Accuracy and Reproducibility of Cytology Triage in a HPV-Based Primary Screening Setting: A Revision of 384 Pap Tests.

INTRODUCTION: After the transition toward the HPV-based screening protocol, which has led to an increase in sensitivity, and in order to bring the specificity back to acceptable values, cytology underwent a change of approach, becoming a triage test. For these reasons, in the Tuscany region (after the recommendations of the GISCi document), it was decided to reduce, as much as possible, the use of ASC-US category in cytology triage, classifying these morphological cases as negative for intraepithelial lesion or malignancies (NILM) or LSIL, basing on the grade of nuclear atypia. So, in Italy, in a cytology triage context (HPV primary screening), a modified Bethesda system (TBS) is currently used. The aim of this study was to evaluate the performance of the review activity of 384 cytology triage cases and of the cervical cancer screening indicators (sensitivity and specificity for CIN2+ lesions) using the TBS 2014 or the modified TBS. MATERIALS AND METHODS: 384 HPV positive cases at one-year recall (192 with a cytology result of NILM both at baseline and at one-year recall; 192 with a cytology result of NILM at baseline but abnormal at one-year recall), all with a histologically confirmed result (128 CIN2+, 256 ≤ CIN1), were selected, and their baseline Pap tests were reviewed in blind mode by 5 expert cytologists. RESULTS: The cytological results of NILM were confirmed for 92.5% and 83.8% of cases using TBS 2014 or modified TBS, respectively. 20/128 CIN2+ cases could have been reported at the baseline cytology triage, causing an anticipatory effect and an improvement in sensitivity of the screening protocol at baseline (+15.6%). Using TBS 2014, the number of false positives more than tripled with respect to the modified TBS 2014, with a significant increase in unnecessary colposcopies (+11.4%). CONCLUSION: This work demonstrated that a greater expertise of cytologists, acquired during the following 3 years of experience with cytological triage, and a strong IQC system could lead to the identification of a significant number of lesions reported to baseline rather than at one-year recall (diagnostic anticipation).

CD44v8-10 is a marker for malignant traits and a potential driver of bone metastasis in a subpopulation of prostate cancer cells.

OBJECTIVE: Bone metastasis is a clinically important outcome of prostate carcinoma (PC). We focused on the phenotypic and functional characterization of a particularly aggressive phenotype within the androgen-independent bone metastasis-derived PC3 cell line. These cells, originated from the spontaneous conversion of a CD44-negative subpopulation, stably express the CD44v8-10 isoform (CD44v8-10pos) and display stem cell-like features and a marked invasive phenotype in vitro that is lost upon CD44v8-10 silencing. METHODS: Flow cytometry, enzyme-linked immunoassay, immunofluorescence, and Western blot were used for phenotypic and immunologic characterization. Real-time quantitative polymerase chain reaction and functional assays were used to assess osteomimicry. RESULTS: Analysis of epithelial-mesenchymal transition markers showed that CD44v8-10pos PC3 cells surprisingly display epithelial phenotype and can undergo osteomimicry, acquiring bone cell phenotypic and behavioral traits. Use of specific siRNA evidenced the ability of CD44v8-10 variant to confer osteomimetic features, hence the potential to form bone-specific metastasis. Moreover, the ability of tumors to activate immunosuppressive mechanisms which counteract effective immune responses is a sign of the aggressiveness of a tumor. Here we report that CD44v8-10pos cells express programmed death ligand 1, a negative regulator of anticancer immunity, and secrete exceptionally high amounts of interleukin-6, favoring osteoclastogenesis and immunosuppression in bone microenvironment. Notably, we identified a novel pathway activated by CD44v8-10, involving tafazzin (TAZ) and likely the Wnt/TAZ axis, known to play a role in upregulating osteomimetic genes. CONCLUSIONS: CD44v8-10 could represent a marker of a more aggressive bone metastatic PC population exerting a driver role in osteomimicry in bone. A novel link between TAZ and CD44v8-10 is also shown.

Stem-like and highly invasive prostate cancer cells expressing CD44v8-10 marker originate from CD44-negative cells.

In human prostate cancer (PCa), the neuroendocrine cells, expressing the prostate cancer stem cell (CSC) marker CD44, may be resistant to androgen ablation and promote tumor recurrence. During the study of heterogeneity of the highly aggressive neuroendocrine PCa cell lines PC3 and DU-145, we isolated and expanded in vitro a minor subpopulation of very small cells lacking CD44 (CD44neg). Unexpectedly, these sorted CD44neg cells rapidly and spontaneously converted to a stable CD44high phenotype specifically expressing the CD44v8-10 isoform which the sorted CD44high subpopulation failed to express. Surprisingly and potentially interesting, in these cells expression of CD44v8-10 was found to be induced in stem cell medium. CD44 variant isoforms are known to be more expressed in CSC and metastatic cells than CD44 standard isoform. In agreement, functional analysis of the two sorted and cultured subpopulations has shown that the CD44v8-10pos PC3 cells, resulting from the conversion of the CD44neg subpopulation, were more invasive in vitro and had a higher clonogenic potential than the sorted CD44high cells, in that they produced mainly holoclones, known to be enriched in stem-like cells. Of interest, the CD44v8-10 is more expressed in human PCa biopsies than in normal gland. The discovery of CD44v8-10pos cells with stem-like and invasive features, derived from a minoritarian CD44neg cell population in PCa, alerts on the high plasticity of stem-like markers and urges for prudency on the approaches to targeting the putative CSC.

Integrin α7 Is a Functional Marker and Potential Therapeutic Target in Glioblastoma.

Functionally relevant markers of glioblastoma stem-like cells (GSCs) have potential for therapeutic targeting to treat this aggressive disease. Here we used generation and screening of thousands of monoclonal antibodies to search for receptors and signaling pathways preferentially enriched in GSCs. We identified integrin α7 (ITGA7) as a major laminin receptor in GSCs and in primary high-grade glioma specimens. Analyses of mRNA profiles in comprehensive datasets revealed that high ITGA7 expression negatively correlated with survival of patients with both low- and high-grade glioma. In vitro and in vivo analyses showed that ITGA7 plays a key functional role in growth and invasiveness of GSCs. We also found that targeting of ITGA7 by RNAi or blocking mAbs impaired laminin-induced signaling, and it led to a significant delay in tumor engraftment plus a strong reduction in tumor size and invasion. Our data, therefore, highlight ITGA7 as a glioblastoma biomarker and candidate therapeutic target.

Differentiating effects of the glucagon-like peptide-1 analogue exendin-4 in a human neuronal cell model.

Glucagon-like peptide-1 (GLP-1) is an insulinotropic peptide with neurotrophic properties, as assessed in animal cell models. Exendin-4, a GLP-1 analogue, has been recently approved for the treatment of type 2 diabetes mellitus. The aim of this study was to morphologically, structurally, and functionally characterize the differentiating actions of exendin-4 using a human neuronal cell model (i.e., SH-SY5Y cells). We found that exendin-4 increased the number of neurites paralleled by dramatic changes in intracellular actin and tubulin distribution. Electrophysiological analyses showed an increase in cell membrane surface and in stretch-activated-channels sensitivity, an increased conductance of Na(+) channels and amplitude of Ca(++) currents (T- and L-type), typical of a more mature neuronal phenotype. To our knowledge, this is the first demonstration that exendin-4 promotes neuronal differentiation in human cells. Noteworthy, our data support the claimed favorable role of exendin-4 against diabetic neuropathy as well as against different neurodegenerative diseases.