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Plasma circulating tumour DNA (ctDNA) has been suggested to be a viable biomarker of response to treatment in patients with high grade serous ovarian carcinoma (HGSOC). TP53 mutations are present in more than 90% of HGSOCs but somatic variants are distributed across all exonic regions of the gene, requiring next generation sequencing (NGS) technologies for mutational analysis. In this study, we compared the suitability of the Accel (Swift) and Oncomine (ThermoFisher) panels for identification of TP53 mutations in ctDNA of HGSOC patients (N = 10). Only 6 patients (60%) were found to have TP53 mutations using the ACCEL panel but the addition of molecular tags in the Oncomine panel improved ctDNA detection with at least one mutation detected in all cases (100%). Orthogonal validation of the 14 somatic variants found by Oncomine, using droplet digital PCR, confirmed 79% (11/14) of the identified mutations. Overall, the Oncomine panel with unique molecular identifiers (UMI) appears more useful for ctDNA analysis in HGSOC.

Antibodies against programmed death-1 (PD-1), and its ligand, (PD-L1) have been approved recently for the treatment of small-cell lung cancer (SCLC). Although there are previous reports that addressed PD-L1 detection on tumour cells in SCLC, there is no comprehensive meta-analysis on the prevalence of PD-L1 expression in SCLC. We performed a systematic search of the PubMed, Cochrane Library and EMBASE databases to assess reports on the prevalence of PD-L1 expression and the association between PD-L1 expression and overall survival (OS). This meta-analysis included 27 studies enrolling a total of 2792 patients. The pooled estimate of PD-L1 expression was 26.0% (95% CI 17.0–37.0), (22.0% after removing outlying studies). The effect size was significantly heterogeneous (I2 = 97.4, 95% CI: 95.5–98.5, p < 0.0001).Positive PD-L1 expression was a favourable prognostic factor for SCLC but not statistically significant (HR = 0.86 (95% CI (0.49–1.50), p = 0.5880; I2 = 88.7%, p < 0.0001). Begg’s funnel plots and Egger’s tests indicated no publication bias across included studies (p > 0.05). Overall, there is heterogeneity in the prevalence of PD-L1 expression in SCLC tumour cells across studies. This is significantly moderated by factors such as immunohistochemistry (IHC) evaluation cut-off values, and assessment of PD-L1 staining patterns as membranous and/or cytoplasmic. There is the need for large size, prospective and multicentre studies with well-defined protocols and endpoints to advance the clinical value of PD-L1 expression in SCLC.

Circulating tumor DNA (ctDNA) may serve as a surrogate to tissue biopsy for noninvasive identification of mutations across multiple genetic loci and for disease monitoring in melanoma. In this study, we compared the mutation profiles of tumor biopsies and plasma ctDNA from metastatic melanoma patients using custom sequencing panels targeting 30 melanoma‐associated genes. Somatic mutations were identified in 20 of 24 melanoma biopsies, and 16 of 20 (70%) matched‐patient plasmas had detectable ctDNA. In a subgroup of seven patients for whom matching tumor tissue and plasma were sequenced, 80% of the mutations found in tumor tissue were also detected in ctDNA. However, TERT promoter mutations were only detected by ddPCR, and promoter mutations were consistently found at lower concentrations than other driver mutations in longitudinal samples. In vitro experiments revealed that mutations in promoter regions of TERT and DPH3 are underrepresented in ctDNA. While the results underscore the utility of using ctDNA as an alternative to tissue biopsy for genetic profiling and surveillance of the disease, our study highlights the underrepresentation of promoter mutations in ctDNA and its potential impact on quantitative liquid biopsy applications. This study by Calapre et al. critically appraised the concordance of the mutational profile of tissue and plasma biopsies prior to treatment, and the evidence of the impact of cfDNA biogenesis on the loci targeted for ctDNA quantification. Plasma ctDNA appears to be a reliable genetic material for mutational profiling of melanomas, allowing detection of clinically targetable mutations and longitudinal monitoring of late‐stage patients.

Primary endometrial serous carcinoma, known for its aggressive nature and poor prognosis, shares similarities with breast and gastric cancers in terms of potential HER2 overexpression as a therapeutic target. Assessing HER expression is complicated by tumor heterogeneity and discrepancies between primary and metastatic sites. In this study, we retrospectively analyzed HER amplification and expression in 16 pairs of primary endometrial serous carcinoma resections and corresponding metastases. HER2 status was determined using immunohistochemistry (IHC), with criteria based on the percentage and intensity of tumor cell staining. Confirmatory techniques, such as dual in situ hybridization (DISH) and fluorescence in situ hybridization (FISH), were also employed. This study reports on the concordance rates and the presence and pattern of HER2 heterogeneity. Our results showed an 87.5% concordance rate in HER2 amplification status between primary and metastatic sites, with 33% of cases scored as 2+ being amplified. Heterogeneity was observed in 100% of amplified cases and 95% of non-amplified cases on in situ testing, with variations in heterogeneity patterns between techniques. In conclusion, our findings emphasize the importance of testing both primary and metastatic sites or recurrences, with a concordance rate of 87.5%. In addition, a review of the literature and combining the results showed a concordance rate of up to 68%. The presence and pattern of heterogeneity, particularly in cases of mosaic or clustered heterogeneity in the primary tumor, may serve as reliable indicators of concordance, predicting a non-amplified HER2 status in corresponding metastases.

Background Poly‐ADP ribose polymerase inhibitors have been shown to improve progression‐free survival in patients with advanced high‐grade epithelial non‐mucinous ovarian cancers characterized by a deficiency in homologous recombination (HRD). Guidelines recommend all patients with advanced high‐grade epithelial ovarian cancer undergo genomic tumor testing for HRD. Our aim was to evaluate the first year of HRD testing at the statewide Western Australia Gynecologic Cancer Service to assess factors associated with obtaining a diagnostic HRD testing result. Methods Retrospective chart review. Results HRD testing was indicated in 84 patients, and ordered in 79, of which three had non‐diagnostic/inconclusive results, all due to insufficient tumor quantity. One patient had the sample collected using a 20‐gauge core biopsy needle under image guidance, one patient following interval debulking surgery, and one following primary debulking surgery. Of 76 patients with an HRD result, HRD was positive in 29 (38.2%). A somatic BRCA mutation was detected in six of these 29 patients (20.6%) and HRD positive, BRCAwt was detected in 23 of 29 patients (79.4%). All core biopsies with 16‐ and 18‐gauge needles had a diagnostic HRD result. Ten of 11 patients who were treated by neoadjuvant chemotherapy and whose biopsies were obtained at interval cytoreductive surgery had sufficient tumor tissue for testing and had a diagnostic HRD result. All ascitic/pleural fluid samples sent for HRD testing yielded diagnostic results. Conclusions Compliance with HRD testing was high, and only three of 79 (3.8%) patients had non‐diagnostic results.

IntroductionGrades 2 and 3 gliomas (G2/3 gliomas), when combined, are the second largest group of malignant brain tumours in adults. The outcomes for G2/3 gliomas at progression approach the dismal outcomes for glioblastoma (GBM), yet there is a paucity of trials for Australian patients with relapsed G2/3 gliomas compared with patients with GBM. LUMOS will be a pilot umbrella study for patients with relapsed G2/3 gliomas that aims to match patients to targeted therapies based on molecular screening with contemporaneous tumour tissue. Participants in whom no actionable or no druggable mutation is found, or in whom the matching drug is not available, will form a comparator arm and receive standard of care chemotherapy. The objective of the LUMOS trial is to assess the feasibility of this approach in a multicentre study across five sites in Australia, with a view to establishing a national molecular screening platform for patient treatment guided by the mutational analysis of contemporaneous tissue biopsiesMethods and analysisThis study will be a multicentre pilot study enrolling patients with recurrent grade 2/3 gliomas that have previously been treated with radiotherapy and chemotherapy at diagnosis or at first relapse. Contemporaneous tumour tissue at the time of first relapse, defined as tissue obtained within 6 months of relapse and without subsequent intervening therapy, will be obtained from patients. Molecular screening will be performed by targeted next-generation sequencing at the reference laboratory (PathWest, Perth, Australia). RNA and DNA will be extracted from representative formalin-fixed paraffin embedded tissue scrolls or microdissected from sections on glass slides tissue sections following a review of the histology by pathologists. Extracted nucleic acid will be quantified by Qubit Fluorometric Quantitation (Thermo Fisher Scientific). Library preparation and targeted capture will be performed using the TruSight Tumor 170 (TST170) kit and samples sequenced on NextSeq 550 (Illumina) using NextSeq V.2.5 hi output reagents, according to the manufacturer’s instructions. Data analysis will be performed using the Illumina BaseSpace TST170 app v1.02 and a custom tertiary pipeline, implemented within the Clinical Genomics Workspace software platform from PierianDx (also refer to section 3.2). Primary outcomes for the study will be the number of patients enrolled and the number of patients who complete molecular screening. Secondary outcomes will include the proportion of screened patients enrolled; proportion of patients who complete molecular screening; the turn-around time of molecular screening; and the value of a brain tumour specific multi-disciplinary tumour board, called the molecular tumour advisory panel as measured by the proportion of patients in whom the treatment recommendation was refined compared with the recommendations from the automated bioinformatics platform of the reference laboratory testing.Ethics and disseminationThe study was approved by the lead Human Research Ethics Committee of the Sydney Local Health District: Protocol No. X19-0383. The study will be conducted in accordance with the principles of the Declaration of Helsinki 2013, guidelines for Good Clinical Practice and the National Health and Medical Research Council National Statement on Ethical Conduct in Human Research (2007, updated 2018 and as amended periodically). Results will be disseminated using a range of media channels including newsletters, social media, scientific conferences and peer-reviewed publications.Trial registration numberACTRN12620000087954; Pre-results.

Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3–4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics.

Immune checkpoint inhibitors (ICIs) have improved outcomes across several cancers, yet many patients do not respond, highlighting the need for robust predictive biomarkers. Tertiary lymphoid structures (TLS), ectopic lymphoid aggregates that support local antigen presentation and adaptive immune activation, have emerged as potential indicators of favourable prognosis and immunotherapy responsiveness. This review summarises current clinical and translational evidence examining the prognostic and predictive value of TLS in solid malignancies. Studies assessing TLS presence, organisation, and biological function were identified through searches of major scientific databases and evaluated with respect to their association with patient outcomes and responses to ICIs. Across multiple tumour types, TLS correlate with improved survival and enhanced anti‑tumour immune activity. TLS‑rich tumours typically show increased infiltration of effector immune cells and more inflamed tumour microenvironments. Several studies also indicate that TLS maturity, particularly the presence of germinal‑centre‑like features, strengthens their predictive value for ICI benefit. However, substantial variation exists in TLS assessment methods and definitions, limiting comparability and hindering translation into routine clinical use. TLS represent a promising biomarker for prognosis and immunotherapy response. Standardised evaluation methods and prospective clinical validation are essential to enable their integration into personalised treatment strategies.

Immune checkpoint inhibition is an important therapeutic option in patients with non-small cell lung cancer. Programmed cell death ligand-1 (PD-L1) expression may serve as a predictive marker for anti-PD-1/PD-L1 therapies. The relationship between non-small cell lung cancer PD-L1 expression and clinicopathological characteristics remains unclear and there is no population level Australian data. We report the results of PD-L1 testing in patients with non-small cell lung cancer diagnosed at major Western Australian public hospitals served by a single state Pathology provider. We analyzed PD-L1 expression by immunohistochemistry in 241 non-small cell lung cancer specimens using the 22C3 clone on a Dako autostainer platform. Tumor cell PD-L1 expression was scored as Tumor Proportion Score and categorized using pre-specified subsets of  1%, 1–49% and  ≥  50% for correlation with clinicopathologic features. PD-L1 Tumor Proportion Score was  1% in 65 (27%) cases, 1–49% in 100 (41%) cases and  ≥  50% in 76 (32%) cases. PD-L1-positive rate was 92% in squamous cell carcinomas and 67% in adenocarcinomas. PD-L1 Tumor Proportion Score was higher in squamous cell carcinomas ( p   =  0.004) and lower in adenocarcinomas ( p   =  0.003). Of the 196 non-squamous carcinomas, 35% had rat sarcoma viral oncogene homolog ( RAS ) mutations, 13% had epidermal growth factor receptor ( EGFR ) mutations, 2% had anaplastic lymphoma kinase ( ALK ) translocations and 2% had ROS1 translocations. Tumor Proportion Score  ≥  50% was seen in 34% (23/68), 28% (7/25) and 25% (1/4) of RAS , EGFR mutant, and ALK translocated carcinomas, respectively. There was no significant correlation between PD-L1 expression and molecular or genetic abnormalities, or other parameters including age, gender, stage, and smoking status. In our patient cohort, PD-L1 Tumor Proportion Score was significantly higher in squamous cell carcinomas and lower in adenocarcinomas. The overall prevalence of Tumor Proportion Score  ≥  50% is consistent with that reported in clinical trials.

AimBreast carcinoma proliferative activity, histological grade and commercial molecular tests are all important in prognostication and treatment. There is a particular need for improved, standardised techniques for subclassification of grade 2 breast cancers into low-risk and high-risk prognostic groups. In this study we investigated whether gene expression profiling of five proliferation genes was feasible using breast cancer tissue in a clinical setting and whether these profiles could enhance pathological assessment.MethodsExpression of five proliferation gene mRNAs; Ki-67, STK 15, CCNB1, CCND1 and MYBL2, was quantified in 27 breast carcinomas and compared with Ki-67 proliferation index (PI) and Nottingham mitotic score.ResultsExpression of Ki-67, STK15 and MYBL2 mRNA showed moderate Spearman's correlation with Ki-67 PI (p<0.01), but CCND1 and CCNB1 showed weak, non-significant correlation. Individual gene expression did not associate with mitotic score but combined mRNA expression correlated with both Ki-67 PI (p=0.018) and mitotic score (p=0.03; 0.007).ConclusionsThis study confirms mRNA analysis in breast carcinoma formalin-fixed, paraffin-embedded samples is feasible and suggests gene expression profiling, using a small set of five proliferation genes, has potential in aiding histological grading or assessment of proliferative activity of breast cancers. To fully evaluate the clinical applicability of this approach, a larger cohort study with long-term follow-up data is required.