Background: Cervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, although little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. In order to analyze the protein composition of human CVF, we used CVF samples that are routinely collected during colposcopy, but are usually discarded. Since these samples are available in large quantities we aimed to analyze their usefulness for proteomics experiments. The samples were analyzed using different prefractionation techniques (ultrafiltration and C 4 (RP)-LC protein separation) followed by C 18 (RP)-LC peptide separation and identification by MALDI-TOF-TOF mass spectrometry. To determine the reproducibility of this proteomics platform we analyzed three technical replicates. Using spectral counting, protein abundances were estimated in a semiquantitative way. We also compared the results obtained in this study with those from previous studies derived from patients with different physiological conditions in order to determine an overlapping protein set.

Highlights Cancer patients with COVID-19 have a higher morbidity and mortality. Particularly patients with ongoing or recent cancer treatment, metastatic solid tumors and hematological malignancies are at risk. Underlying immunosuppression, elevated cytokine levels, altered expression of the angiotensin converting enzyme (ACE-2) and TMPRSS2, and a prothrombotic status in cancer patients may fuel the effects of a SARS-CoV-2 sepsis. The gene expression level of ACE2 may be an indicator of the susceptibility to SARS-CoV-2 infection, while TMPRSS2 plays a supporting role. Better knowledge of the mechanisms involved may be a tool to identify high risk patients and to prevent severe complications by targeting the involved pathways.

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Vascular endothelial growth factor (VEGF)-A inhibitors exhibit unseen high responses and toxicity in recurrent epithelial ovarian cancer suggesting an important role for the VEGF/VEGFR pathway. We studied the correlation of VEGF signalling and AKT/mTOR signalling. Using a tissue microarray of clinical samples ( N =86), tumour cell immunohistochemical staining of AKT/mTOR downstream targets, pS6 and p4E-BP1, together with tumour cell staining of VEGF-A and pVEGFR2 were semi-quantified. A correlation was found between the marker for VEGFR2 activation (pVEGFR2) and a downstream target of AKT/mTOR signalling (pS6) ( R =0.29; P =0.002). Additional gene expression analysis in an independent cDNA microarray dataset ( N =24) showed a negative correlation ( R =−0.73, P <0.0001) between the RPS6 and the VEGFR2 gene, which is consistent as the gene expression and phosphorylation of S6 is inversely regulated. An activated tumour cell VEGFR2/AKT/mTOR pathway was associated with increased incidence of ascites ( χ 2 , P =0.002) and reduced overall survival of cisplatin–taxane-based patients with serous histology ( N =32, log-rank test, P =0.04). These data propose that VEGF-A signalling acts on tumour cells as a stimulator of the AKT/mTOR pathway. Although VEGF-A inhibitors are classified as anti-angiogenic drugs, these data suggest that the working mechanism has an important additional modality of targeting the tumour cells directly.

Background Inflammatory breast cancer (IBC) is a rare and rapidly progressive form of invasive breast cancer. The aim of this study was to explore the clinical evolution, stromal tumour-infiltrating lymphocytes (sTIL) infiltration and programmed death-ligand 1 (PD-L1) expression in a large IBC cohort. Patients and methods Data were collected prospectively from patients with IBC as part of an international collaborative effort since 1996. In total, 143 patients with IBC starting treatment between June 1996 and December 2016 were included. Clinicopathological variables were collected, and sTIL were scored by two pathologists on standard H&E stained sections. PD-L1 expression was assessed using a validated PD-L1 (SP142) assay. A validation cohort of 64 patients with IBC was used to test our findings. Results Survival outcomes of IBC remained poor with a 5-year overall survival (OS) of 45.6%. OS was significantly better in patients with primary non-metastatic disease who received taxane-containing (neo)adjuvant therapy ( P = 0.01), had a hormone receptor-positive tumour ( P = 0.001) and had lower cN stage at diagnosis ( P = 0.001). PD-L1 positivity on immune cells (42.9%) was higher in IBC than in non-IBC in both our patient samples and the validation cohort. Furthermore, PD-L1 expression predicted pCR ( P = 0.002) and correlated with sTIL infiltration ( P < 0.001). sTIL infiltration of more than 10% of the stroma was a significant predictor of improved OS (HR 0.47, 95% CI 0.27–0.81, P = 0.006) in a multivariate model. Conclusions IBC is characterised by poor survival and high PD-L1 immunoreactivity on sTIL. This suggests a role for PD1/PD-L1 inhibitors in the treatment of IBC. Furthermore, we showed that PD-L1 expression predicts response to neo-adjuvant therapy and that sTIL have prognostic significance in IBC. Electronic supplementary material The online version of this article (10.1186/s13058-019-1108-1) contains supplementary material, which is available to authorized users.

ObjectivesCervicovaginal fluid (CVF) can be considered as a potential source of biomarkers for diseases of the lower female reproductive tract. The fluid can easily be collected, thereby offering new opportunities such as the development of self tests. Our objective was to identify a CVF protein biomarker for cervical cancer or its precancerous state.MethodsA differential proteomics study was set up using CVF samples from healthy and precancerous women. Label-free spectral counting was applied to quantify protein abundances.ResultsThe proteome analysis revealed 16 candidate biomarkers of which alpha-actinin-4 (p = 0.001) and pyruvate kinase isozyme M1/M2 (p = 0.014) were most promising. Verification of alpha-actinin-4 by ELISA (n = 28) showed that this candidate biomarker discriminated between samples from healthy and both low-risk and high-risk HPV-infected women (p = 0.009). Additional analysis of longitudinal samples (n = 29) showed that alpha-actinin-4 levels correlated with virus persistence and clearing, with a discrimination of approximately 18 pg/ml.ConclusionsOur results show that CVF is an excellent source of protein biomarkers for detection of lower female genital tract pathologies and that alpha-actinin-4 derived from CVF is a promising candidate biomarker for the precancerous state of cervical cancer. Further studies regarding sensitivity and specificity of this biomarker will demonstrate its utility for improving current screening programs and/or its use for a cervical cancer self-diagnosis test.

Summary Tumour stromal neovascularization was investigated in 114 invasive and 20 in situ carcinomas of the uterine cervix by staining representative sections with the specific endothelial marker anti CD31 (clone JC/70A, isotope IgG1). A digital image analyser was used to measure the immunoreactivity. The following parameters were determined in the 'hot spots': vessel counts, vessel perimeter and endothelial stained area (expressed per mm2). The results were correlated with clinical and histopathological data. There was no significant relationship between the histopathological findings (tumour histology, tumour differentiation, FIGO stage, presence of lymph node metastasis or lymphovascular space involvement) and the median vessel count. In a univariate analysis all angiogenesis parameters had prognostic value: a higher vascularity was associated with worse prognosis (P < 0.05). Multiple regression analysis showed that vascular permeation (P < 0.001) and the median vessel count (P = 0.005) were the most important prognostic indicators. In the future these criteria may be used for selection of patients for anti-angiogenesis therapy.

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