Cancer Panel Research Articles

CVM-1118 (foslinanib), a 2-phenyl-4-quinolone derivative, promotes apoptosis and inhibits vasculogenic mimicry via targeting TRAP1.

CVM-1118 (foslinanib) is a phosphoric ester compound selected from 2-phenyl-4-quinolone derivatives. The NCI 60 cancer panel screening showed CVM-1125, the major active metabolite of CVM-1118, to exhibit growth inhibitory and cytotoxic effects at nanomolar range. CVM-1118 possesses multiple bioactivities, including inducing cellular apoptosis, cell cycle arrest at G2/M, as well as inhibiting vasculogenic mimicry (VM) formation. The TNF receptor associated protein 1 (TRAP1) was identified as the binding target of CVM-1125 using nematic protein organization technique (NPOT) interactome analysis. Further studies demonstrated CVM-1125 reduced the protein level of TRAP1 and impeded its downstream signaling by reduction of cellular succinate levels and destabilization of HIF-1α. The pharmacogenomic biomarkers associated with CVM-1118 were also examined by Whole Genome CRISPR Knock-Out Screening. Two hits (STK11 and NF2) were confirmed with higher sensitivity to the drug in cell knock-down experiments. Biological assays indicate that the mechanism of action of CVM-1118 is via targeting TRAP1 to induce mitochondrial apoptosis, suppress tumor cell growth, and inhibit vasculogenic mimicry formation. Most importantly, the loss-of-function mutations of STK11 and NF2 are potential biomarkers of CVM-1118 which can be applied in the selection of cancer patients for CVM-1118 treatment. CVM-1118 is currently in its Phase 2a clinical development.

Comparison of genetic variation between primary colorectal cancer and metastatic peritoneal cancer.

Among cancer metastases by primary colorectal cancer (CRC), peritoneal metastasis is the second most common metastatic lesion after liver metastasis. In treating metastatic CRC, it is very important to differentiate targeted-therapy and chemotherapy according to the characteristics of each lesion because the genetic variation of the primary and metastatic lesions are different. However, there are few studies of genetic characteristics on peritoneal metastasis caused by primary CRC, so molecular-level studies are continuously required. We propose an appropriate peritoneal metastasis treatment policy by identifying the genetic characteristics between primary CRC and synchronous peritoneal metastatic lesions. Primary CRC and synchronous peritoneal metastasis samples were analyzed in pairs from six patients using Comprehensive Cancer Panel (409 cancer-related genes, Thermo Fisher Scientific, USA) and next-generation sequencing (NGS). The mutations were commonly found on the KMT2C and THBS1 genes in both primary CRC and peritoneal metastasis. The PDE4DIP gene was mutated in all cases except for on a sample of peritoneal metastasis. As a result of analysis using the mutation database, we confirmed that the gene mutations of primary CRC and the peritoneal metastasis derived from it showed the same tendency, although we did not accompany the gene expression level or epigenetic study. It is thought that the treatment policy through molecular genetic testing of primary CRC can also be applied to peritoneal metastasis treatment. Our study is expected to be the basis for further peritoneal metastasis research.

Hybrid-seq deciphers the complex transcriptional profile of the human BRCA1 DNA repair associated gene

ABSTRACT Breast Cancer Gene 1 (BRCA1) is a tumour suppressor protein that modulates multiple biological processes including genomic stability and DNA damage repair. Although the main BRCA1 protein is well characterized, further proteomics studies have already identified additional BRCA1 isoforms with lower molecular weights. However, the accurate nucleotide sequence determination of their corresponding mRNAs is still a barrier, mainly due to the increased mRNA length of BRCA1 (~5.5 kb) and the limitations of the already implemented sequencing approaches. In the present study, we designed and employed a multiplexed hybrid sequencing approach (Hybrid-seq), based on nanopore and semi-conductor sequencing, aiming to detect BRCA1 alternative transcripts in a panel of human cancer and non-cancerous cell lines. The implementation of the described Hybrid-seq approach led to the generation of highly accurate long sequencing reads that enabled the identification of a wide spectrum of BRCA1 splice variants (BRCA1 sv.7 – sv.52), thus deciphering the transcriptional landscape of the human BRCA1 gene. In addition, demultiplexing of the sequencing data unveiled the expression profile and abundance of the described BRCA1 mRNAs in breast, ovarian, prostate, colorectal, lung and brain cancer as well as in non-cancerous human cell lines. Finally, in silico analysis supports that multiple detected mRNAs harbour open reading frames, being highly expected to encode putative protein isoforms with conserved domains, thus providing new insights into the complex roles of BRCA1 in genomic stability and DNA damage repair.

Genomic profiling in advanced NSCLC in Italy: First results of NERoNE (NSCLC Emilia Romagna Next Generation Sequencing Evaluation) project.

e21077 Background: The NERoNE Project evaluates the use of Next Generation Sequencing (NGS) in molecular profiling of advanced NSCLC (aNSCLC) patients (pts) in three Oncology Units (OU) of Emilia Romagna Region aiming to clarify the impact of such extensive tool in characterizing different NSCLC subpopulations. Methods: Since January 2020 to December 2022, 970 sequential aNSCLC pts from three OU (AUSL-IRCCS of Reggio Emilia, Modena University Hospital and IRST Meldola) were profiled by NGS. Samples from Modena and Reggio Emilia were analyzed at Modena University Hospital Molecular Pathology Lab using Myriapod IL- 56G, Cancer Panel DNA, Cancer Panel RNA, Oncomine DX Target Test Assay, Oncomine Focus Assay. Samples from IRST Meldola were processed at its Molecular Diagnostic Unit by Myriapod NGS cancer panel DNA e RNA, Oncomine Focus Assay and Oncomine Comprehensive Assay v3.We considered molecular alterations with available target therapies in Italy: EGFR mutations (mut) (exon 18,19,20,21), KRAS mut, BRAF V600E mut, ALK, ROS1, RET and NTRK rearrangements, MET exon 14 skipping mut, ErbB2 mut, FGFR 1-3 mut. PDL1 expression was assessed by standard immunohistochemistry on 486 pts. Results: Among 970 pts, 501 with at least one mut (52%) were considered for analysis: 266 were female (53%), mean age was 69 ± 11 years (min-max: 33-94) and 473 pts (94%) had adenocarcinoma. Molecular features are listed in table 1. Distribution of mut among ages, categorized as < 60, 60-70 and > 70 y, was similar, EGFR activating mut were most prevalent in females; KRAS mut (G12C and non G12C) were most prevalent in males; 41 pts had co-mutations (8.2%): the most frequent co-mut were EGFR mut (27%). PDL1 expression was < 1% 129 (27%), 1-49% 211 (43,4%) and ≥50% 146 pts (30%). The majority of pts with EGFR activating mut had PDL1 < 50%. Conclusions: NERoNE evaluates data from NGS profiling of three OU mapping key actionable molecular alterations in a large population of aNSCLC also accounting for PDL1 status. This preliminary analysis will be the starting point for further evaluations in order to improve the personalized approach to aNSCLC. [Table: see text]

Caveat emptor: A descriptive analysis of guideline-recommended biomarker inclusion in targeted NGS panels for metastatic non-small cell lung cancer (mNSCLC).

3124 Background: Next generation sequencing (NGS)-based panels are routinely used in the diagnosis of mNSCLC, guidelines recommending assessing 12 genes for small variants (SV), (EGFR, BRAF, KRAS, MET, ERBB2); fusions (F), (ALK, ROS1, RET, NTRK1, NTRK2, NTRK3); and copy number variants (CNV), (MET) to assess eligibility for targeted drug therapies. Reimbursement is often limited to targeted panels; yet, a comprehensive review of targeted panels’ ability to capture all recommended biomarkers has not been reported. Methods: Targeted panels were classified as ≤50 and 51-100 genes. We searched the DEX Diagnostics Exchange Registry for NGS-based lung cancer and solid tumor targeted panels. Each panel was compared against the 12 recommended genetic variants. If the type of genetic analysis (SV/F/CNV) was unclear, labs were contacted for clarification. Fusion assays by PCR or FISH with reflex testing to NGS were included. Results: 30 targeted NGS panels from 21 commercial labs were identified; 13 (43%) were specific to lung cancer. Six of 13 panels assessing fusions used FISH or PCR not NGS alone. 12 panels reported only SV, 8 small SV/F, 5 SV/CNS, and 5 all three variant types. Only 1 of the 30 panels (51-100 genes) captured all 12 biomarkers with correct variant types. Of 20 panels reporting ALK, RET, and ROS1, only 11(55%) reported fusions. Of the 5 reporting all three variant types, NTRK fusions were most commonly absent. On average, solid tumor panels sequenced twice as many genes (51 vs. 24) but were no more likely to capture all recommended biomarkers ((1/17 (6%) vs. 0/13 (0%), (p>0.05)). The average biomarkers captured in lung vs. solid tumor panels were 7.9 and 6.6, respectively (p>0.05), while in ≤50 gene panels vs. 51-100 gene panels, 7.0 and 7.6, respectively (p>0.05). Conclusions: Of 30 commercially available NGS panels with <100 genes, only one captured all recommended gene variant types for mNSCLC. Failure to capture all biomarkers was independent of panel size or specificity to lung cancer. NGS panels including small variants, fusions, and copy number variants assessed more biomarkers. Medical oncologists and clinical pathologists ordering targeted NGS panels for the management of patients with mNSCLC must ensure they assess all biomarkers relevant to targeted therapies. Insurers reimbursing targeted panels should acknowledge the inadequacy of <100 gene panels alone to provide guideline-concordant molecular diagnostic testing. [Table: see text]

Using real-world evidence to evaluate broad panel vs. sequential single and narrow panel sequencing in members with non-small cell lung cancer (NSCLC).

e18892 Background: The clinical benefits of biomarker testing for patients with non-small cell lung cancer (NSCLC) are substantial, as increased use of appropriate targeted therapies is associated with improved survival rates. Next generation sequencing (NGS) testing has become increasingly important as it enables the identification of multiple biomarkers simultaneously and efficiently while minimizing the number of biopsies required. The purpose of this study is to assess the total cost of care (TCOC) of broad panel sequencing (BPS) ( > 5 genes) and narrow panel sequencing (NPS) in NSCLC patients. Methods: This study evaluated members with a NSCLC diagnosis undergoing biomarker testing insured by a large commercial fully-insured and Medicare health plan in the United States from January 1, 2021, to December 31, 2021. Due to the complex and inconsistent practices of billing among lab vendors, claims were analyzed and classified using supervised machine learning models to identify the correct test category from Concert Genetics (CG) test taxonomy. Test categories within CG test taxonomy were then mapped to the higher-level classifications of BPS and NPS. CG’s BPS categories included: Cell-Free Circulating Tumor DNA Lung Cancer Panel Tests, Cell-Free DNA Cancer Profiling Panel Tests (5-50 genes), Cell-Free DNA Cancer Profiling Panel Tests (≥ 51 genes), Lung Cancer Diagnostic Algorithmic Tests, Lung Cancer Panel Tests, Tumor-Type Agnostic Molecular Solid Tumor Profiling Panel Tests (≥ 51 genes), Tumor-Type Agnostic Solid Tumor Molecular Profiling Panel Tests (5-50 genes), Tumor-Type Agnostic Solid Tumor Molecular Profiling Panels Tests with IHC and Cytogenetic Analyses. Claims matched to other categories were considered NPS. No price concessions were allowed for external vendors to account for cost differences when evaluating TCOC. Results: In this study, 356 members were evaluated; members were on average 72.5 years (std = 7.5), predominately male (n = 199; 55.9%) with Medicare (n = 332; 93.3%) coverage. These members underwent BPS testing (n = 151; 42.4%) and were NCCN treatment compliant (n = 221; 62.1%). The most common comorbidities identified for these members were hyperlipidemia (n = 286; 80.3%) and hypertension (n = 281; 78.9%). After adjusting for age, gender, and insurance type, the TCOC for patients undergoing BPS testing was $1,750 more than NPS testing (p = 0.03). Conclusions: There was a difference in TCOC between BPS and NPS testing. Standardized billing processes need to be implemented to evaluate clinical outcomes to adequately compare their clinical utility and identify the influence of explanatory variables on costs.

Accuracy of a simplified sequencing and bioinformatics platform on genetic variant detection in patients with prostate cancer.

e17061 Background: Consensus guidelines suggest that genetic testing is clinically indicated for prostate cancer (PCa) patients who meet certain family history, stage and histopathological grade criteria. Current next generation sequencing (NGS) diagnostics rely on capital and maintenance intensive sequencers which require highly trained technical personnel to operate. This may restrict availability of testing where specimen transport or access to personnel/capital is limited. We sought to compare results obtained using a simpler sequencer coupled with a bioinformatics platform which requires minimal maintenance and capital cost with those from more traditional NGS equipment. Methods: We compared results in detection of 10 genes recommended by NCCN PCa guidelines (ATM, BRCA1, BRCA2, CHEK2, MLH1, MSH2, MSH6, PALB2, PSM2, and HOXB13) between the Oxford Nanopore Technologies MinION to the Illumina NextSeq sequencer. All samples were sequenced in a depth greater than 50x. Bioinformatics for the Illumina sequencer was performed using Illumina’s TruSight Hereditary Cancer panel. As the MinION reads long DNA fragments, and cancer diagnostics is focused on short exon sequencing, we used the CorrSeq Dx by Foresee Genomics to create long DNA molecules consisting of several tandem repeats of short DNA fragments separated by sequences recognized by automated bioinformatics software. Statistical analysis was performed using Cohen’s kappa test. This IRB-exempt study was conducted in accordance with the Declaration of Helsinki. Results: We detected using 380 variants in 13 patients on the Illumina platform and 379 (3 false negatives, 2 false positives) variants on the same patients using the MinION sequencer with CorrSeq Dx assay. The positive predictive value and sensitivity for the simplified platform was 99.5% (375/(375+2)) and 99.2% (375/(375+3)), respectively. The demonstrated accuracy of the simplified process was 99.9% (Cohen's kappa > 0.99). Conclusions: Thisl data suggests that a simpler sequencing solution with appropriate bioinformatics could allow clinical laboratories with limited technical and capital resources to perform germline and possibly somatic testing in-house; use of this technology may also serve to reduce costs for labs currently performing these tests. While some labs or clinics can outsource their NGS diagnostics if such commercial options are available, these options may not be possible where transport of specimens is logistically difficult or if access to technology is limited. A simpler process for NGS testing could improve access to guideline-based care in both remote and economically disadvantaged areas, resulting in better treatment outcomes as well as increased global healthcare equity. Further testing of this technology in prostate cancer as well as other disease states is warranted to fully understand its potential impact on patient care.

DelPHI: A novel program to accelerate access to precision oncology in the community oncology setting.

e18713 Background: It has been well recognized that the rapid advances in precision oncology have not fully been translated into improved access for community cancer programs in the USA, where as much as 95% of oncological assessment and treatment occurs. To address this growing gap, Protean BioDiagnostics and Roche Information Solutions have created a novel project to improve the Delivery of Precision Health Information (DelPHI) to community-based oncology programs using an innovative precision oncology testing system (MAPS™) integrated with a digital decision support and telemedicine framework (navify). The project seeks to bridge the clinical and administrative gaps to help bring precision oncology to the community. Methods: In an initial implementation of DelPHI, 28 cases were analyzed from a community oncology clinic. Analyses included immunohistochemistry (IHC) testing, rapid molecular testing, and a 523-gene DNA, 55-gene RNA comprehensive cancer panel. These cases were grouped together and queried for IA actionability alterations, potentially actionable alterations, potentially germline, pathogenic variants, time to each step, diagnosis changes, and noted if there were any inadequate case materials. Results: Based on the 28 patients that were run (13 lung, 4 breast, 1 prostate, 2 colorectal, 2 gastric, 1 esophagus, 1 melanoma, 2 bladder, 1 glioma, 1 poorly differentiated carcinoma), 50 total actionable alterations were detected. 90% of the patients had at least one potentially actionable alteration, 46% of patients had a IA actionability alteration, and 29% of patients had a potentially pathogenic germline variant that may warrant further testing. We identified 2/28 patients with incorrect diagnoses that were corrected after testing. 23/28 patients underwent testing via the large, comprehensive panel, and 16 of these 23 had additional targets. 3 individuals had inadequate tissue to analyze, and 1 patient had an ALK expression imbalance that was not seen in other tests, which may be due to heterogeneity of the sample; both of these issues may benefit from liquid biopsies. Conclusions: Precision medicine can be delivered to community cancer centers using a combined integrated molecular testing system coupled with a digital knowledge support framework leading to improved speed of patient testing and uncovering of actionable biomarkers. Numerous challenges were identified, including delays in accessing tissue and limited tissue availability. These challenges could be overcome with alternative testing approaches, such as liquid biopsy and education to improve strategies for tissue collection and management.

Validation of a breast cancer polygenic risk score in 15,490 Brazilians using exome sequencing.

10631 Background: Brazil has a highly admixed population with significant Southern European, African and Native American genetic ancestries. Approximately 75% of the population has two or more continental ancestries. Therefore, breast cancer polygenic risk scores (PRS) developed in predominantly European populations requires validation in the Brazilian population. Compared with genotyping arrays and whole genome sequencing, affordable and scalable whole exome sequencing (WES) allows genotyping of both rare and common variants in the targeted regions and imputation of common variants not directly captured. In this study we validate a previously developed 3820-variant PRS using WES in 15,490 Brazilians. Methods: The cohort consisted of 6,362 women with a history of breast cancer (mean age 49.6, stddev 11.6) and 9,128 unphenotyped adults as controls (mean age 41.6, stddev 13.3). WES were performed on germline DNA and variants were called and curated. Imputation was done using the 1000 Genomes Phase 3 reference panel for variants falling outside the WES captured regions. After excluding subjects with known pathogenic or likely pathogenic variants in BRCA1, BRCA2, PALB2, PTEN, or TP53, and first or second-degree relatives of the probands, 14,577 individuals remained in the final analysis (5,730 cases and 8,847 controls). We restricted PRS calculation to 2,893 of the original 3,820 variants with an estimated global imputation accuracy of 96%. Odds ratios (OR) were adjusted using the top 10 principal components. Results: The breast cancer PRS showed a highly significant correlation with cancer risk (p-value = 7.1e-81, OR = 1.43, 95% confidence interval (CI) = 1.38-1.48 per PRS standard deviation). Compared with women in the middle deciles, those in the highest 10% had an OR of 1.93, while those in the lowest 10% had an OR of 0.52 (Table). Conclusions: In this study we show that WES can capture 76% of the variants included in a previously developed and validated breast cancer PRS, and the calculated PRS is correlated with breast cancer risk in the admixed Brazilian population. This opens the door for clinical studies into the application of PRS in Brazil, particularly in women without mutations in breast cancer high-penetrance genes. In addition, our WES-based PRS strategy can be quickly, affordably, and seamlessly adopted by labs that already offer Hereditary Breast Cancer panels that use WES as a backbone. [Table: see text]

Utilization of germline testing in patients with early-onset pancreatic cancer (EOPC).

e16300 Background: EOPC is associated with significant life years lost. Approximately 5%-10% of pancreatic adenocarcinoma (PAC) have an underlying genetic predisposition and national guidelines recommend testing in all patients with PAC. Certain germline mutations such as BRCA2 and PALB2 predict response to specific treatments including platinum and targeted therapies. It is currently unknown to what extent patients with EOPC pursue recommended germline testing. We examined the utilization of germline testing in patients with EOPC who were referred to genetics. Methods: A retrospective review of patients with EOPC at the Levine Cancer Institute (LCI), defined as ≤60 years of age at time of diagnosis, who were referred and seen by genetics between January 2019 and December 2021 was performed. Germline testing was performed using multi-gene cancer panels. Positive germline testing was considered as any known pathogenic or likely pathogenic variant not including variants of unknown significance (VUS). Results: We identified 69 patients with EOPC who were evaluated by genetics. The median age was 55 years (range 25-60) with 36 (52%) male, 33 (48%) female, 48 (70%) white, 16 (23%) Black, 61 (89%) with ECOG 0-1, 36 (52%) current or prior smoking history, and 28 (42%) presenting with metastatic disease. As shown, out of 69 patients, 10 (14%) declined testing or had unknown results, 30 (43%) had negative germline testing, 29 (42%) had genetic testing that revealed 19 (65%) had VUS, and 10 (34%) had pathogenic variants. The most frequently seen pathogenic variants were ATM (30%), BRCA1 (30%), BRCA2 (10%), MLH1 (10%), RAD51C+ (10%) and CFTR (10%). Conclusions: Our data suggest that most patients with EOPC who are evaluated undertake germline testing. Given the predictive nature of certain pathogenic mutations on PAC treatment options, characteristics of patients with EOPC who decline testing should be further explored. There is need for larger studies to confirm the high incidence of pathogenic variants observed in our cohort of EOPC patients. [Table: see text][Table: see text]

IDENTIFICATION OF GENOMIC BIOMARKERS OF DISEASE PROGRESSION AND SURVIVAL IN NEWLY‐DIAGNOSED PRIMARY CNS LYMPHOMA

Introduction: There is a significant unmet need for robust genomic biomarkers that identify patients with newly-diagnosed primary CNS lymphoma (PCNSL) at high risk of progression, both during standard methotrexate-based induction as well as after dose-intensive consolidation. Methods: We performed targeted next-generation sequencing of 529 cancer genes of formalin-fixed, paraffin-embedded tumors from 74 patients with newly diagnosed primary CNS diffuse large B-cell lymphoma using the UCSF500 Cancer Panel and the Institute for Human Genetics CLIA Laboratory. The assay detects structural variants including single nucleotide variants, small insertions and deletions, and copy-number alterations (CNAs). Sixty-four EBV-negative PCNSL patients treated with a standard methotrexate, temozolomide, rituximab (MTR) induction were available for complete analysis. Results: We identified frequent mutations involving MYD88 (77%), CD79B (41.9%), ETV6 (27%) and BTG1 (25.7%) as well as frequent CNA involving 6q loss (62.5%), 9p loss (34.4%) and 12 gain (31.3%). Three genomic alterations were most significantly associated with early disease progression in PCNSL: (1) Alterations at 6p21.3, predominantly copy neutral loss of heterozygosity (CN-LOH) at the HLA Class I (HLA-A, B, C) and Class II loci (HLA-DR, DP, DQ, TAP1) as well as homozygous deletion of HLA Class II; (2) Mutations of tumor suppressor genes BTG1 and ETV6. Overall, these genomic alterations were detected in 28 out of 31 progression events in the cohort. The subgroup with either CN-LOH or homozygous deletion at 6p had the earliest progression, followed by the subgroup with mutations at either BTG1/ETV6 and no 6p CN-LOH or homozygous deletion, compared to no alterations at these 3 loci. 10 of 18 tumors with these alterations at 6p also contained BTG1 and/or ETV6 mutations. Survival analysis demonstrates that the subgroup with 6p CN-LOH or homozygous deletion had the earliest progression, followed by the subgroup with BTG1/ETV6 mutations compared to no alterations at these 3 loci (PFS p = 3.7e-5, OS p = 0.0017, logrank test, Figure 1). Multivariate Cox proportional hazard analysis demonstrated a significant 3.24-fold increased risk of progression with 6p CN-LOH/homozygous deletion or BTG1/ETV6 mutations (p = 3.5e-5, adjusted by Age, KPS, IELSG, MSKCC, IOL, Maintenance, and Consolidation). The research was funded by: National Cancer Institute NIH R01CA139-83-01A1 Keywords: aggressive B-cell non-Hodgkin lymphoma, diagnostic and prognostic biomarkers, genomics, epigenomics, and other-omics No conflicts of interests pertinent to the abstract.

Comprehensive analysis of patients with cancer with a positive CHEK2 test: Are we getting closer to a genotype/phenotype correlation?

10588 Background: The CHEK2 gene encodes a protein kinase that acts as a tumor suppressor with an important role in DNA damage repair. This gene is included in most hereditary breast, ovarian and prostate cancer panels, but the clinical actionability of CHEK2 pathogenic/likely pathogenic variants (PVs) may be challenging. Indeed, decisions concerning clinical surveillance and/or risk reducing procedures as well as screening for multiple associated cancer risks are not consensual. Our objective was to identify a potential genotype/phenotype association regarding CHEK2 PV identified in our population. Methods: Study group included all patients (pts) who tested positive for CHEK2 PVs between July 2000 and June 2022; control group included pts without PVs after multigene panel testing (wt) between January 2020 and June 2022. PVs were categorized as: truncating (includes nonsense, frameshift, splice site variants, gross deletions and duplications) and not truncating (missense and in-frame deletions). Clinical characteristics from both groups included gender, personal and family cancer history and tumor characteristics. Results: From 8604 pts tested, we identified 101 CHEK2 and 1506 wt pts. CHEK2 pts were mainly women (95; 92%) and Portuguese (94; 93.1%). All non-Portuguese pts had Eastern Europe ancestry. CHEK2 pts were either heterozygous (94;93%), homozygous (3; 3%) or had digenic inheritance (4; 6%: both a CHEK2 and a BRCA1/2 PV). Compared to wt pts, heterozygous CHEK2 pts were younger at 1st cancer diagnosis (44.5 yrs vs 47 yrs) and had more multiple primary cancers (OR: 1.8; p: 0.02). CHEK2 breast cancer was mostly but not exclusively estrogen receptor positive (69/78; 88,4%) and Her2 negative (54/69; 78%). Two BC cases had triple negative phenotype. The most frequent PVs detected were c.349A > G (25 pts), c.1100del (24 pts) and c.319+2T > A (20 pts). Considering heterozygous PVs, 57 were truncating and 37 non-truncating, with c. 1100del and c.349A > G being the most frequent, in each group, respectively. For truncating versus non-truncating PVs there was no difference in phenotype regarding breast (OR: 0.69; p: 0.15), CRC (OR: 0.94; p: 0.91), gastric (OR: 0.76; p: 0.65), thyroid (OR: 1.3; p: 0,69) and kidney cancer (OR: 0.36; p: 0.27). Further stratification of PVs according to cancer type revealed a correlation of CRC with c.1100del (OR: 4.57; p: 0.0025) and c.349A > G (OR: 5.49; p: 0.0003), while gastric, kidney and thyroid cancers correlated with c.349A > G (OR: 5.8; p: 0.0069), c.1100del (OR: 26.86; p: 0.02) and c.319+2T > A (OR: 3.31; p: 0.05) PVs, respectively. Conclusions: This analysis suggests a potential genotype-phenotype correlation regarding CHEK2 pathogenic variants, regardless of its truncating potential. The study of clinical outcomes will further contribute to determine how this information may influence the clinical and risk management of CHEK2 pts and their families.

BRCA1 VUS: A functional analysis to differentiate pathogenic from benign variants identified in clinical diagnostic panels for breast cancer.

Genetic testing for susceptibility genes through next‑generation sequencing (NGS) has become a widely used technique. Using this, a number of genetic variants have been identified, several of which are variants of unknown significance (VUS). These VUS can either be pathogenic or benign. However, since their biological effect remains unclear, functional assays are required to classify their functional nature. As the use of NGS becomes more mainstream as a diagnostic tool in clinical practice, the number of VUS is expected to increase. This necessitates their biological and functional classification. In the present study, a VUS was identified in the BRCA1 gene (NM_007294.3:c.1067A>G) in two women at risk for breast cancer, for which no functional data has been reported. Therefore, peripheral lymphocytes were isolated from the two women and also from two women without the VUS. DNA from all samples were sequenced by NGS of a breast cancer clinical panel. Since the BRCA1 gene is involved in DNA repair and apoptosis, the functional assays chromosomal aberrations, cytokinesis‑blocked micronucleus, comet, γH2AX, caspase and TUNEL assays were then conducted on these lymphocytes after a genotoxic challenge by ionizing radiation or doxorubicin to assess the functional role of this VUS. The micronucleus and TUNEL assays revealed a lower degree of DNA induced‑damage in the VUS group compared with those without the VUS. The other assays showed no significant differences between the groups. These results suggested that this BRCA1 VUS is likely benign, since the VUS carriers were apparently protected from deleterious chromosomal rearrangements, subsequent genomic instability and activation of apoptosis.

Identification of Genomic Alterations in Thai Patients With Colorectal Cancer Using Next-Generation Sequencing-Based Multigene Cancer Panel.

Introduction Colorectal cancer (CRC) is one of the leading causes of death and illness in the general population. Although the incidence of CRC is steadily decreasing worldwide, it is being diagnosed more in individuals under 50 years of age. Multiple disease-causing variants have been reported to be involved in the development of CRC. This study aimed to investigate the molecular and clinical characteristics of Thai patients with CRC. Methods NGS-based multigene cancer panel testing was performed on 21 unrelated patients. Target enrichment was performed using a custom-designed Ion AmpliSeq on-demand panel. Thirty-six genes associated with CRC and other cancer were analyzed for variant detection. Results Sixteen variants (five nonsense, eight missense, two deletions, and one duplication) in nine genes were identified in 12 patients. Eight (66.7%) patients harboring disease-causing deleterious variants in genes APC, ATM, BRCA2, MSH2, and MUTYH. One of the eight patients also carried additional heterozygous variants in genes ATM, BMPR1A, and MUTYH. In addition, four patients carried variants of uncertain significance in genes APC, MLH1, MSH2, STK11, and TP53. Among all detected genes, APC was the most frequent causative gene observed in CRC patients, which is consistent with previous reports. Conclusion This study demonstrated the comprehensive molecular and clinical characterization of CRC patients. These findings showed the benefits of using multigene cancer panel sequencing for pathogenic gene detection and showed the prevalence of genetic aberrations in Thai patients with CRC.

Comprehensive analysis to construct a novel immune-related prognostic panel in aging-related gastric cancer based on the lncRNA‒miRNA-mRNA ceRNA network.

Introduction: Gastric cancer (GC) is the fifth frequent malignancy and is responsible for the third leading cause of cancer-related deaths. Gastric cancer is an aging-related disease, with incidence and mortality rates increasing with aging. The development of GC is affected by lncRNAs, miRNAs, and mRNAs at the transcriptional and posttranscriptional levels. This study aimed to establish a prognostic panel for GC based on competing endogenous RNA (ceRNA) networks. Methods: RNA sequences were obtained from the TCGA database. Different expressions of RNAs were scrutinized with the EdgeR package. The ceRNA network was built using the starBase database and the Cytoscape. The prognostic panel was constituted with the LASSO algorithm. We developed a nomogram comprising clinical characteristic and risk score. The receiver operating characteristic (ROC) was used to evaluate the accuracy of the nomogram prediction. Hub RNAs expressions were detected by qPCR, immunohistochemistry and western blot respectively. Clinical relevance and survival analyses were analyzed. The relationship between RNAs and immune infiltrations, as well as immune checkpoints, was analyzed and evaluated using the CIBERSORT, TIMER and TISIDB databases. Results: Four DElncRNAs, 21 DEmiRNAs and 45 DEmRNAs were included in the ceRNA network. A 3-element panel (comprising lncRNA PVT1, hsa-miR-130a-3p and RECK) with poor overall survival (OS) was established and qPCR was applied to validate the expressions of hub RNAs. Hub RNAs were firmly associated with T, M, and N stage. The CIBERSORT database showed that the high lassoScore group exhibited a significantly high ratio of resting memory CD4+ T cells, M2 macrophages and a significantly low ratio of activated memory CD4+ T cells and M1 macrophages. According to the TIMER database, this panel was linked to immune infiltrations and immune cell gene markers. TISIDB database indicated that RECK was positively correlated with immune checkpoints (including CD160, CD244, PDCD1, and TGFBR1). Discussion: A novel triple prognostic panel of GC constructed based on the ceRNA network was associated with clinical prognostic, clinicopathological features, immune infiltrations, immune checkpoints and immune gene markers. This panel might provide potential therapeutic targets for GC and more experimental verification research is needed.

Benzimidazole carbamate induces cytotoxicity in breast cancer cells via two distinct cell death mechanisms

Metastatic breast cancer (mBC) is responsible for >90% of breast cancer-related deaths. Microtubule-targeting agents (MTAs) are the front-line treatment for mBC. However, the effectiveness of MTAs is frequently limited by the primary or acquired resistance. Furthermore, recurrent mBC derived from cancer cells that survived MTA treatment are typically more chemoresistant. The overall response rates for the second- and third-line MTAs in mBC patients previously treated with MTAs are 12–35%. Thus, there is an ongoing search for novel MTAs with a distinct mode of action that can circumvent chemoresistance mechanisms. Our results show that methyl N-(6-benzoyl-1H-benzimidazol-2-yl)carbamate (BCar), a microtubule-disrupting anthelmintic that binds to the colchicine binding site separate from the binding sites of clinically used MTAs, has the potential to treat MTA-resistant mBC. We have comprehensively evaluated the cellular effects of BCar in a panel of human breast cancer (BC) cell lines and normal breast cells. BCar effects on the clonogenic survival, cell cycle, apoptosis, autophagy, senescence, and mitotic catastrophe were measured. Approximately 25% of BCs harbor mutant p53. For this reason, the p53 status was included as a variable. The results show that BC cells are >10x more sensitive to BCar than normal mammary epithelial cells (HME). p53-mutant BC cells are significantly more sensitive to BCar treatment than p53 wild-type BC cells. Furthermore, BCar appears to kill BC cells primarily via either p53-dependent apoptosis or p53-independent mitotic catastrophe. When compared to docetaxel and vincristine, two clinical MTAs, BCar is fairly innocuous in HME cells, providing a much wider therapeutic window than docetaxel and vincristine. Together, the results strongly support the notion that BCar-based therapeutics may serve as a new line of MTAs for mBC treatment.

Cancer gene analysis of liquid-based cytology specimens using next-generation sequencing: A technical report of bimodal DNA- and RNA-based panel application.

As liquid-based cytology (LBC) specimens harbor high-quality DNA, genomic analysis using LBC specimens is beneficial for integrative diagnosis. This study aimed to clarify the feasibility of LBC specimens for a bimodal application of DNA- and RNA-based next-generation sequencing (NGS) panels. LBC specimens were prepared from cultured human cancer HEC59 cells using commercially available fixatives (Cellprep, CytoRich Red, and SurePath solutions), and were subjected to NGS for a feasibility study. Clinical LBC specimens of thyroid and salivary gland tumors were prepared using CytoRich Red solution. After DNA and RNA extraction, NGS analyses were performed in a single run using combined DNA- and RNA-based custom-made cancer panels for the detection of gene mutations and fusions. High-quality DNA and RNA were obtained, and the expected gene mutations and fusions were detected in HEC59 cells using all types of LBC fixatives. Most available clinical cases (18 out of 20) exhibited pathogenic gene mutations (15 cases) and fusion genes (3 cases) using the bimodal DNA- and RNA-based panels. Overall, 18 cases (90%) showed oncogenic mutations or fusion genes of diagnostic values. Simultaneous application of bimodal DNA- and RNA-based gene panels was useful in NGS analysis using residual LBC specimens for integrative diagnosis. Residual LBC specimens for genomic analysis, including fusion gene analysis, are particularly useful for obtaining genomic information before surgical resection.

Abstract #1412835: A Case of Cowden Syndrome

Cowden syndrome is part of the PTEN Hamartoma Tumor Syndrome spectrum. It is characterized by the presence of multiple hamartomas on the skin and mucous membranes and an increased risk of developing cancers of the thyroid, breast, uterus, kidney, colon, rectum and skin. Most cases are caused by genetic changes in the phosphatase and tensin homolog (PTEN) gene, inherited in an autosomal dominant manner. It is rare with an estimated prevalence of 1 in 200,000 to 250,000. A 36-year-old female presented to the Veteran Affairs Endocrinology clinic for follow up of thyroid cancer. She had undergone a total thyroidectomy 9 years prior due to a large goiter, with pathology revealing papillary thyroid cancer. She received radioactive iodine ablation afterwards. Thyroid ultrasound showed a heterogenous soft tissue density measuring 1.8 x 1.1 x 1.6 cm in the left thyroid bed. Fine needle biopsy of the mass confirmed recurrent papillary thyroid cancer. Her medical history includes endometriosis with right ovary cyst for which she underwent total hysterectomy with bilateral salpingo-oophorectomy, benign colon polyps status post resection, gallbladder polyps status post cholecystectomy, bilateral breast masses (measuring 0.5 cm on the right and 0.8 cm on the left) and multiple skin lesions. Family history is significant for multiple benign skin lesions and colon polyps in her father and breast cancer and endometriosis in her paternal aunts. Due to her extensive personal and family history of tumors, a comprehensive hereditary cancer panel was sent out. Results came back positive for the PTEN gene, indicating a diagnosis of Cowden syndrome. She was referred to oncology for cancer surveillance, dermatology for regular skin checks and surgery for prophylactic mastectomy. She does not plan to have children, and she was advised to notify her first-degree family to have genetic testing done. Patients with Cowden syndrome may present to the Endocrinology clinic for management of non-medullary thyroid cancer, multinodular goiter or Hashimoto’s thyroiditis and may be noted to have multiple skin and mucous membrane lesions. They may report a history of breast tumors, endometrial abnormalities and polyps as in this case. After diagnosis is confirmed by genetic testing, cancer surveillance is the major focus of medical management given the high risk of malignancy.

Early Detection of Prostate Cancer: AUA/SUO Guideline Part I: Prostate Cancer Screening.

The summary presented herein covers recommendations on the early detection of prostate cancer and provides a framework to facilitate clinical decision-making in the implementation of prostate cancer screening, biopsy, and follow-up. This is Part I of a two-part series that focuses on prostate cancer screening. Please refer to Part II for discussion of initial and repeat biopsies as well as biopsy technique. The systematic review utilized to inform this guideline was conducted by an independent methodological consultant. The systematic review was based on searches in Ovid MEDLINE and Embase and Cochrane Database of Systematic Reviews (January 1, 2000-November 21, 2022). Searches were supplemented by reviewing reference lists of relevant articles. The Early Detection of Prostate Cancer Panel developed evidence- and consensus-based guideline statements to provide guidance in prostate cancer screening, initial and repeat biopsy, and biopsy technique. Prostate-specific antigen (PSA)-based prostate cancer screening in combination with shared decision-making (SDM) is recommended. Current data regarding risk from population-based cohorts provide a basis for longer screening intervals and tailored screening, and the use of available online risk calculators is encouraged.

Updates to Advanced Prostate Cancer: AUA/SUO Guideline (2023).

In 2022 the American Urological Association (AUA) requested an Update Literature Review (ULR) to incorporate new evidence generated since the 2020 publication of this guideline. The resulting 2023 Guideline Amendment addresses updated recommendations for patients with advanced prostate cancer. The ULR addressed 23 of the original 38 guideline statements and included an abstract-level review of eligible studies published since the 2020 systematic review. Sixteen studies were selected for full text review. The current summary presents the updates made to the Guideline as a result of that new literature. The Advanced Prostate Cancer Panel amended evidence- and consensus-based statements based on an updated review to aid clinicians in the management of patients with advanced prostate cancer. These statements are detailed herein. This Guideline Amendment provides a framework designed to improve a clinician's ability to treat patients diagnosed with advanced prostate cancer with the most current evidence-based information. Further research and publication of high-quality clinical trials will be essential to continue to improve the quality of care for these patients.