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Simple Summary PD-1/PD-L1 axis blockade immunotherapy leverages on assessment of tumour PD-L1 status by immunohistochemistry for identifying cancer patients’ who would benefit most from this therapy. Because some PD-L1 negative cancer patients also benefit from the treatment, it has been postulated that spatial heterogeneity of PD-L1 expression in tumour tissue may reduce the predictive value of the test. Here we demonstrate for the first time the presence of PD-L1+ circulating tumor–derived microvesicles in the blood stream of high grade serous ovarian cancer patients and propose the assessment of tumor-derived circulating PD-L1+ microvesicles to be inserted in predictive algorithms for complementing tumour PD-L1 status. Abstract Background: Ovarian cancer (OC) has recently attracted attention for the use of PD-1/PD-L1 axis blocking agents, with durable activity reported only in a subset of patients. The most used biomarker for sensitivity to the PD-1/PD-L1 axis blockade is tumour PD-L1 status by immunohistochemistry. However, patient stratification using this method suffers from intrinsic heterogeneity of OC, likely contributing to the unsatisfactory results obtained so far. Cells communicate with each other by releasing microvesicles (MVs) that carry parental cell surface features. Thus, we hypothesised that PD-L1+ tumour cells (TC) and infiltrating PD-L1+ leukocytes should shed MVs carrying surface PD-L1 that may serve as a proxy for the whole tumour PD-L1 status. Results: We showed for the first time the presence of measurable amounts of TC- and leukocyte-derived PD-L1+ MVs (range: 1.4–178.8 MVs/μL and 6.2–504.8 MVs/μL, respectively) in the plasma of high-grade serous OC (HGSOC) patients (n = 63), using a sensitive flow cytometry platform. The concentration of PD-L1+ MVs of either origin did not associate with the PD-L1 status of TCs and leukocytes in the tumour biopsies, suggesting that the circulating PD-L1+ MVs also included ones from locations not selected for immunohistochemistry analysis and represented the PD-L1 status of the whole tumour mass. In this study, we also describe the serendipitous discovery of circulating PD-L1+ MVs of platelet origin (10.3–2409.6 MVs/μL). Conclusions: The enumeration of circulating PD-L1+ MVs in HGSOC patients may provide a novel direction for assessing the tumour PD-L1 status and contribute to HGSOC patient stratification for immunotherapy interventions. The presence of circulating PD-L1+ MVs of platelet origin, a finding not yet reported in HGSOC patients, warrants further studies.

Intense research is being conducted using flow cytometers available in clinically oriented laboratories to assess extracellular vesicles (EVs) surface cargo in a variety of diseases. Using EVs of various sizes purified from the HT29 human colorectal adenocarcinoma cell line, we report on the difficulty to assess small and medium sized EVs by conventional flow cytometer that combines light side scatter off a 405 nm laser with the fluorescent signal from the EVs general labels Calcein-green and Calcein-violet, and surface markers. Small sized EVs (~70 nm) immunophenotyping failed, consistent with the scarcity of monoclonal antibody binding sites, and were therefore excluded from further investigation. Medium sized EVs (~250 nm) immunophenotyping was possible but their detection was plagued by an excess of coincident particles (swarm detection) and by a high abort rate, both factors affected the measured EVs concentration. By running samples containing equal amounts of Calcein-green and Calcein-violet stained medium sized EVs, we found that swarm detection produced false double positive events, a phenomenon that was significantly reduced, but not totally eliminated, by sample dilution. Moreover, running highly diluted samples required long periods of cytometer time. Present findings raise questions about the routine applicability of conventional flow cytometers for EV analysis.

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The standard-of-care (SOC) first-line therapy for ovarian cancer (OC) patients is plagued with high relapse rates. Several studies indicated the immune system’s prominent role changing the disease course in OC patients. Chemo-immunotherapy regimens, currently being explored, include oregovomab, which is a monoclonal antibody specific for the OC associated antigen carbohydrate/cancer antigen 125 (CA125) that yielded promising results when administered together with SOC in a previous study. The QPT-ORE-002 multi-site phase II randomized study demonstrated that in patients with advanced OC, oregovomab combined with first-line SOC improved overall and progression-free survival, compared to SOC alone. The study included an Italian cohort in which we demonstrated that adding oregovomab to SOC resulted in increased patient numbers with amplified CA125-specific CD8+T lymphocytes/ml peripheral blood counts, which might explain the improved therapeutic effect of SOC + oregovomab over SOC alone. Predictive for oregovomab efficacy was a less suppressive immune environment at baseline as indicated by low numbers of circulating myeloid-derived suppressor cells, subset type 4, and a low neutrophil-and-monocyte to lymphocyte ratio.

BackgroundIdentifying the patients who may benefit the most from immune checkpoints inhibitors remains a great challenge for clinicians. Here we investigate on blood serum amyloid A (SAA) as biomarker of response to upfront pembrolizumab in patients with advanced non-small-cell lung cancer (NSCLC).MethodsPatients with PD-L1 ≥ 50% receiving upfront pembrolizumab (P cohort) and with PD-L1 0–49% treated with chemotherapy (CT cohort) were evaluated for blood SAA and radiological response at baseline and every 9 weeks. Endpoints were response rate (RR) according to RECIST1.1, progression-free (PFS) and overall survival (OS). The most accurate SAA cut-off to predict response was established with ROC analysis in the P cohort.ResultsIn the P Cohort (n = 42), the overall RR was 38%. After a median follow-up of 18.5 months (mo), baseline SAA ≤ the ROC-derived cut-off (29.9 mg/L;n = 28/42.67%) was significantly associated with higher RR (53.6 versus 7.1%; OR15, 95% CI 1.72–130.7,p = 0.009), longer PFS (17.4 versus 2.1 mo;p p 29.9 mg/L. In multivariate analysis, low SAA positively affects PFS (p = 0.001) and OS (p = 0.048) irrespective of ECOG PS, number of metastatic sites and pleural effusion. SAA monitoring (n = 40) was also significantly associated with survival endpoints: median PFS 17.4 versus 2.1 mo and median OS not reached versus 7.2 mo when SAA remained low (n = 14) and high (n = 12), respectively. In the CT Cohort (n = 30), RR was not affected by SAA level (p > 0.05) while low SAA at baseline (n = 17) was associated with better PFS (HR 0.38, 95% CI 0.16–0.90,p = 0.006) and OS (HR 0.25, 95% CI 0.09–0.67,p ConclusionLow SAA predicts good survival outcomes irrespective of treatment for advanced NSCLC patients and higher likelihood of response to upfront pembrolizumab only. The strong prognostic value might be exploited to easily identify patients most likely to benefit from immunotherapy. A further study (FoRECATT-2) is ongoing to confirm results in a larger sample size and to investigate the effect of SAA on immune response in vitro assays.