e17548 Background: Clinically actionable variants (CAVs) in BRCA1 and BRCA2 determine eligibility for targeted therapy in high grade serous ovarian cancer (HGSOC). However, RNA based biomarkers indicating BRCA status remain limited. Circular RNAs (circRNAs) are non coding RNAs with regulatory roles in gene expression. Their stability and detectability in human biofluids make circRNAs attractive liquid biopsy candidates. This study examined whether circRNA expression varies by BRCA background and assessed translational potential. Methods: This was a translational biomarker study examining circRNA expression across distinct BRCA backgrounds in high grade serous ovarian cancer. Three HGSOC cell lines representing BRCA1 mutant, BRCA2 mutant, and BRCA wildtype backgrounds were analysed. The primary endpoint was identification of circRNAs differentially expressed according to BRCA status. circRNA profiling was performed using the Arraystar circRNA microarray platform. Differential expression was assessed using normalised signal intensity, a fold change threshold ≥2, and associated statistical outputs, with adjustment for multiple testing. Candidate circRNAs were prioritised based on fold change, reproducibility, and annotation quality. Selected circRNAs were validated using divergent primers and quantitative PCR across the HGSOC panel, a chemotherapy resistant model, and clinical tumour samples stratified by BRCA status. Results: Heatmaps and volcano plots showed circRNA profiles were driven mainly by cell line specific patterns. However, at the individual transcript level, distinct circRNAs demonstrated BRCA associated differences. Five circRNAs were prioritised for validation. Three circRNAs (hsa_circ_400223, hsa_circ_008026, and hsa_circ_003300) were upregulated in both BRCA1 and BRCA2 mutant models compared with wildtype. hsa_circ_100059 was predominantly expressed in the BRCA1 mutant cell line, while hsa_circ_025636 was highly expressed in the BRCA2 mutant cell line. In clinical samples, hsa_circ_025636, a RASSF8 derived circRNA, showed higher expression in a subset of BRCA mutant cases relative to wildtype (p<0.05). Conclusions: This study provides evidence that circRNAs may serve as biomarkers of BRCA status in HGSOC. While global circRNA profiles were not defined by BRCA genotype, specific circRNAs demonstrated BRCA associated expression. The RASSF8 derived circRNA hsa_circ_025636 is a promising candidate and warrants evaluation in larger cohorts alongside functional studies.

Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionized the management of BRCA1- and BRCA2-associated ovarian cancer (OC). In 2020, Ireland implemented a nationwide, oncology-led pathway for mainstreamed BRCA1/2 testing in patients with OC. This study evaluated the pathway and characterised the BRCA1/2 landscape in an Irish cohort of patients with OC. Samples collected between January 2020 and December 2023 were tested in two national molecular diagnostic laboratories. Single-molecule molecular inversion probe sequencing was used to detect single nucleotide variants, while multiplex ligation-dependent probe amplification assessed large genomic rearrangements. Variants were classified according to the American College of Medical Genetics and Genomics and Association for Clinical Genomic Science 2020 guidelines and CanVIG-UK specifications. In total, 535 patients were included, with a median age of 65.5 years (range, 27-89 years). Of these, 455/535 (85.0%) underwent germline BRCA1/2 (gBRCA) testing using peripheral blood, and 360/535 (67.2%) underwent tumour BRCA1/2 (tBRCA) testing, resulting in 292/535 (54.6%) patients having paired testing. Among gBRCA-tested patients, 10.3% (47/455) had a clinically actionable variant (CAV), while 2.1% (10/455) had a variant of uncertain significance. Of the 262 patients who underwent paired testing and had negative gBRCA results, 9.5% (25/262) had a somatic BRCA CAV. Recurrent germline BRCA CAVs were identified in regional clusters, including BRCA1 c.5266dup, BRCA1 c.1175_1214del, and BRCA2 c.4398_4402del. This nationwide real-world study demonstrates that mainstreamed germline and somatic BRCA1/2 testing is feasible in Ireland and reports the prevalence of BRCA CAVs in a cohort of patients with OC. Although the observed prevalence of germline BRCA CAVs was lower than anticipated, it is consistent with UK real-world data. The identification of recurrent, regionally clustered germline variants further enhances the characterisation of the Irish genomic landscape.

e17558 Background: Most patients with high grade serous ovarian cancer (HGSOC) ultimately relapse with chemotherapy resistant disease, and resistance to Poly (ADP ribose) polymerase inhibitors (PARPi) is increasingly recognised. The mechanisms underlying inherent and acquired resistance remain poorly understood, and no validated biomarkers exist to guide treatment beyond BRCA status. circularRNAs (circRNAs) are stable non-coding RNAs with regulatory roles in gene expression and may capture transcriptional reprogramming associated with treatment resistance. This study examined circRNA expression in chemotherapy and PARPi resistant ovarian cancer (OC) models to identify candidate biomarkers of resistance. Methods: Chemotherapy (carboplatin and paclitaxel) and PARPi resistant (olaparib) COV362 (BRCA1 mutant) sublines were generated using progressive dose escalation. Resistance was confirmed using CCK-8 and BrdU proliferation assays. This was a preclinical, exploratory biomarker discovery study. The primary endpoint was the identification of circRNAs differentially expressed between parental (drug-sensitive), chemotherapy-resistant and PARPi-resistant cell lines. circRNA expression profiling was performed using the Arraystar circRNA microarray platform. Differential expression was assessed using normalised signal intensity, fold-change thresholds and associated statistical outputs, with adjustment for multiple testing. Candidate circRNAs were prioritised based on magnitude of differential expression and annotation quality, and were validated using divergent primers and quantitative PCR. Results: Chemotherapy and PARPi resistant COV362 sublines were successfully established over 10 and 14 months, respectively. circRNA profiling revealed distinct segregation of parental (drug sensitive), chemotherapy and PARPi resistant cell lines using hierarchical clustering. Although global circRNA patterns remained broadly conserved, each model showed a distinct set of upregulated transcripts. At the individual transcript level, however, a subset of circRNAs demonstrated resistance-associated differences and were prioritised for further evaluation. hsa_circ_0085803 (p <0.05) was upregulated in the chemotherapy resistant model, whereas hsa_circ_0003258 (p <0.05) was predominately expressed in the PARPi resistant mode compared to matched parental cell line. Conclusions: Distinct circRNA signatures were associated with chemotherapy and PARPi resistance in COV362, with hsa_circ_0085803 and hsa_circ_0003258 identified as candidate biomarkers of resistance. These findings support the potential of circRNAs as indicators of treatment resistance in HGSOC.

Supplementary Table 1. All validation results and evaluation of intratumoral heterogeneity.

BACKGROUND:We report a pilot clinical trial investigating the feasibility of liquid biopsy genotyping (LBG) at the time of radiological suspicion of advanced NSCLC, incorporating a micro-cost model (MCM). (PLAsma Genomic Testing in Advanced NSCLC; The PLAN trial, ClinicalTrials.gov Identifier: NCT05542485). METHODS:Patients with a radiologic suspicion of stage III-IV lung cancer were recruited from four cancer centres in Ireland between August 2023 and July 2024. LBG was performed using the Archer LiquidplexTM NGS assay. The MCM considered staff time, consumables and capital costs and savings from avoidance of repeat tissue biopsy genotyping (TBG) or inappropriate systemic therapy such as immunotherapy for EGFR + NSCLC. RESULTS:A total of 138 patients were enrolled in the study with 38 excluded from the primary analysis (Squamous=16; SCLC = 22). Of patients that were eligible, LBG was completed in 100 % (100/100). TBG was completed in 68 % (68/100; insufficient tissue 20 %; 20/100; declining ECOG PS 12 %; 12/100). Repeat tissue biopsies were avoided in 12 % (12/100) of patients due to available LBG reports. The median calendar days from LBG to receipt of genomic report was 21 days shorter for LBG (z = -6.8, p < 0.01) versus TBG, as a median (range: 1-104 days). For evaluable paired cases with both TBG and LBG available (n = 68), concordance was 90 % (61/68). LBG resulted in detection of 5 actionable variants. LBG (€1135) was less than half the cost of TBG (€2404). LBG also resulted in overall cost savings of €20,288 (reduced TBG; use of immunotherapy). CONCLUSIONS:LBG reduces the time to genomic diagnosis in patients with newly diagnosed NSCLC compared to tissue genotyping, identifies actionable variants not reported in tissue, and results in overall cost savings.

Purpose HPV driven oropharyngeal squamous cell carcinoma (OPSCC) proffers a more favorable prognosis in contrast to its HPV negative counterparts. Consequently, studies have analyzed de-intensification measures of current treatment practices to minimize the associated morbidity. However, 25% of patients with HPV positive OPSCC experience a locoregional recurrence (LR) and/or distant metastasis (DM). This study aimed to determine if patient demographics, tumor characteristics and specific HPV genotyping were attributable to HPV positive OPSCC who develop LR and/or DM based on data collated from the largest Head and Neck Cancer center in the Republic of Ireland, with a mean follow up period of 11.16 years. Methods Patient demographics was obtained from HPV positive OPSCC patients from 3 Dublin Hospitals diagnosed over a 10-year period. In addition, HPV genotyping via Ion Torrent Next Generation Sequencing (NGS) and mutations analysis using smMIP panel based NGS was conducted on the cohort archival tumor DNA. Results 67 patients met the criteria for inclusion and had a mean follow-up period of 11.16 years. 28.36% developed LR and/or DM. A statistically significant correlation was identified linking age at diagnosis (p = 0.047), excessive alcohol consumption (p = 0.017) and a higher clinical stage (p = 0.01) with LR and/or DM.HPV 16 was the most prevalent genotype (93.65%), with dual HPV infection illustrated in 4 cases. BRAF, EGFR, ERBB2, KIT, KRAS, NRAS, PDGFRA somatic mutations were not identified in the recurrence cohort. Conclusion With the ongoing focus to reduce treatment for patient with HPV positive OPSCC, upfront recognition of patients at greater risk of LR and/or DM is essential. In the era of personalized medicine, it is hoped that interrogation of archival tumor tissue via similar techniques used in this study may yield pioneering findings with a clinical and prognostic significance.

BACKGROUND:ROS1 fusion is a relatively low prevalence (0.6-2.0%) but targetable driver in lung adenocarcinoma (LUAD). Robust and low-cost tests, such as immunohistochemistry (IHC), are desirable to screen for patients potentially harboring this fusion. The aim was to investigate the prevalence of ROS1 fusions in a clinically annotated European stage I-III LUAD cohort using IHC screening with the in vitro diagnostics (IVD)-marked clone SP384, followed by confirmatory molecular analysis in pre-defined subsets. METHODS:Resected LUADs constructed in tissue microarrays, were immunostained for ROS1 expression using SP384 clone in a ready-to-use kit and Ventana immunostainers. After external quality control, analysis was performed by trained pathologists. Staining intensity of at least 2+ (any percentage of tumor cells) was considered IHC positive (ROS1 IHC + ). Subsequently, ROS1 IHC + cases were 1:1:1 matched with IHC0 and IHC1 + cases and subjected to orthogonal ROS1 FISH and RNA-based testing. RESULTS:The prevalence of positive ROS1 expression (ROS1 IHC + ), defined as IHC 2+/3+, was 4 % (35 of 866 LUADs). Twenty-eight ROS1 IHC + cases were analyzed by FISH/RNA-based testing, with only two harboring a confirmed ROS1 gene fusion, corresponding to a lower limit for the prevalence of ROS1 gene fusion of 0.23 %. They represent a 7 % probability of identifying a fusion among ROS1 IHC + cases. Both confirmed cases were among the only four with sufficient material and H-score ≥ 200, leading to a 50 % probability of identifying a ROS1 gene fusion in cases with an H-score considered strongly positive. All matched ROS1 IHC- (IHC0 and IHC1 + ) cases were also found negative by FISH/RNA-based testing, leading to a 100 % probability of lack of ROS1 fusion for ROS1 IHC- cases. CONCLUSIONS:The prevalence of ROS1 fusion in an LUAD stage I-III European cohort was relatively low. ROS1 IHC using SP384 clone is useful for exclusion of ROS1 gene fusion negative cases.

NSCLC is the leading cause of cancer-related mortality worldwide, including in Ireland. While historically there has been a lack of treatment options, the identification of genomic 'targets' through NGS of patient's NSCLC tumours has resulted in a rapid expansion of targeted treatment options for selected patients. This retrospective study aims to identify the proportion of patients with NSCLC in Ireland whose tumours harbour actionable genomic alterations.

5031 Background: Mismatch repair (MMR) deficiency and microsatellite instability (MSI) are predictive biomarkers for immunotherapy response. The best approach to identify patients with such tumors is unclear in prostate cancer. Methods: This study included men diagnosed with primary prostate cancer during prospective follow-up of the Health Professionals Follow-up Study (HPFS) and Physicians’ Health Study (PHS). The highest-grade/index lesions of tumor tissue from radical prostatectomy (95%) or transurethral resections of the prostate were mounted in triplicate on tissue microarrays. Immunohistochemistry for the MMR proteins MLH1, MSH2, MSH6, and PMS2 was performed, with scoring as MMR-deficient requiring a visible staining in a non-tumor internal positive control of the same case. For validation, a polymerase chain reaction-based MSI assay (Idylla MSI Test, Biocartis) was performed on tumor DNA of MMR-deficient cases and a selection of MMR-intact cases. Results: The study included 1016 men with prostate cancer. Tumor stage was predominantly pathologically localized (71% stage pT1/2, 18% T3a/b) with a full distribution of Gleason scores, including 20% Gleason score 6 (grade group 1), 36% 3+4 (grade group 2), 23% 4+3 (grade group 3), 8% 8 (grade group 4) and 13% 9-10 (grade group 5). MMR tumor scoring could be performed for MLH1 in 747 cases (75% of those with tumor tissue), for MSH2 in 903 cases (90%), for MSH6 in 708 cases (74%), and for PMS2 in 703 cases (72%). The remaining tumors were unevaluable due to lack of non-tumor tissue necessary as an internal positive control. Of the 903 tumors evaluable for MSH2 protein loss, 4 tumors had loss of MSH2 (prevalence 0.4%, 95% confidence interval [CI] 0.2–1.1%), and 3 of 708 evaluable tumors had concomitant loss of MSH6 (prevalence 0.4%, 95% CI 0.1–1.2%). No tumor had loss of MLH1 or PMS2. The MMR-deficient tumors had Gleason scores of 3+4, 8 (two cases), and 9–10. Tumor DNA of 1 of the 4 MMR-deficient tumors met the manufacturer’s cut-off for MSI-high; two additional MMR-deficient cases had non-zero repeats, with good reproducibility between tumor cores and technical replicates. One MSH2-deficient tumor and 6 DNA samples from 3 MMR-intact cases had repeat scores of zero. Conclusions: In this nationwide prospective study, MMR deficiency was rare in primary, surgically treated prostate cancer. This low prevalence contrasts with hospital-based studies of MMR deficiency and MSI that may have represented tumors with certain clinical features. The low prevalence and the need for an internal positive control for reliable scoring calls into question to what extent immunohistochemistry-based screening for MMR deficiency on limited tissue specimens, such as prostate biopsies, is routinely feasible.

Introduction: The identification of genomic "targets" through next-generation sequencing (NGS) of patient's NSCLC tumors has resulted in a rapid expansion of targeted treatment options for selected patients. This retrospective study aims to identify the proportion of patients with advanced NSCLC in the Republic of Ireland whose tumors harbor actionable genomic alterations through broad NGS panel testing. Methods: Institutional review board approval was obtained before study initiation. Patients with NSCLC whose tumors underwent genomic testing through the largest available NGS panel at a nationally funded Cancer Molecular Diagnostics laboratory (St. James's Hospital) between June 2017 and June 2022 were identified. Patient demographics and tumor-related data were collected by retrospective review from all cancer centers in Ireland, referring to the Cancer Molecular Diagnostics laboratory. A total of 203 (9%) tumor samples were excluded due to insufficient neoplastic cell content. Genomic data were collected through retrospective search of Ion Reporter software. The spectrum and proportion of patients with oncogenic driver mutations were evaluated using descriptive statistics (SPSS version 29.0). Results: In total, 2052 patients were identified. Patients were referred from 23 different hospital sites and all four geographic regions (Leinster = 1091, 53%; Munster = 763, 37.2%; Connacht = 191, 9.3%; Ulster = 7, 0.3%). Median age was 69 (range: 26-94) years; 53% were male. The most common tumor histologic subtype was adenocarcinoma (77%, n = 1577). An actionable genomic alteration was identified in 1099 cases (53%), the most common of which was KRAS (n = 657, 32%). Less frequently, NSCLC tumors harbored the following: MET exon 14 skipping (n = 53, 2.6%), MET amplification (n = 26, 1.3%), EGFR (n = 181, 8.8%), HER2 (n = 35, 1.7%), and BRAF (n = 72, 3.5%) mutations. Fusions were detected in 76 patients (3.7%) including ALK (n = 44, 58%), RET (n = 11, 14.5%), ROS1 (n = 16, 21%), and FGFR3 (n = 5, 6.6%), whereas no NTRK fusion was identified. Coalterations were detected in 114 patients (5.6%), the most common of which was KRAS/PIK3CA (n = 19, 17%), EGFR/PIK3CA (n = 10, 8.5%), and KRAS/IDH1 (n = 9, 8%). Other co-alterations of interest identified included KRAS G12A/ROS1 fusion (n = 1) and KRAS G12C/BRAF G469A (n = 2).

The island of Ireland comprises the Republic of Ireland and Northern Ireland which forms part of the United Kingdom. It is situated in the North Atlantic Ocean in North-Western Europe (Fig. 1). The census population of Ireland in 2022 reported a growing population of more than 7 million for the entire island and 5.1 million in the Republic, the highest population since 1841.1

The liquid biopsy has the potential to improve patient care in the diagnostic and therapeutic setting in non-small cell lung cancer (NSCLC). Consented patients with epidermal growth factor receptor (EGFR) positive disease (n = 21) were stratified into two cohorts: those currently receiving EGFR tyrosine kinase inhibitor (TKI) therapy (n = 9) and newly diagnosed EGFR TKI treatment-naïve patients (n = 12). Plasma genotyping of cell-free DNA was carried out using the FDA-approved cobas® EGFR mutation test v2 and compared to next generation sequencing (NGS) cfDNA panels. Circulating tumor cell (CTC) numbers were correlated with treatment response and EGFR exon 20 p.T790M. The prognostic significance of the neutrophil to lymphocyte ratio (NLR) and lactate dehydrogenase (LDH) was also investigated. Patients in cohort 1 with an EGFR exon 20 p.T790M mutation progressed more rapidly than those with an EGFR sensitizing mutation, while patients in cohort 2 had a significantly longer progression-free survival (p = 0.04). EGFR exon 20 p.T790M was detected by liquid biopsy prior to disease progression indicated by computed tomography (CT) imaging. The cobas® EGFR mutation test detected a significantly greater number of exon 20 p.T790M mutations (p = 0.05). High NLR and derived neutrophil to lymphocyte ratio (dNLR) were associated with shorter time to progression and worse survival outcomes (p < 0.05). High LDH levels were significantly associated with shorter time to disease progression (p = 0.03). These data support the use of liquid biopsy for monitoring EGFR mutations and inflammatory markers as prognostic indicators in NSCLC.

Aims Since its emergence, significant interest surrounds the use of SARS-CoV-2 serological tests as an alternative or as an adjunct to molecular testing. However, given the speed of this pandemic, paralleled with the pressure to develop and provide serological tests in an expediated manner, not every assay has undergone the rigorous evaluation that is usually associated with medical diagnostic assays. We aimed to examine the performance of several commercially available SARS-CoV-2 IgG antibody assays among participants with confirmed COVID-19 disease and negative controls. Methods Serum taken between day 17 and day 40 post onset of symptoms from 41 healthcare workers with RT-PCR confirmed COVID-19 disease, and pre-pandemic serum from 20 negative controls, were tested for the presence of SARS-CoV-2 IgG using 7 different assays including point-of-care (POC) and laboratory-based assays. Results Assay performance varied. The lab-based Abbott diagnostics SARS-CoV-2 IgG assay proved to be the assay with the best positive and negative predictive value, and overall accuracy. The POC Nal von Minden GmbH and Biozek assays also performed well. Conclusion Our research demonstrates the variations in performance of several commercially available SARS-CoV-2 antibody assays. These findings identify the limitations of some serological tests for SARS-CoV-2. This information will help inform test selection and may have particular relevance to providers operating beyond accredited laboratories.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease. Cell-of-origin classification in DLBCL has identified activated B cell (ABC) and germinal center B cell (GCB) as two major subtypes. Patients with the ABC subtype show reduced overall survival with standard therapies. Development of a quantitative RT-PCR-based lymphoma cell-of-origin (LCOO) assay to determine ABC, GCB, and unclassifiable subtypes in formalin-fixed, paraffin-embedded tissue (FFPET) DLBCL samples is reported. The LCOO classifier was trained on two DLBCL cohorts with validation performed by using an analytical grade assay in an independent cohort of 60 FFPET DLBCL samples. In the validation cohort, LCOO classification was 88.1%, 84.7%, and 84.7% concordant with microarray, immunohistochemistry (Hans classification), and Lymphoma Subtyping Test, respectively. Importantly, LCOO and Lymphoma Subtyping Test assays commonly assigned subtypes in 17 (94.4%) of 18 ABC samples and 34 (89.5%) of 38 GCB DLBCL samples from this cohort. Progression-free survival and overall survival of ABC and GCB subtypes, as classified by all platforms, were not significantly different in the validation cohort. LCOO classification using publicly available microarray gene expression from two independent data sets (414 fresh frozen and 474 FFPET DLBCL biopsies) revealed a significantly worse outcome for the ABC subtype compared with that of the GCB subtype. Thus, a sensitive, reproducible, LCOO assay developed on an easy to standardize quantitative RT-PCR platform may be an important clinical tool for DLBCL cell-of-origin classification.

In advanced disease, the presence of an EGFR mutation confers a more favourable prognosis and strongly predicts for sensitivity to EGFR tyrosine kinase inhibitors (TKIs). Clinical studies have demonstrated that ∼20% of biopsies are inadequate for molecular testing due to insufficient tumour tissue. Tissue genotyping is further limited by often inaccessible tissue, heterogeneity and risks associated with serial tumour biopsies. More than 60% of patients who initially respond to EGFR TKI therapy develop resistance due to the emergence of the T790M or other resistance mutations.

Introduction: RET gene fusions are established oncogenic drivers in 1% of NSCLC. Accurate detection of advanced patients with RET fusions is essential to ensure optimal therapy choice. We investigated the performance of fluorescence in situ hybridization (FISH) as a diagnostic test for detecting functional RET fusions. Methods: Between January 2016 and November 2019, a total of 4873 patients with NSCLC were routinely screened for RET fusions using either FISH (n = 2858) or targeted RNA next-generation sequencing (NGS) (n = 2015). If sufficient material was available, positive cases were analyzed by both methods (n = 39) and multiple FISH assays (n = 17). In an independent cohort of 520 patients with NSCLC, whole-genome sequencing data were investigated for disruptive structural variations and functional fusions in the RET and compared with ALK and ROS1 loci. Results: FISH analysis revealed RET rearrangement in 48 of 2858 cases; of 30 rearranged cases double tested with NGS, only nine had a functional RET fusion. RNA NGS yielded RET fusions in 14 of 2015 cases; all nine cases double tested by FISH had RET locus rearrangement. Of these 18 verified RET fusion cases, 16 had a split signal and two a complex rearrangement by FISH. By whole-genome sequencing, the prevalence of functional fusions compared with all disruptive events was lower in the RET (4 of 9, 44%) than the ALK (27 of 34, 79%) and ROS1 (9 of 12, 75%) loci. Conclusions: FISH is a sensitive but unspecific technique for RET screening, always requiring a confirmation using an orthogonal technique, owing to frequently occurring RET rearrangements not resulting in functional fusions in NSCLC. (C) 2021 International Association for the Study of Lung Cancer. Published by Elsevier Inc.