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.
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.
Background
Identifying 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).
Methods
Patients 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.
Results
In 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
< 0.0001) and OS (not reached versus 7.2mo;
p
< 0.0001) compared with SAA > 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
< 0.001).
Conclusion
Low 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.
Immediate hypersensitivity reactions (iHSRs) to taxanes are observed in 6% and 4% of gynecologic and breast cancer patients, respectively. Drug desensitization is the only option, as no comparable ...alternative therapy is available. Surfactants in the taxane formulation have been implicated in the immunopathogenesis of iHSRs, although sporadic skin test (ST) positivity and iHSRs to nab-paclitaxel have suggested the involvement of the taxane moiety and/or IgE-mediated pathomechanisms. In vitro diagnostic tests might offer insights into mechanisms underlying iHSRs to taxanes. The aim of the present study was to address this unmet need by developing a novel basophil activation test (BAT). The study included patients (
= 31) undergoing paclitaxel/carboplatin therapy. Seventeen patients presented with iHSRs to paclitaxel (iHSR-Tax
), and eleven were tolerant (iHSR-Tax
). Fourteen patients presented with iHSRs to carboplatin (iHSR-Pl
), and fourteen were tolerant (iHSR-Pl
). The BAT median stimulation index (SI) values were 1.563 (range, 0.02-4.11;
= 11) and -0.28 (range -4.88-0.07,
= 11) in iHSR-Tax
and iHSR-Tax
, respectively. The BAT median SI values were 4.45 (range, 0.1-26.7;
= 14) and 0 (range, -0.51-1.65;
= 12) in iHSR-Pl
and iHSR-Pl
, respectively. SI levels were not associated with iHSR severity grading. Comparing BAT results in iHSR-Tax
and iHSR-Tax
showed the area under the receiver operator characteristic (ROC) curve to be 0.9752 (
= 0.0002). The cutoff calculated by the maximized likelihood ratio identified 90.91% of iHSR-Tax
patients and 90.91% of iHSR-Tax
patients. Comparing BAT results for iHSR-Pl
and iHSR-Pl
showed the area under the ROC curve to be 0.9286 (
= 0.0002). The cutoff calculated by the maximized likelihood ratio identified 78.57% of iHSR-Pl
patients and 91.67% of iHSR-Pl
patients. Most iHSR-Tax
patients for which ST was available (10/11) scored ST-negative and BAT-positive, whereas most iHSR-Pl
patients for which ST was available (14/14) scored both BAT- and ST-positive. This suggested the intervention of non-IgE-mediated mechanisms in iHSR-Tax
patients. Consistent with this view, an in silico molecular docking analysis predicted the high affinity of paclitaxel to the degranulation-competent MRGPRX2 receptor. This hypothesis warrants further in vitro investigations. In conclusion, the present study provides preliminary proof-of-concept evidence that this novel BAT has potential utility in understanding mechanisms underlying iHSRs to taxanes.
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.
Purpose
Hyaluronan (HA)-receptors (mainly CD44 and RHAMM) are overexpressed in a wide variety of cancers including ovarian tumors, and HA-bioconjugates have been developed to enhance selective entry ...of cytotoxic drugs into HA receptor-expressing cancerous cells. Here, we evaluated the potential application of a new HA-paclitaxel bioconjugate, ONCOFID-P, for intraperitoneal (IP) treatment of ovarian cancer.
Methods
In vitro cytotoxic effect of ONCOFID-P was first assessed on CD44(+) OVCAR-3 and SKOV-3 human ovarian cancer cell lines. Studies were performed in female Balb/c athymic mice IP implanted with OVCAR-3 or SKOV-3 and treated with IP ONCOFID-P, and IP and intravenous (IV) free paclitaxel, at their maximum tolerated dose (MTD 168, 80 and 80 mg/kg, total dose, respectively). The potential detrimental effect of the IP ONCOFID-P and IP free paclitaxel on hematopoiesis was also assessed on peripheral blood, bone marrow and spleen.
Results
Results show that ONCOFID-P cytotoxicity against both OVCAR-3 and SKOV-3 cell lines was somewhat less effective than free paclitaxel. Conversely, in in vivo experiments, IP treatment with ONCOFID-P was overall more effective than IV and IP free paclitaxel in inhibiting intra-abdominal tumor dissemination, abrogating ascites, prolonging survival and curing mice. ONCOFID-P and IP free paclitaxel were equivalent in terms of myelotoxicity, although the former was administered at a two-fold higher dose.
Conclusions
Present data strongly support the development of ONCOFID-P for locoregional treatment of ovarian cancer.
The possibility to study solid tumors through the analysis of extracellular vesicles in biological fluids is one of the most exciting and rapidly advancing field in cancer research. The extracellular ...vesicles are tiny sacs released in both physiological and pathological conditions and can be used to monitor the evolution of several pathological states, including neoplastic diseases. Indeed, these vesicles carry biological informations and can affect the behavior of recipient cells by transferring proteins, DNA, RNA, and microRNA. In this review the authors analyze the methods to collect biological fluid samples (urine, plasma/serum, and cell supernatant), and to isolate and quantify extracellular vesicles highlighting advantages and drawbacks. Moreover, the authors provide an overview on the adoption and the advantages of the methods (such as digital PCR, next generation sequencing, reverse‐phase protein microarrays, flow‐cytometry, etc.) most frequently used to analyze the molecular content of extracellular vesicles. Despite the great scientific interest on this topic, there is still a great uncertainty about which is the best method for the collection, isolation, quantification, and molecular evaluation of these vesicles and a standardization is needed. The features of EVs make them ideal candidates for liquid biopsy‐based biomarkers. However, the small size of EVs makes their analysis very difficult and requires multiple advanced technologies, being therefore a limitation.
The possibility to study solid tumors through the analysis of extracellular vesicles in biological fluids is one of the most exciting and rapidly advancing field in cancer research. Despite the great scientific interest on this topic, there is still a great uncertainty about which is the best method for the collection, isolation, quantification, and molecular evaluation of these vesicles and a standardization is needed. The features of EVs make them ideal candidates for liquid biopsy‐based biomarkers. However, EVs analysis is very difficult and requires multiple advanced technologies, being therefore a limitation.
To assess how neoadjuvant chemoradiation regimens modulate the immune system state in tumor-draining lymph nodes (TDLN), in the setting of advanced cervical cancer.
Tumor-draining lymph nodes of ...patients undergoing chemotherapy only (nonirradiated, NI-TDLN) and chemoradiation with lower-dose (39.6 Gy, LD-TDLN) and higher-dose radiation (50 Gy, HD-TDLN) were analyzed by multicolor flow cytometry.
Enlarging our previous data, LD-TDLN showed features overall indicative of an enhanced antitumor response as compared with NI-TDLN, namely a significant Th1 and Tc1 polarization and a lower amount of the potent CD4(+)Foxp3(+)CD25(high) regulatory T cell (Treg) subset identified by neuropilin-1 expression. Conversely, compared with NI-TDLN, HD-TDLN showed features overall indicative of an impaired antitumor response, namely a significantly inverted CD4/CD8 cell ratio, a higher Nrp1(+)Treg frequency, and a higher frequency of CCR4(+)Treg, a Treg subset facilitated in migrating out from TDLN to suppress the immune response against distant cancer cells. Moreover, the Th1 and Tc1 polarization induced by LD radiation was lost, and there was an unfavorable tolerogenic/immunogenic dendritic cell ratio compared with LD-TDLN.
Even minor differences in radiation dose in neoadjuvant regimens for locally advanced cervical cancer are crucial for determining the balance between a tolerogenic and an efficacious antitumor immune response in TDLN. Because most of the anticancer immune response takes place in TDLN, the present findings also emphasize the importance of chemoradiation protocols in the context of immunotherapeutic trials.
Class III β-tubulin (TUBB3) overexpression in ovarian cancer (OC) associates with poor prognosis. We investigated whether TUBB3 overexpression elicited anti-TUBB3 antibody production in OC patients ...and whether these antibodies may have diagnostic and prognostic impact. The presence of serum anti-TUBB3 antibodies was investigated in 49 untreated OC patients and 44 healthy individuals by an in-house developed ELISA that used recombinant TUBB3 as the antigen. Receiver operating characteristic (ROC) curves were generated to assess the diagnostic accuracy of the assay. Anti-TUBB3 antibodies discriminated OC patients and healthy individuals with excellent sensitivity and specificity (91.8% and 90.9%, respectively). In multivariate analysis, anti-TUBB3 antibody level emerged as an independent prognostic factor for progression free and overall survival. The ELISA was then optimized using a biotin-labeled TUBB3 C-terminal peptide424-450 instead of recombinant TUBB3 as the antigen and streptavidin-coated plates. The diagnostic role of the anti-TUBB3 antibodies was studied in an independent series of 99 OC patients and 80 gynecological benign disease patients. ROC-curve analysis showed a valuable diagnostic potential for serum anti-TUBB3 antibodies to identify OC patients with higher sensitivity and specificity (95.3% and 97.6%, respectively). Overall, our results provide evidence that preoperative anti-TUBB3 antibody level is a promising diagnostic and prognostic biomarker for the management of OC patients.
PDF
