) genes occurred with one exception of a HIV positive patient exclusively in HPV-independent SCC with a frequent co-occurrence of TP53 and CDKN2A mutations (28/77; 42%). Mutations in multiple genes occurred in 9 (11%) of 79 HPV-induced SCC versus 47 (62%) of 77 HPV-independent SCC (χ2; P < .001). More than one mutation per gene (multi hits) was characteristic for HPV-independent SCC in 14 (18%) of 77 compared with only 3 (4%) of 79 HPV-induced SCC (χ2; P < .001). The total number of mutations in HPV-induced penile SCC (47 mutations) was significantly lower than that in HPV-independent SCC (143 mutations; Welsh test; P < .001). The presence of somatic driver gene mutations did not correlate with the age of patients, histology, or tumor stage of the primary SCC in either etiologic group, suggesting that acquisition of driver gene mutations is an early event after invasion. This large cohort analysis identified characteristic differences in mutational landscapes for the 2 etiologies. While genetic mutations in tumor suppressor genes drive HPV-independent penile carcinogenesis, oncogenic action of E6 and E7 substitute for mutations in HPV-induced SCC. A subgroup of patients with advanced SCC may be candidates for targeted therapy and clinical trials, although the majority of advanced penile SCC remain a therapeutic challenge.

Prenatal Diagnosis of Costello Syndrome Using Noninvasive Prenatal Single Gene Testing With Maternal Blood

Introduction Costello syndrome (CS) is a rare disorder that affects multiple organ systems, which is usually diagnosed postnatally, except for 2 cases diagnosed prenatally from chromosome analysis by amniocentesis. We present the first case of prenatally diagnosed CS via maternal blood testing. Case Description A 38-year-old woman was referred for genetic screening at 28 weeks of gestational age (GA) due to polyhydramnios and shortened limbs seen on prenatal ultrasound. Cell-free DNA, noninvasive prenatal single gene screening was positive for G12S HRAS mutation, which is indicative of CS. The infant was delivered via elective repeat cesarean section at 36 weeks and 6 days GA, was admitted to the neonatal intensive care unit for respiratory distress. Physical examination was significant for macrocephaly, wide depressed nasal bridge, short neck, rhizomelic limb shortening, micropenis, and bilateral equinovarus deformity of the feet. Echocardiography on the first day showed severe pulmonary hypertension. Brain MRI showed a possible cavernous mass lesion and small arachnoid cyst over the left cerebellar hemisphere. Postnatal gene-targeted testing confirmed CS diagnosis with pathogenic variants. The infant was managed for respiratory distress, hypoglycemia, jaundice, and feet deformities. He was subsequently transferred to another hospital at 45 weeks of age for gastrostomy tube placement due to persistent difficulty with oral feeding. Discussion CS affects multiple organ systems. Clinical presentations includes short stature; macrocephaly; coarse facial features, loose and soft skin; hypotonia and joint laxity; cardiac involvement with congenital heart defect, feeding difficulty, and developmental delay. The phenotypic spectrum is wide and can range from mild features to early lethal complications mostly as a result of severe progressive Hypertrophic Cardiomyopathy (HCM) and respiratory compromise. CS is an autosomal dominant genetic disorder caused by mutations in the HRAS gene, but almost all cases have new mutations with no family history. Disease course depends on cardiac and predisposition to benign and malignant tumor complications. Treatment requires a multidisciplinary team directed toward the specific clinical findings. Conclusion To our knowledge, this is the first reported case of prenatally diagnosed CS via maternal blood testing. Molecular diagnosis in the prenatal period allows neonatal care providers to prepare for potential complications in the postnatal period.