Relevant Mutations Research Articles

Abstract 2795: A food effect and esomeprazole drug-drug interaction study of RLY-4008, a highly selective FGFR2 inhibitor, in healthy subjects

Abstract Background/Introduction: Effective therapies for FGFR2-driven cholangiocarcinoma (CCA) and other advanced solid tumors remain unmet medical needs. RLY-4008 is a potent, highly selective and irreversible FGFR2 inhibitor designed to target both primary FGFR2 oncogenic alterations and clinically relevant resistance mutations. RLY-4008 is currently being studied in a Phase 1/2 clinical trial to treat patients with FGFR2-driven CCA. To guide RLY-4008 dosing in cancer patients, we report here results from a clinical study to assess the effect of food and proton pump inhibitor (PPI) esomeprazole (ESO) on the pharmacokinetics (PK) of RLY-4008 in healthy subjects (HS). Methods: This was a randomized, open-label, three-period, fed and fasted two-treatment crossover and fixed sequence, drug-drug interaction study to assess the effect of food and ESO on RLY-4008 PK. Twenty-four HS were enrolled to receive single oral doses of 50 mg RLY-4008 (fasted or fed with a high fat, high calorie meal) or RLY-4008 with ESO (fasted), with ≥7 days washout between each treatment. RLY-4008 PK was assessed up to 120 hours post dose. The effect of food and ESO was assessed using log-transformed RLY-4008 Cmax and AUC by linear mixed-effect modeling and calculating point estimates and associated 90% confidence intervals (CI) of geometric least squares mean ratios (LSGMR). Safety and tolerability were assessed through discharge/end of study. Results: Twenty-four HS were enrolled and completed the study. The median Tmax of RLY-4008 was similar when administered fasted, with food or with ESO. LSGMRs of Cmax, AUC(0-tlast) and AUC (0-inf) between RLY-4008 administered with food and fasted were 87%, 105%, and 106%, respectively, and the associated 90% CIs were 77-97%, 100-110%, and 100-110%, respectively. The LSGMRs and associated 90% CIs of AUCs were fully contained within 80% to 125%, while the lower bound of 90% confidence of Cmax LSGMR was below 80%. The 13% reduction in RLY-4008 Cmax with food was not considered clinically relevant. LSGMRs of Cmax, AUC(0-tlast) and AUC (0-inf) between RLY-4008 administered with ESO and alone were 110%, 113%, and 113%, respectively, and the associated 90% CIs were 102-119%, 109-117%, and 109-117%, respectively, and were fully contained within 80% to 125%. RLY-4008 was safe and well tolerated in HS when administered fasted, fed or with ESO. Conclusion: Food and ESO does not have a clinically relevant effect on RLY-4008 PK in HS, indicating that RLY-4008 can be dosed with or without food and can be dosed with PPIs, H2-blockers and anti-acids in cancer patients. Citation Format: Jinshan Shen, Rishikesh Sawant, Rick Blakesley, Kai Yu Jen, Fabien Ricard, Xiaoyan Li, Cassandra Key, Djuro Karanovic. A food effect and esomeprazole drug-drug interaction study of RLY-4008, a highly selective FGFR2 inhibitor, in healthy subjects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2795.

Abstract 2115: Zero-waste molecular diagnostics from biopsy needle wash water

Abstract Background: Over the past decade, the WHO classification of brain tumors has increasingly integrated molecular diagnostics into official classification criteria. However, biopsy tissue is precious due to high morbidity associated with repeat biopsies, and usually earmarked for histology, leaving little or no tissue for clinical molecular diagnostics. We have identified that the fluid used to wash biopsy needles after cores contains excess tumor DNA and cells that can be diagnostically useful. Additional research of tumor DNA and tumor tissue recovered from biopsy needle washes provides an opportunity for improvement in the diagnosis and treatment for brain tumor patients, while utilizing material that is otherwise thrown away. Methods: In two cases, surgeons were instructed to save 5-15ml of fluid used to wash biopsy needles. Isothermal targeted amplification of H3.3 and H3.1 histone genes was performed on raw wash fluid. Resulting amplicons were sequenced using an Oxford Nanopore MinION. DNA extracted from wash fluid was subjected to NGS sequencing via the Illumina TruSight Oncology 500 panel and compared to a CLIA certified NGS sequencing results from clinically indicated biopsy tissue. Results: For case 1, 5ul of raw wash fluid was subjected to rapid Loop-mediated Isothermal Amplification (LAMP) targeting the H3.1 and H3.3 K27M histone mutations and immediately sequenced using a MinION sequencer via rapid library preparation. Sequencing revealed a H3.3 K27M histone mutation present in wash fluid at 12% variant allele fraction (VAF). The variant call (99.99% CI) was available 1hr5mins after acquisition of material. For case 2, 5ml of wash fluid was processed using a cell-free DNA extraction kit resulting in ~250ng of recovered DNA. A LAMP assay targeting the H3.3 K27M mutation was performed on extracted DNA from wash fluid. Sequencing revealed an H3.3 K27M histone mutation present at 55% VAF, which was later corroborated using digital droplet PCR. Clinically indicated tissue from both biopsies was submitted for cancer panel sequencing in a CLIA certified setting. DNA extracted from wash fluid from both cases was subjected to Illumina TruSight Oncology 500 panel sequencing. In both cases, the panel was able to corroborate all SNV mutations reported by clinical sequencing that were also covered by the panel. Accrual of needle wash specimens is ongoing, with multiple monthly procedures. Conclusions: Large amounts of diagnostically useful material is present in biopsy needle wash fluid, which is usually discarded. Isothermal assays coupled with MinION sequencing can rapidly identify clinically relevant point mutations directly from wash fluid (e.g. IDH1), and enough DNA was recovered from wash fluid from both case-studies to submit for targeted cancer panel sequencing. These results suggest biopsy needle wash fluid should be considered a first-class diagnostic resource in any cancer. Citation Format: Jack Wadden, Vishal John, Seongbae Kong, Kait Verbal, Amy K. Bruzek, Wajd Al-Holou, Jason A. Heth, Hugh Garton, Carl Koschmann. Zero-waste molecular diagnostics from biopsy needle wash water [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2115.

Abstract 4019: Preclinical activity of ELVN-002: A potent, selective, and irreversible HER2 and pan-HER2 mutant small molecule inhibitor for the treatment of HER2 driven malignancies

Abstract Mutation or amplification of the human epidermal growth factor receptor 2 (HER2) gene has been identified in numerous solid tumor types, including non-small cell lung cancer (NSCLC), breast, and colorectal cancer. These genetic alterations are believed to confer elevated HER2 activity resulting in oncogenic transformation. In NSCLC, an estimated 3% of patients harbor activating mutations in the HER2 gene. Enhertu (T-DXd), a HER2-specific antibody-drug conjugate was recently approved as an important therapy for this patient population. However, for patients who are not candidates for T-DXd or have discontinued treatment due to an adverse event or disease progression, there exist few therapeutic options. Accordingly, HER2 inhibitors have the potential to provide meaningful therapeutic benefit to these patients. Due to significant structural homology between EGFR and HER2, most investigational small molecule HER2 kinase inhibitors are dual EGFR/HER2 inhibitors and are substantially dose-limited by EGFR-related toxicities in patients. These toxicities likely limit their clinical activity. Currently, tucatinib is the only HER2-selective small molecule inhibitor approved for metastatic breast cancer in combination with trastuzumab and capecitabine. However, it lacks sufficient potency against several of the key clinically relevant mutations, including HER2 YVMA, the most common exon 20 insertion mutation, prevalent in HER2 mutant NSCLC. ELVN-002 is a potent, irreversible inhibitor of HER2 with a >100-fold selectivity over EGFR. ELVN-002 potently inhibits the phosphorylation of HER2 in cell lines endogenously expressing HER2 or in those engineered to express specific clinically relevant HER2 mutants. This activity readily translates into marked anti-proliferative activity against cell lines dependent upon HER2 for their growth and survival. In addition, ELVN-002 is highly selective for HER2 and HER2 mutants versus wild-type EGFR as demonstrated via assessment of both EGFR-driven proliferation and EGFR phosphorylation in cells. Furthermore, ELVN-002 is highly active in mouse models of HER2-driven cancers, including subcutaneous models driven by wild type or mutant HER2. ELVN-002 is also active in an NCI-N87 wild-type HER2 intracranial model. In all models tested, ELVN-002 treatment is well tolerated and resulted in tumor regressions at exposures believed to be clinically achievable. Due to its selectivity and breadth of activity versus the clinically relevant HER2 mutants, ELVN-002 has the potential to be an effective treatment for HER2 mutant NSCLC as well as other HER2 mutant amplified solid tumors, including metastatic HER2 positive breast cancer. Additionally, ELVN-002 could provide a meaningful therapeutic option for patients with CNS metastases. ELVN-002 has been selected for clinical development. Citation Format: Monette Aujay, Amanda J. Broad, Stefan D. Gross, Li Ren, Joseph P. Lyssikatos, Samuel Kintz, Qi Wang, Helen Collins. Preclinical activity of ELVN-002: A potent, selective, and irreversible HER2 and pan-HER2 mutant small molecule inhibitor for the treatment of HER2 driven malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4019.

Abstract 1386: Ag5 is highly potent, mitochondrially targeted agent with a differentiated mechanism of action that promises to be efficacious in solid cancers

Abstract Mitochondrial remodeling in cancer is important for tumorigenesis, metastasis and linked totreatment resistance and poor prognosis. Dysfunctional mitochondria are also driven by oncogenes (e.g. KRAS, MYC) resulting in an increased oxidation state triggering the antioxidant response in order to decrease ROS levels, and to trigger apoptosis when the oxidative stress cannot be overcome. So far, targeting cancer cell energy metabolism has been unsuccessful due to tumor evolution, low therapeutic index and redundancy of protective mechanisms. Taken together, the clinical sequelae of mitochondrial remodeling represent one of the largest areas of unmet need in cancer therapy. We are developing Ag5, a highly potent, mitochondrially targeted agent with a novel mechanism of action that promises to be efficacious in solid cancers. Ag5 selectively kills cancer cells by catalyzing the oxidation of thiol groups of members ofthese pathways, a rapid mechanism of action that can be reversed with ROS scavengers (e.g. NAC). As a consequence, Ag5 will preferentially kill cancer cells, but will spare normal cells due to their REDOX homeostasis. We characterized Ag5 efficacy with a range of in vitro viability assays and showed potency against a panel of established cancer cell lines with an IC50 in the low nM range, whereas Ag5 activity was significantly lower in non-malignant cells. Ag5 sensitivity was not only correlated with high ROS levels as measured by mitoSOX, but also with target protein oxidation (especially the mitochondrially expressed PRDX3), cardiolipin and cytochrome c release, thus highlighting the mitochondrial targeting of Ag5. Several well characterised gene mutations lead to alterations in mitochondrial antioxidant function which sensitise the tumors to Ag5, either by decreasing endogenous antioxidant synthesis or increasing reliance on antioxidants for cell survival. For example, ARID1A damaging mutations directly lead to decreased glutathione synthesis and are found in many solid cancers (e.g. NSCLC, CRC and clear cell ovarian cancers). Experimental data with pairs of ARID1A wild-type or KO cell lines showed strong sensitization to Ag5 activity when ARID1A is knocked out. Furthermore, Ag5 was shown to be effective in reducing tumor growth in KRAS G12C mutated NSCLC and other relevant cell lines in viability assays and an orthotopic in vivo model. Combination treatment with KRAS, SOS1 and SHP2 inhibitors demonstrated not only Ag5 synergy, but also superior efficacy when Ag5 was included over the combination of these inhibitors alone. In summary, Ag5 is a novel and innovative therapeutic candidate shown to be safe and effective in preclinical studies. Based on experimental data with clinically relevant mutations, we hypothesize that Ag5 is ideally investigated in enriched patient populations of lung cancerand other solid cancers. Citation Format: Martin Treder. Ag5 is highly potent, mitochondrially targeted agent with a differentiated mechanism of action that promises to be efficacious in solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1386.

Abstract 2302: Detection of tumor-associated methylation and mutation signatures for early colorectal cancer diagnosis

Abstract Introduction: The ability to identify epigenetic signatures, together with genetic alterations, has expanded the translational utility of genomic sequencing in clinical settings. Epigenetic changes may appear at very early stages before diagnosis. Thus, they could be used as epigenetic biomarkers for early detection or risk of cancer development. Circulating cell-free DNA (cfDNA) profiling has emerged as a promising biomarker for cancer diagnosis and prognosis. Herein, we developed targeted next-generation sequencing (NGS) based assays for assessing multiple DNA methylated and mutated regions in both low-quantity (plasma cfDNA) and low-quality formalin-fixed paraffin-embedded (FFPE) tissue for colorectal cancer (CRC) detection. Methods: For the OptiSeqTM CRC methylation panel, we assessed the methylation status of CpG dinucleotides in 42 genes. With this panel, we profiled 6 CRC samples (matching cfDNA and FFPE), 2 primary colon tumors with adjacent normal, and 6 advanced adenomas (AA) FFPE tissue DNA. For healthy individuals, we profiled 12 normal colon FFPE samples and 31 plasma cfDNA (26 matching buffy coat DNA samples). For the OptiSeqTM CRC mutation panel, the mutation status was assessed in 43 genes. Here, we profiled 6 CRC samples (matching cfDNA and FFPE), 19 CRC plasma cfDNA, 4 AA FFPE and 42 healthy individuals’ plasma cfDNA (26 matching buffy coat DNA samples). The analytical and clinical performance for both panels (separately and combined) were calculated using advanced adenoma (AA), CRC, and cancer-free individuals. Results: For CRC cfDNA samples, clinical samples testing showed that the methylation panel had a sensitivity of 50% (95% CI: 13.9-86.0) and specificity of 100% (95% CI: 86.2-100) while the mutation panel yielded the sensitivity 88% (95% CI: 67.7-96.8) and specificity 100% (95% CI: 89.5-100). Combining the mutation panel with the methylation panel, the sensitivity increased to 100% (95% CI: 51.6-100) and specificity to 100% (95% CI: 65.1-93.3). For AA FFPE samples, the methylation panel analysis yielded a sensitivity of 100% (95% CI: 39.5-100.0) and a specificity of 100% (95% CI: 67.8-100). Conclusion: We have developed and validated the targeted NGS-based panels to detect clinically relevant mutation and methylation patterns and demonstrate its potential as a highly sensitive assay for early CRC diagnosis. Citation Format: Pushpinder Bains, Mikhail Kulak, Zhen Cui, Alisha Babu, Stella Tran, Bongyong Lee, Aiguo Zhang, Michael Sha. Detection of tumor-associated methylation and mutation signatures for early colorectal cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2302.

Abstract 1439: In vivo characterization of a selective FGFR2 inhibitor with potency against gatekeeper and molecular brake mutations

Abstract Fibroblast growth factor receptor (FGFR) mutations, fusions, and rearrangements have been linked to the pathogenesis of multiple tumor types. FGFR2 alterations have emerged as oncogenic drivers across a variety of tumor types, including cholangiocarcinoma, lung, endometrial, and uterine cancer. Approved FGFR inhibitors have demonstrated responses in patients that harbor FGFR genetic alterations but show reduced activity in patients with gatekeeper and molecular brake mutations. In addition, pan-FGFR inhibitors are associated with dose-limiting hyperphosphatemia linked to the inhibition of FGFR1. To address these issues, a novel series of reversible FGFR2 inhibitors with activity against clinically relevant mutations and selectivity over FGFR1 has been identified. The inhibition of FGFR2 phosphorylation in engineered cell lines demonstrated activity toward wild-type FGFR2, molecular brake mutations N549H/K, and gatekeeper mutations V564I/F/L. Selectivity of these inhibitors against FGFR1 and the broader kinome could lead to fewer dose limiting toxicities compared to approved FGFR inhibitors. Additionally, maintaining potency against molecular brake and gatekeeper mutations may result in significantly improved clinical response. In vivo characterization to be presented includes dose escalating rodent pharmacokinetics (PK), second species PK, and pharmacodynamics (PD) in tumor bearing mice. Most importantly, efficacy in a mouse tumor xenograft model at a dose that does not show elevated plasma phosphorus levels in the rat hyperphosphatemia model will be presented. Herein, the in vitro and in vivo characterization of a representative selective reversible FGFR2 inhibitor is described. Citation Format: John Fischer, Karyn Bouhana, Mark Chicarelli, Brad Fell, Jennifer Fulton, Anna Guarnieri, Leyla Haygood, Ravi Jalluri, Amber Johnson, Brent Mclean, Max Mejia, Rob Rieger, John Robinson, Mareli Rodriguez, Francis Sullivan, Yang Wang, Shannon Winski, YeYun Zhou. In vivo characterization of a selective FGFR2 inhibitor with potency against gatekeeper and molecular brake mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1439.

Comparison between Three Different Techniques for the Detection of EGFR Mutations in Liquid Biopsies of Patients with Advanced Stage Lung Adenocarcinoma

Oncogenic mutations in the EGFR gene are targets of tyrosine kinase inhibitors (TKIs) in lung adenocarcinoma (LC) patients, and their search is mandatory to make decisions on treatment strategies. Liquid biopsy of circulating tumour DNA (ctDNA) is increasingly used to detect EGFR mutations, including main activating alterations (exon 19 deletions and exon 21 L858R mutation) and T790M mutation, which is the most common mechanism of acquired resistance to first- and second-generation TKIs. In this study, we prospectively compared three different techniques for EGFR mutation detection in liquid biopsies of such patients. Fifty-four ctDNA samples from 48 consecutive advanced LC patients treated with TKIs were tested for relevant EGFR mutations with Therascreen® EGFR Plasma RGQ-PCR Kit (Qiagen). Samples were subsequently tested with two different technologies, with the aim to compare the EGFR detection rates: real-time PCR based Idylla™ ctEGFR mutation assay (Biocartis) and next-generation sequencing (NGS) system with Ion AmpliSeq Cancer Hotspot panel (ThermoFisher). A high concordance rate for main druggable EGFR alterations was observed with the two real-time PCR-based assays, ranging from 100% for T790M mutation to 94% for L858R variant and 85% for exon 19 deletions. Conversely, lower concordance rates were found between real-time PCR approaches and the NGS method (L858R: 88%; exon19-dels: 74%; T790M: 37.5%). Our results evidenced an equivalent detection ability between PCR-based techniques for circulating EGFR mutations. The NGS assay allowed detection of a wider range of EGFR mutations but showed a poor ability to detect T790M.

Different miRNAs Related to FBXW7 Mutations or High Mitotic Indices Contribute to Rectal Neuroendocrine Tumors: A Pilot Study.

Recent studies suggest that miRNA may be involved in the development of rectal neuroendocrine tumors (NETs). We explored the frequency of clinicopathologically relevant mutations and miRNA expression in rectal NETs to examine molecular profiles related to prognosis and behavior. Twenty-four eligible specimens with endoscopically excised rectal NETs were selected. Next-generation sequencing and an miRNA expression assay were used to evaluate the expression profile relevant to common genetic mutations in rectal NETs. Kyoto Encyclopedia of Genes and Genomes analysis predicted that the possible target signaling pathways were correlated with dysregulated miRNAs. Nineteen rectal NETs harbored more than one mutation in the 24 cancer-related genes. Seven miRNAs (hsa-miR-769-5p, hsa-miR-221-3p, hsa-miR-34a-5p, hsa-miR-181c-5p, hsa-miR-1246, hsa-miR-324-5p, and hsa-miR-361-3p) were significantly down-regulated in tumors harboring the FBWX7 mutation. Unsupervised hierarchical clustering analysis showed that up-regulation of these seven miRNAs may result in high mitotic indices, indicating the role of miRNAs in tumor progression. Among the down-regulated miRNAs, hsa-miR-769-5p was strongly correlated with extracellular matrix-receptor interaction and lysine degradation. Among the clinicopathological factors, up-regulated hsa-miR-3934-5p was linked to an increased mitotic count. No change in miRNA expression was associated with a tumor size >1 cm, lymphovascular invasion, or Ki-67 index. In summary, we identified different miRNA signatures involved in FBXW7 mutations or high mitotic indices in rectal NETs, which may play a critical role in tumor behavior.

LACE 2.0: an interactive R tool for the inference and visualization of longitudinal cancer evolution

BackgroundLongitudinal single-cell sequencing experiments of patient-derived models are increasingly employed to investigate cancer evolution. In this context, robust computational methods are needed to properly exploit the mutational profiles of single cells generated via variant calling, in order to reconstruct the evolutionary history of a tumor and characterize the impact of therapeutic strategies, such as the administration of drugs. To this end, we have recently developed the LACE framework for the Longitudinal Analysis of Cancer Evolution.ResultsThe LACE 2.0 release aimed at inferring longitudinal clonal trees enhances the original framework with new key functionalities: an improved data management for preprocessing of standard variant calling data, a reworked inference engine, and direct connection to public databases.ConclusionsAll of this is accessible through a new and interactive Shiny R graphical interface offering the possibility to apply filters helpful in discriminating relevant or potential driver mutations, set up inferential parameters, and visualize the results. The software is available at: github.com/BIMIB-DISCo/LACE.

Assessment of the Mutation Profile of Tonsillar Squamous Cell Carcinomas Using Targeted Next-Generation Sequencing.

Data regarding driver mutation profiles in tonsillar squamous cell carcinomas (TSCCs) remain scarce, limiting the understanding of its pathogenesis and unexpected behavior in the updated staging system. We investigated the incidence of clinically relevant mutations and their contribution in the prognosis of the condition, and their association with human papillomavirus (HPV) infection and adjuvant therapy. We subjected 43 surgically resected TSCC samples to targeted next-generation sequencing, determined their HPV status using polymerase chain reaction, and performed The Cancer Genomic Atlas and Gene Set Enrichment analyses. Thirty-five TSCC samples (81.4%) showed at least one oncogenic/likely oncogenic mutation among twenty-nine cancer-related genes. The top five mutated genes were TP53 (46.5%), PIK3CA (25.6%), PTEN (18.6%), EGFR (16.3%), and SMAD4 (14.0%). The EGFR pathway was the most frequently affected (51.2%), followed by the p53 (48.8%), PI3K (39.5%), and RTK (34.9%) pathways. The gene set enrichment analysis confirmed that the genes involved in signal transduction, such as growth factor receptors and second messengers, EGFR tyrosine kinase inhibitors, and PI3K signaling pathways, were mostly related with TSCCs. TP53 mutation was an independent prognostic factor predicting worse overall survival in the adjuvant therapy group. RTK mutations were related to survival in all patients and in the HPV-positive group, but multivariate analyses showed no significance. In conclusion, oncogenic/likely oncogenic mutations were relatively high in TSCCs, and TP53 and RTK mutations may be candidate predictors for poor prognosis in the adjuvant therapy and HPV-positive groups, respectively, under the updated staging system.

Identification of Clinically Relevant HIV Vif Protein Motif Mutations through Machine Learning and Undersampling.

Human Immunodeficiency virus (HIV) and its clinical entity, the Acquired Immunodeficiency Syndrome (AIDS) continue to represent an important health burden worldwide. Although great advances have been made towards determining the way viral genetic diversity affects clinical outcome, genetic association studies have been hindered by the complexity of their interactions with the human host. This study provides an innovative approach for the identification and analysis of epidemiological associations between HIV Viral Infectivity Factor (Vif) protein mutations and four clinical endpoints (Viral load and CD4 T cell numbers at time of both clinical debut and on historical follow-up of patients. Furthermore, this study highlights an alternative approach to the analysis of imbalanced datasets, where patients without specific mutations outnumber those with mutations. Imbalanced datasets are still a challenge hindering the development of classification algorithms through machine learning. This research deals with Decision Trees, Naïve Bayes (NB), Support Vector Machines (SVMs), and Artificial Neural Networks (ANNs). This paper proposes a new methodology considering an undersampling approach to deal with imbalanced datasets and introduces two novel and differing approaches (MAREV-1 and MAREV-2). As theses approaches do not involve human pre-determined and hypothesis-driven combinations of motifs having functional or clinical relevance, they provide a unique opportunity to discover novel complex motif combinations of interest. Moreover, the motif combinations found can be analyzed through traditional statistical approaches avoiding statistical corrections for multiple tests.

Real world rates of genetic referrals and germline testing in patients with prostate adenocarcinoma at an academic cancer center in southern New Jersey.

114 Background: Prostate adenocarcinoma is the second leading cause of cancer-related death in men in the United States. A major risk factor is family history, with prostate cancer in a first-degree relative effectively doubling a patient’s risk. However, relying on family history alone fails to identify many patients with clinically relevant cancer-related germline mutations, which can have important prognostic and therapeutic implications for patients and their families. In 2018, the NCCN recommended expanding the germline testing to include all men with high risk, very high risk, regional or metastatic prostate cancer. We sought to identify the rate of genetic testing within this patient population at the MD Anderson Cancer Center at Cooper University Hospital. Methods: The institution database was queried to identify patients with the ICD-10 diagnosis of prostate adenocarcinoma between 01-2019 and 12-2020; the inclusion criteria were age 18 years or older, biopsy-proven prostate adenocarcinoma, and qualification to receive germline genetic testing per NCCN guidelines. A total of 201 patients were screened and 97 cases were analyzed. Results: Of the 97 patients who qualified for genetic testing, 53 were referred to genetics (54.6%). 27 of the 53 patients who met with our genetics team underwent testing (50.9%). Of the 27 patients tested, nine patients (33.3%) were found to have clinically relevant cancer-related germline mutations with therapeutic implications for the patient and/or screening recommendations for the patient and their family members. Conclusions: Overall, the proportion of patients who qualified for and completed genetic testing was low at 27.8%. Of those who completed testing, the rate of detection of a significant germline mutation was high at 33.3%, indicating that a significant proportion of those untested may carry a genetic mutation of clinical importance. Based on these results, we developed an electronic Best Practice Alert that notifies providers if a patient qualifies for a genetic risk evaluation based on his prostate cancer risk. We implemented this system, with reported high utilization demonstrating feasibility of this tool. Preliminary data shows improved rates of referral among prostate cancer patients.

A phase II trial of risk-enabled therapy after initiating neoadjuvant chemotherapy for bladder cancer (RETAIN).

438 Background: Cisplatin-based neoadjuvant chemotherapy (NAC) followed by cystectomy or chemoradiation is the standard of care for urothelial carcinoma (UC) patients with MIBC. Both cystectomy and chemoradiation have short and long-term toxicity and QOL implications. Mutations in DNA damage repair/response genes are associated with pathologic downstaging after NAC. We hypothesized that a combination of biomarker selection and clinical staging would identify patients prospectively for a cystectomy or chemoradiation avoidance algorithm. Methods: We conducted a single-arm, phase II, non-inferiority trial (NCT02710734) to evaluate a risk-adapted approach for MIBC. Patients with cT2-T3N0M0 UC underwent NAC with accelerated MVAC. Pre-NAC TURBT specimens were sequenced (Caris) for mutations in ATM, ERCC2, FANCC or RB1. Patients with ≥ 1 mutations and no clinical evidence of disease by restaging TUR, urine cytology and imaging post-NAC began pre-defined active surveillance (AS). Remaining patients underwent bladder-directed therapy. The primary endpoint was metastasis-free survival (MFS) at 2 years for the entire cohort. The risk-adapted approach would be declared non-inferior to the standard of care if the lower bound of an exact 1-sided 95% CI for MFS was > 64%. The study required 70 patients with a 4.5% type I error and 81.6% power. Results: 71 (ITT) patients were enrolled over 33 months at four academic centers. Median age was 70 years (47-83), 74% were male, 92% Caucasian, 81% ECOG PS 0 and 79% cT2. 90% completed 3 cycles of MVAC with 17% grade 3-4 TRAEs. In the ITT population, 33 (46%) had a relevant mutation and 26 (37%) began AS. With a median follow-up of 41 months, 47 patients (66%) are metastasis-free (CI 54%-77%). The 2-year MFS for the ITT patients (primary endpoint) was 72% (lower bound exact 1-sided 95% CI=62%). On post hoc analysis, the 2-year MFS was 65% in the AS group (CI 44%-83%) and 76% (CI 60%-87%) in the remaining patients (P=0.42). In the AS group, 18 patients (69%) had some UC recurrence, 8 had a cystectomy, 2 chemoradiation, and 13 (50%) are metastasis-free with an intact bladder. Of the 10 patients (38%) on AS who developed metastatic disease, 9 recurred with localized disease first. The 2-year OS is 83% (CI 72%-90%) and 89% (CI 68%-96%) in the ITT and AS groups, respectively. Associations between mutation presence and MFS or UC recurrence were not observed. Conclusions: The 72% 2-year MFS rate in this MIBC cohort treated with a risk-adapted approach did not satisfy the pre-specified non-inferiority condition. 38% of AS patients developed metastatic disease, with most patients first recurring locally in the bladder. With a median follow-up of 41 months, although 50% of AS patients have been able to avoid cystectomy without metastatic disease, further refinement of this approach is necessary. Clinical trial information: NCT02710734 .

Molecular and Genetic Characterization of Hepatitis B Virus (HBV) among Saudi Chronically HBV-Infected Individuals.

The study aimed to characterize the genotype and subgenotypes of HBV circulating in Saudi Arabia, the presence of clinically relevant mutations possibly associated with resistance to antivirals or immune escape phenomena, and the possible impact of mutations in the structural characteristics of HBV polymerase. Plasma samples from 12 Saudi Arabian HBV-infected patients were analyzed using an in-house PCR method and direct sequencing. Saudi patients were infected with mainly subgenotype D1. A number of mutations in the RT gene (correlated to antiviral resistance) and within and outside the major hydrophilic region of the S gene (claimed to influence immunogenicity and be related to immune escape) were observed in almost all patients. Furthermore, the presence of mutations in the S region caused a change in the tertiary structure of the protein compared with the consensus region. Clinical manifestations of HBV infection may change dramatically as a result of viral and host factors: the study of mutations and protein-associated cofactors might define possible aspects relevant for the natural and therapeutic history of HBV infection.

Phase 1/1b first-in-human study of IDRX-42, a novel oral tyrosine kinase inhibitor (TKI), in patients with metastatic and/or unresectable gastrointestinal stromal tumors (GISTs).

TPS483 Background: The majority of GISTs are driven by constitutively activated kinases, either KIT or platelet-derived growth factor-alpha ( PDGFRA), and respond to TKI treatment (eg, imatinib, sunitinib, regorafenib, ripretinib). Most GISTs develop heterogeneous resistance to TKI during treatment, usually driven by secondary mutations in the primary kinase oncogene ( KIT or PDGFRA). Novel therapies are needed to prevent emergence of resistant subclones or to treat TKI-resistant GISTs. IDRX-42 (formerly M4205) is a potent, highly selective, orally administered small-molecule TKI targeting disease-specific oncogenic drivers and a range of clinically relevant resistance mutations of KIT. Nonclinical studies of IDRX-42 have demonstrated robust antitumor activity in patient and cell line-derived GIST xenograft models, including models with relevant KIT mutations and TKI resistance mutations (De Sutter et al AACR 2022). Methods: IDRX-42-001 is a phase 1/1b, first-in-human, open-label, multicenter study to evaluate single-agent IDRX-42 in participants with metastatic and/or surgically unresectable GISTs. Phase 1 dose escalation utilizes a 3+3 design to evaluate safety, tolerability, PK, and preliminary antitumor activity of IDRX-42 to establish the MTD and RP2D. Phase 1b will consist of exploratory cohorts with selected lines of prior therapy to assess the antitumor activity of IDRX-42 and further characterize its safety profile at RP2D. Eligible participants are ≥18 yr old with ECOG PS 0–1, documented pathogenic mutation in KIT or any PDGFRA mutation other than exon 18 mutations, and ≥1 measurable lesion per mRECIST v1.1 by investigator assessment. Phase 1 dose-escalation participants must have documented progression on at least imatinib. For phase 1b exploratory cohorts, cohort 1 participants must have progressed on imatinib only (2nd line); cohort 2 participants must have progressed on imatinib and sunitinib (3rd line) or progressed on imatinib, sunitinib, and an additional agent (ie, regorafenib or ripretinib; 4th line); cohort 3 participants must have progressed on imatinib, sunitinib, regorafenib, and ripretinib (≥5th line). IDRX-42 will be initiated at 120 mg PO QD in 28-d cycles until disease progression, unacceptable toxicity, or other reason. Primary endpoints are to determine the nature, incidence, & severity of IDRX-42 TEAEs and DLTs (phase 1), and to evaluate safety and determine ORR per investigator assessed mRECIST v1.1 (phase 1b). Secondary efficacy endpoints include PK parameters, DoR, PFS, and time to response related to IDRX-42. Exploratory endpoints will evaluate potential predictive and pharmacodynamic markers using ctDNA, as well as changes in KIT, PDGFRA, and other relevant gene mutant allele fractions and their correlation with clinical outcomes. Enrollment is ongoing (NCT05489237). Clinical trial information: NCT05489237 .

The Endoscopic Ultrasound Molecular Analysis of Pancreatic Cancer (EUMEPC) study.

751 Background: Pancreatic cancer carries a dismal prognosis and limited treatment options. Attempts to implement precision therapies have proved difficult to date, in part due to challenges obtaining high quality genetic material from pancreatic biopsies. Endoscopic ultrasound (EUS) is a common diagnostic procedure, but limited data is available on the clinical utility of these biopsies for comprehensive molecular profiling (CMP) to inform therapeutic choices. Methods: This study aimed to enrol up to 150 patients undergoing EUS biopsies for pancreatic cancer, in order to complete targeted DNA/RNA sequencing on 100. The primary outcome was to characterise the proportion of patients able to undergo CMP on their diagnostic EUS biopsies, with fresh-frozen biopsies preferred but FFPE specimens accepted if there was no alternative. Secondary endpoints included the proportion of patients with clinically relevant molecular findings as determined by Molecular Tumour Board (MTB) discussion, proportion of patients initiating targeted therapies, and quantitative/qualitative analysis on molecular material derived from fresh-frozen versus FFPE EUS biopsies. Results: 109 patients have been enrolled since May 2020, and molecular profiling has been completed for 102 patients. 2 patients (1.8%) were excluded due to having no available biopsies, and 5 biopsies (4.7%) were unable to be sequenced due to inadequate quality metrics. Common oncogenes were detected at roughly expected frequencies, with mutations in KRAS occurring in 91 (89.2%), TP53 in 67 (65.7%), SMAD4 in 11 (10.8%) and CDKN2A in 7 (6.8%). Median tumour mutation burden (TMB) in this cohort was low at 3.1Mut/Mb although 7 patients (6.9%) had a high TMB (defined as >10Mut/Mb), of which 3 (2.9%) had markedly hypermutated tumours (>200Mut/Mb). All processed samples were microsatellite stable. Therapeutically relevant mutations were detected in 21 (20.6%) including RNF43 in 7 patients (6.9%), KRAS G12C and BRCA in 3 patients each (2.9%), in addition to BRAF V600E in 2 (1.9%), and CHEK2 and BARD1 mutations in 1 each (0.9%). High TMB was observed in 7 patients (6.9%). To date, 5 patients (4.9%) have commenced on targeted therapies, with one patient notably experiencing a complete response to targeted therapy which has now been sustained for >12 months. Conclusions: This real-world study confirms the utility of endoscopic biopsies as a valuable and reliable source of genetic material for clinically relevant molecular tumour profiling. Secondary analyses are underway to further characterise treatment and survival implications in this cohort.

Plasticity and Stereotypic Rewiring of the Transcriptome Upon Bacterial Evolution of Antibiotic Resistance.

Bacterial evolution of antibiotic resistance frequently has deleterious side effects on microbial growth, virulence, and susceptibility to other antimicrobial agents. However, it is unclear how these trade-offs could be utilized for manipulating antibiotic resistance in the clinic, not least because the underlying molecular mechanisms are poorly understood. Using laboratory evolution, we demonstrate that clinically relevant resistance mutations in Escherichia coli constitutively rewire a large fraction of the transcriptome in a repeatable and stereotypic manner. Strikingly, lineages adapted to functionally distinct antibiotics and having no resistance mutations in common show a wide range of parallel gene expression changes that alter oxidative stress response, iron homeostasis, and the composition of the bacterial outer membrane and cell surface. These common physiological alterations are associated with changes in cell morphology and enhanced sensitivity to antimicrobial peptides. Finally, the constitutive transcriptomic changes induced by resistance mutations are largely distinct from those induced by antibiotic stresses in the wild type. This indicates a limited role for genetic assimilation of the induced antibiotic stress response during resistance evolution. Our work suggests that diverse resistance mutations converge on similar global transcriptomic states that shape genetic susceptibility to antimicrobial compounds.

Integrated Decision-Making in the Treatment of Colon-Rectal Cancer: The Case of KRAS-Mutated Tumors.

In recent years, precision medicine has taken an increasing place in various branches of medical oncology, including colorectal cancer. Among the potentially relevant mutations for this cancer is the KRAS mutation, initially defined as "untargetable"; today, we see the birth of new molecules that target one of the variants of the KRAS mutation, KRAS G12C, having a significant impact on the therapeutic options for other malignancies, such as metastatic lung cancer. This fundamental step forward has stimulated scientific research on other potential targets of KRAS, both indirect and direct, and combination treatments aiming to overcome the mechanisms of resistance to these drugs that decrease in efficacy in colorectal cancer. What was once a negative predictive marker of response to anti-EGFR drugs today has become a potential target for targeted treatments. In turn, the prognostic role of the mutation has become extremely interesting, making it a potentially useful element in therapeutic decision-making, not only regarding oncological treatments but also in a more complex and complete manner within a global vision of the patient, involving other figures on the multidisciplinary team, such as surgeons, radiotherapists, and interventional radiologists.

Mutation Testing in Evolving Systems: Studying the Relevance of Mutants to Code Evolution

Context:When software evolves, opportunities for introducing faults appear. Therefore, it is important to test the evolved program behaviors during each evolution cycle. However, while software evolves, its complexity is also evolving, introducing challenges to the testing process. To deal with this issue, testing techniques should be adapted to target the effect of the program changes instead of the entire program functionality. To this end,commit-aware mutation testing, a powerful testing technique, has been proposed. Unfortunately, commit-aware mutation testing is challenging due to the complex program semantics involved. Hence, it is pertinent to understand the characteristics, predictability, and potential of the technique.Objective:We conduct an exploratory study to investigate the properties ofcommit-relevant mutants, i.e., the test elements of commit-aware mutation testing, by proposing a general definition and an experimental approach to identify them. We thus aim at investigating the prevalence, location, and comparative advantages of commit-aware mutation testing over time (i.e., the program evolution). We also investigate the predictive power of several commit-related features in identifying and selecting commit-relevant mutants to understand the essential properties for its best-effort application case.Method:Our commit-relevant definition relies on the notion of observational slicing, approximated by higher-order mutation. Specifically, our approach utilizes the impact of mutants, effects of one mutant on another in capturing and analyzing the implicit interactions between the changed and unchanged code parts. The study analyses millions of mutants (over 10 million), 288 commits, five (5) different open-source software projects involving over 68,213 CPU days of computation and sets a ground truth where we perform our analysis.Results:Our analysis shows that commit-relevant mutants arelocated mainly outside of program commit change(81%), suggesting a limitation in previous work. We also note that effective selection of commit-relevant mutants has the potential of reducing the number of mutants by up to 93%. In addition, we demonstrate that commit relevant mutation testing is significantly more effective and efficient than state-of-the-art baselines, i.e., random mutant selection and analysis of only mutants within the program change. In our analysis of the predictive power of mutants and commit-related features (e.g., number of mutants within a change, mutant type, and commit size) in predicting commit-relevant mutants, we found that mostproxy features do not reliably predict commit-relevant mutants.Conclusion:This empirical study highlights the properties of commit-relevant mutants and demonstrates the importance of identifying and selecting commit-relevant mutants when testing evolving software systems.

Hereditary diffuse gastric cancer in a Japanese family with CDH1 mutation three case reports

BackgroundGermline pathogenic variants in the E-cadherin gene CDH1 cause hereditary diffuse gastric cancer (HDGC), which is an autosomal dominant cancer syndrome, accounting for 1–3% of all gastric cancers. HDGC harboring a CDH 1 variant is extremely rare in Japan.MethodIn this study we report the clinical courses of three cases with HDGC from a single Japanese family.ResultsThe proband exhibited advanced and metastatic gastric cancer, and was found to have a previously reported heterozygous frameshift variant in CDH1 (NM_004360.3:c.1009_1010del:p.Ser337Phefs*12). Five at-risk relatives underwent presymptomatic molecular testing after careful genetic counseling, and three were molecularly diagnosed as positive for the variant. Esophagogastroduodenoscopy was performed in these relatives revealing abnormal small pale mucosal patches, small ulcerative lesion and no abnormal findings. Moreover, random and targeted biopsies were compatible with pathological diagnosis of HDGC in the three cases, all of which underwent total prophylactic gastrectomy.ConclusionIt is critical for the assessment and management of HDGC patients to be actively offered a multidisciplinary and familial-oriented approach. Notably, genetic screening in suspected individuals and familial members is a determining piece for a higher detection rate and the identification of clinical relevant mutations in both low and high-incidence gastric cancer countries.