GJB5 association with BRAF mutation and survival in cutaneous malignant melanoma.

BACKGROUND: Gap-junctional intercellular communication is crucial for epidermal cellular homeostasis. Inability to establish melanocyte-keratinocyte contact and loss of the intercellular junction's integrity may contribute to melanoma development. Connexins, laminins and desmocollins have been implicated in the control of melanoma growth, where their reduced expression has been reported in metastatic lesions. OBJECTIVES: The aim of this study was to investigate connexin 31·1 (GJB5) expression and identify any association with BRAF mutational status, prognosis of patients with melanoma and mitogen-activated protein kinase (MAPK) inhibitor (MAPKi) treatment. METHODS: GJB5 expression was measured at RNA and protein level in melanoma clinical samples and established cell lines treated (or not) with BRAF and MEK inhibitors (MEKi), as well as in cell lines which developed MAPKi resistance. Findings were further validated and confirmed by analysis of independent datasets. RESULTS: Our analysis reveals significant downregulation of GJB5 expression in metastatic melanoma lesions compared with primary ones and in BRAF-mutated vs. BRAF-wildtype (BRAFWT ) melanomas. Likewise, GJB5 expression is significantly lower in BRAFV600E compared with BRAFWT cell lines and increases on MAPKi treatment. MAPKi-resistant melanoma cells display a similar expression pattern compared with BRAFWT cells, with increased GJB5 expression associated with morphological changes. Enhancement of BRAFV600E expression in BRAFWT melanoma cells significantly upregulates miR-335-5p expression with consequent downregulation of GJB5, one of its targets. Furthermore, overexpression of miR-335-5p in two BRAFWT cell lines confirms specific GJB5 protein downregulation. Reverse transcriptase quantitative polymerase chain reaction analysis also revealed upregulation of miR-335 in BRAFV600E melanoma cells, which is significantly downregulated in cells resistant to MEKi. Our data were further validated using the TCGA_SKCM dataset, where BRAF mutations associate with increased miR-335 expression and inversely correlate with GJB5 expression. In clinical samples, GJB5 underexpression is also associated with patient overall worse survival, especially at early stages. CONCLUSIONS: We identified a significant association between metastases/BRAF mutation and low GJB5 expression in melanoma. Our results identify a novel mechanism of gap-junctional protein regulation, suggesting a prognostic role for GJB5 in cutaneous melanoma.

High BRAF variant allele frequencies are associated with distinct pathological features and responsiveness to target therapy in melanoma patients.

BACKGROUND: BRAF mutant melanoma patients are commonly treated with anti-BRAF therapeutic strategies. However, many factors, including the percentage of BRAF-mutated cells, may contribute to the great variability in patient outcomes. PATIENTS AND METHODS: The BRAF variant allele frequency (VAF; defined as the percentage of mutated alleles) of primary and secondary melanoma lesions, obtained from 327 patients with different disease stages, was assessed by pyrosequencing. The BRAF mutation rate and VAF were then correlated with melanoma pathological features and patients' clinical characteristics. Kaplan-Meier curves were used to study the correlations between BRAF VAF, overall survival (OS), and progression-free survival (PFS) in a subset of 62 patients treated by anti-BRAF/anti-MEK therapy after metastatic progression. RESULTS: A highly heterogeneous BRAF VAF was identified (3%-90%). Besides being correlated with age, a higher BRAF VAF level was related to moderate lymphocytic infiltration (P = 0.017), to melanoma thickness according to Clark levels, (level V versus III, P = 0.004; level V versus IV, P = 0.04), to lymph node metastases rather than cutaneous (P = 0.04) or visceral (P = 0.03) secondary lesions. In particular, a BRAF VAF >25% was significantly associated with a favorable outcome in patients treated with the combination of anti-BRAF/anti-MEK drug (OS P = 0.04; PFS P = 0.019), retaining a significant value as an independent factor for the OS and the PFS in the multivariate analysis (P = 0.014 and P = 0.003, respectively). CONCLUSION: These results definitively support the role of the BRAF VAF as a potential prognostic and predictive biomarker in melanoma patients in the context of BRAF inhibition.

Preclinical immunotherapy with Cytokine-Induced Killer lymphocytes against epithelial ovarian cancer.

Despite improvements in surgery and medical treatments, epithelial ovarian cancer (EOC) remains the most lethal gynaecological malignancy. Aim of this study is to investigate the preclinical immunotherapy activity of cytokine-induced killer lymphocytes (CIK) against epithelial ovarian cancers, focusing on platinum-resistant settings. We generated CIK ex vivo starting from human peripheral blood samples (PBMCs) collected from EOC patients. Their antitumor activity was tested in vitro and in vivo against platinum-resistant patient-derived ovarian cancer cells (pdOVCs) and a Patient Derived Xenograft (PDX), respectively. CIK were efficiently generated (48 fold median ex vivo expansion) from EOC patients; pdOVCs lines (n = 9) were successfully generated from metastatic ascites; the expression of CIK target molecules by pdOVC confirmed pre and post treatment in vitro with carboplatin. The results indicate that patient-derived CIK effectively killed autologous pdOVCs in vitro. Such intense activity was maintained against a subset of pdOVC that survived in vitro treatment with carboplatin. Moreover, CIK antitumor activity and tumor homing was confirmed in vivo within an EOC PDX model. Our preliminary data suggest that CIK are active in platinum resistant ovarian cancer models and should be therefore further investigated as a new therapeutic option in this extremely challenging setting.

Predicting locally advanced rectal cancer response to neoadjuvant therapy with 18F-FDG PET and MRI radiomics features.

PURPOSE: Pathological complete response (pCR) following neoadjuvant chemoradiotherapy or radiotherapy in locally advanced rectal cancer (LARC) is reached in approximately 15-30% of cases, therefore it would be useful to assess if pretreatment of 18F-FDG PET/CT and/or MRI texture features can reliably predict response to neoadjuvant therapy in LARC. METHODS: Fifty-two patients were dichotomized as responder (pR+) or non-responder (pR-) according to their pathological tumor regression grade (TRG) as follows: 22 as pR+ (nine with TRG = 1, 13 with TRG = 2) and 30 as pR- (16 with TRG = 3, 13 with TRG = 4 and 1 with TRG = 5). First-order parameters and 21 second-order texture parameters derived from the Gray-Level Co-Occurrence matrix were extracted from semi-automatically segmented tumors on T2w MRI, ADC maps, and PET/CT acquisitions. The role of each texture feature in predicting pR+ was assessed with monoparametric and multiparametric models. RESULTS: In the mono-parametric approach, PET homogeneity reached the maximum AUC (0.77; sensitivity = 72.7% and specificity = 76.7%), while PET glycolytic volume and ADC dissimilarity reached the highest sensitivity (both 90.9%). In the multiparametric analysis, a logistic regression model containing six second-order texture features (five from PET and one from T2w MRI) yields the highest predictivity in distinguish between pR+ and pR- patients (AUC = 0.86; sensitivity = 86%, and specificity = 83% at the Youden index). CONCLUSIONS: If preliminary results of this study are confirmed, pretreatment PET and MRI could be useful to personalize patient treatment, e.g., avoiding toxicity of neoadjuvant therapy in patients predicted pR-.

CD44v6 as innovative sarcoma target for CAR-redirected CIK cells.

Purpose of our study was to explore a new immunotherapy for high grade soft tissue sarcomas (STS) based on cytokine-induced killer cells (CIK) redirected with a chimeric antigen receptor (CAR) against the tumor-promoting antigen CD44v6. We aimed at generating bipotential killers, combining the CAR specificity with the intrinsic tumor-killing ability of CIK cells (CAR+.CIK). We set a patient-derived experimental platform. CAR+.CIK were generated by transduction of CIK precursors with a lentiviral vector encoding for anti-CD44v6-CAR. CAR+.CIK were characterized and assessed in vitro against multiple histotypes of patient-derived STS. The anti-sarcoma activity of CAR+.CIK was confirmed in a STS xenograft model. CD44v6 was expressed by 40% (11/27) of patient-derived STS. CAR+.CIK were efficiently expanded from patients (n = 12) and killed multiple histotypes of STS (including autologous targets, n = 4). The killing activity was significantly higher compared with unmodified CIK, especially at low effector/target (E/T) ratios: 98% vs 82% (E/T = 10:1) and 68% vs 26% (1:4), (p<0.0001). Specificity of tumor killing was confirmed by blocking with anti-CD44v6 antibody. CAR+.CIK produced higher amounts of IL6 and IFN-γ compared to control CIK. CAR+.CIK were highly active in mice bearing subcutaneous STS xenografts, with significant delay of tumor growth (p<0.0001) without toxicities. We report first evidence of CAR+.CIK's activity against high grade STS and propose CD44v6 as an innovative target in this setting. CIK are a valuable platform for the translation of CAR-based strategies to challenging field of solid tumors. Our findings support the exploration of CAR+.CIK in clinical trials against high grade STS.

Favourable prognostic role of histological regression in stage III positive sentinel lymph node melanoma patients.

BACKGROUND: Sentinel lymph node (SLN)-positive melanoma patients are a heterogeneous group of patients with survival rates ranging from ∼20 to over 80%. No data are reported concerning the role of histological regression on survival in stage III melanoma. METHODS: The study included 365 patients with positive SLN from two distinct hospitals. The model was developed on patients from 'AOU Città della Salute e della Scienza di Torino', and externally validated on patients from IRCCS of Candiolo. Survival analyses were carried out according to the presence of regression and adjusted for all other prognostic factors. RESULTS: Among patients followed at 'AOU Città della Salute e della Scienza di Torino' (n=264), the median follow-up time to death or censoring (whatever two events occurred earlier) was 2.7 years since diagnosis (interquartile range: 1.3-5.8). In all, 79 patients died from melanoma and 11 from other causes. Histological regression (n=43) was associated with a better prognosis (sub-HR=0.34, CI 0.12-0.92), whereas the other factors above showed an inverse association. In the external validation, the concordance index was 0.97 at 1 year and decreased to 0.66 at 3 years and to 0.59 at 5 years. Adding histological regression in the prognostic model increased the discriminative ability to 0.75 at 3 years and to 0.62 at 5 years. Finally, using a cutoff of 20% for the risk of death led to a net re-classification improvement of 15 and 11% at 3 and 5 years after diagnosis, respectively. CONCLUSIONS: Histological regression could lead to an improvement in prognostic prediction in patients with stage III-positive SLN melanoma.

Surgical treatment of primary melanoma of the umbilicus with sentinel lymph node biopsy and plastic reconstruction: case report and review of the literature.

UNLABELLED: AIMS, PATIENTS AND METHODS: The umbilical melanoma is rare, and the surgical treatment can create difficulties for both radical excision and plastic reconstruction. Our aims are to present a case of primary melanoma of the umbilicus and to discuss the best surgical treatment, as well as review the relevant literature. RESULTS: Surgical excision of primary melanoma of the umbilicus must be carried out to reach the peritoneum. Sentinel lymph node biopsy must be carried as well as plastic reconstruction. CONCLUSION: Despite the progress in new medical therapy for melanoma, suitable surgical excision is, at present, the only treatment able to improve patient prognosis. In this report we describe the surgical treatment and plastic reconstruction of a case of umbilical melanoma.

CD31 and CD34 expression as immunohistochemical markers of endothelial transdifferentiation in human cutaneous melanoma.

INTRODUCTION: Vasculogenic mimicry, as previously described in aggressive melanoma, is characterized by the de novo generation of intratumoral patterned vascular channels, composed of PAS-positive basement membrane in the absence of endothelial cells, providing additional microcirculation, in support to the classic tumoral angiogenesis. METHODS: We investigated the immunohistochemical expression of two endothelial markers, CD31 and CD34, in tumoral cells of 60 melanomas (45 primary cutaneous and 15 metastatic) as possible evidence of vasculogenic mimicry. In addition we investigated the relationship between CD31 and CD34 expression and three pathological markers such as Clark's level, and skin ulceration, predictive of melanoma's aggressive behaviour, and mitotic index. RESULTS: No cases of common melanocytic nevi immunoreacted with CD31 or CD34. Random CD31 immunoreactivity was present in 6% of Clark's level I/II, 50% of Clark's level III and 80% Clark's level IV/V. CD34 was negative in Clark's level I/II but randomly stained the 20% and 55% of level III and IV/V respectively. 66% (10/15) of metastatic melanomas were CD31 positive showing a canalicular immunostaining pattern, conversely CD34 expression was never found. 7/8 cutaneous ulcerated melanomas immunostained for CD31 and 4/8 for CD34. CD31 immunostained 88% high/intermediate MI, and 53% of low MI melanomas. CD34 decorated the 29% of high/intermediate and 38% of low MI melanomas. CONCLUSIONS: CD31 and CD34 immunoreactivity closely parallel both the different morphologic steps of melanocytic tumor progression and the presence of histological parameters related to the aggressive behaviour. Their expression could be related to endothelial transdifferentiation of melanoma cells although a consequent functional role has not been demonstrated yet.

Breastfeeding and perceived changes in the appearance of the breasts: a retrospective study.

AIM: To investigate the association between breastfeeding and the perception that women have of changes in the appearance of their breasts. METHODS: Four hundred and ninety-six Italian women were interviewed in three health centres 18 mo (SD 3.4 mo, range 12.6-23.1 mo) after the birth of their first baby in May 2002. Information was collected on pregnancy, infant feeding and bra cup size before pregnancy. The main outcome measures were self-reported changes in the appearance of the breasts (enlargement or reduction in breast size and loss of firmness) and bra cup size at the time of the interview. RESULTS: Seventy-three percent of the mothers reported that their breasts were different compared with before pregnancy; enlargement and loss of firmness representing the most common changes. The prevalence of changes among the mothers who had and had not breastfed was 75% and 69%, respectively (relative risk: 1.09, 95% CI: 0.96-1.23). Bra cup size before pregnancy was neither associated with the frequency of breastfeeding nor with the occurrence of changes in the appearance of the breasts. CONCLUSIONS: In Italy, mothers frequently report that the size and the shape of their breasts have changed after childbirth, but these changes do not seem to be associated with breastfeeding.

Developing follicles of the spotted ray Torpedo marmorata express different glycoside residues in relation to granulosa differentiation and vitelline envelope formation.

Lectins constitute a class of proteins/glycoproteins that specifically bind to terminal glycoside residues. The present investigation aimed to identify lectin-binding sites in developing follicles of Torpedo marmorata. Using eleven lectins (WGA, GSI-A4, GSI-B4, PSA, UEA-I, PNA, MPA, Con-A, DBA, LCA, BPA, SBA), we demonstrated that the biochemical nature and the distribution of carbohydrate residues significantly change during oogenesis in the granulosa cells and the vitelline envelope. In fact, a progressive appearance of surface glycoproteins bearing terminated ss-GlcNAc O-linked side chains was observed in the granulosa during the differentiation of pyriform-like cells from the small ones via intermediate cells simultaneously with a significant reduction of the D-Gal chains present in their nucleus. Glycoproteins bearing ss-GlcNAc O-linked side chains were first evident on the surface of small cells in contact with the oocyte, then on the intermediate ones, and finally on pyriform-like cells. The distribution pattern of such glycoproteins over the differentiated granulosa cells remained unchanged during the subsequent stages of the oocyte growth so granulosa cells preserved the same sugar distribution pattern. Furthermore, a progressive loss of D-Gal residues was evident in the nucleus of granulosa cells. In fact, staining for D-Gal was intense in the nucleus of small follicle cells and progressively reduced till disappearing in differentiated pyriform-like cells. Conversely, the small follicle cells located under the basal lamina were devoid of ss-GlcNAc residues, and the nuclear content in D-Gal remained unchanged. This finding strongly suggests that surface glycoproteins containing ss-GlcNAc residues, and the nuclear content in D-Gal might be related to the differentiation of pyriform-like cells. The present investigation also demonstrates that the content of the sugar residues of the vitelline envelope (VE) changes during oocyte growth, suggesting that pyriform-like cells may contribute to its formation.

Down syndrome and breastfeeding.

AIM: The aim of the study was to investigate the frequency of breastfeeding among children with Down syndrome. METHODS: The mothers of 560 children with Down syndrome attending four university hospitals in Italy were interviewed and the neonatal clinical records retrieved. Information was collected on the type of infant feeding and on why some mothers had not breastfed their children. Two groups of healthy children whose feeding habits had been previously investigated were recruited as control subjects (1601 and 714, respectively). A paediatrician in each hospital was interviewed about the neonatal admission policy of children with Down syndrome. RESULTS: Among the 560 Down children, 246 (44%) were admitted to the neonatal unit. Compared with the two control groups, children with Down syndrome were significantly more frequently bottle-fed (57% vs 15% and 24%, respectively, odds ratio 7.5, 95% CI 6.0-9.4 and 4.2, 95% CI 3.3-5.4. respectively). Only 30% of infants admitted to the neonatal unit were breastfed. The main reasons reported by the mothers for not having breastfed were infants' illness in infants who had been admitted to the neonatal unit and frustration or depression, perceived milk insufficiency and difficulty with suckling for those babies who had not been admitted to the unit. The paediatricians reported that the admission of a baby with Down syndrome to the neonatal unit could sometimes take place not for medical reasons, but for diagnostic work-up or for a more appropriate diagnosis and to maintain communication with the family. CONCLUSIONS: Down syndrome babies are less frequently breastfed compared with healthy children. Support in breastfeeding should become a relevant point of health supervision for children with Down syndrome.

[Littoral hemangioma of the spleen]. / Emantioma littorale della milza.

The authors report a rare case of littoral hemangioma of the spleen (LHS) accompanied by a revision of the literature on the argument. A male 65-year-old patient was referred to their attention with suspected ultrasonographic diagnosis of lymphoma with a splenic localisation. The complete CT diagnosis led to suspected splenic angioma. During surgery, anatomopathological analysis of the biopsy revealed LHS. The pathological anatomy showed lesions ranging in size from small foci to large nodules which almost completely replaced the splenic parenchyma. These areas were made up of vascular canals or axes that imitate splenic sinuses and have irregular lumen, often appearing as papillary projections and cyst-like spaces; they are bordered by high (cylindrical) endothelial cells that project into the vascular lumen and reveal hemophagocytosis; there is very little mitotic activity. The patient was discharged 7 days after surgery. The authors underline the extreme rarity of this neoplasm and the virtual absence of symptoms, although some cases report signs of hypersplenism, including platelet deficiency and anemia. The diagnostic iter must take care to exclude other pathologies affecting the spleen, including lymphoma, metastases and primary malignant splenic tumours. Lastly, a differential diagnosis must be made with the malignant variant, littoral hemangiosarcoma of the spleen.

Iron status in breast-fed infants.

Iron status of 30 infants who had been breast fed until their first birthday and who had never received cow milk, medicinal iron, or iron-enriched formula and cereals was investigated; 30% were anemic at 12 months of age. The duration of exclusive breast-feeding was significantly longer among nonanemic infants (6.5 vs 5.5 months). None of the infants who were exclusively breast fed for 7 months or more and 43% of those who were breast fed for a shorter time were anemic. Infants who were exclusively breast fed for a prolonged period had a good iron status at 12 and 24 months.