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AimsGene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated.MethodsCell lines harbouring EML4(13)–ALK(20) and SLC34A2(4)–ROS1(32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides.ResultsFour (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms.ConclusionsReference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches.

AimsLung cancer predictive biomarker testing is essential to select advanced-stage patients for targeted treatments and should be carried out without delays even during health emergencies, such as the coronavirus (COVID-19) outbreak.MethodsFifteen molecular laboratories from seven different European countries compared 4 weeks of national lockdown to a corresponding period in 2019, in terms of tissue and/or plasma-based molecular test workload, analytical platforms adopted, number of cases undergoing programmed death-ligand1 (PD-L1) expression assessment and DNA-based molecular tests turnaround time.ResultsIn most laboratories (80.0%), tissue-based molecular test workload was reduced. In 40.0% of laboratories (6/15), the decrease was >25%, and in one, reduction was as high as 80.0%. In this instance, a concomitant increase in liquid biopsy was reported (60.0%). Remarkably, in 33.3% of the laboratories, real-time PCR (RT-PCR)-based methodologies increased, whereas highly multiplexing assays approaches decreased. Most laboratories (88.9%) did not report significant variations in PD-L1 volume testing.ConclusionsThe workload of molecular testing for patients with advanced-stage lung cancer during the lockdown showed little variations. Local strategies to overcome health emergency-related issues included the preference for RT-PCR tissue-based testing methodologies and, occasionally, for liquid biopsy.

AimThe present work aims to evaluate the presence of stromal tumour-infiltrating lymphocytes (TILs) and programmed cell death-ligand 1 (PDL1) expression in breast carcinomas and their correlation with available clinicopathological features.MethodsTwo independent series of invasive breast cancer (IBC), one including ductal carcinoma in situ (DCIS) pair-matched cases, were selected, and quantification of TILs was accomplished in each case. Immunohistochemistry was also performed to evaluate the expression of PDL1.ResultsIn both cohorts evaluated, increased stromal TILs and PDL1 expression were present in about 10% of IBCs, being significantly associated with each other and both with grade 3 and triple-negative subtype. We observed a similar distribution of stromal TILs and PDL1 expression between DCIS and IBC. Finally, we observed that increased stromal TILs and PDL1 expression were significantly associated with cancer stem cell (CSC) markers, basal cell markers and vimentin expression. Interestingly, in IBC cases with vimentin expression, increased stromal TILs, as well as decreased PDL1 expression, disclosed a better clinical outcome, independently of the main classical BC prognostic factors.ConclusionsWe have confirmed the association of stromal TILs and PDL1 expression with aggressive forms of BC and that both are already found in in situ stages. We also showed that stromal TILs and PDL1 expression are associated with clinical outcome in cases enriched for a mesenchymal immunophenotype. We describe for the first time a close relationship between CSC markers and PDL1 expression.