Association of melanoma, neural system tumors and germ line mutations at the 9p21 region in the CDKN2A, CDKN2B and CDKN2BAS genes has been reported in a small number of families worldwide and described as a discrete syndrome in melanoma families registered as a rare disease, the melanoma-astrocytoma syndrome.
Recurrent glioblastomas (GBs) are highly aggressive tumors associated with a 6-8 mo survival rate. In this study, we evaluated the possible benefits of an immunotherapeutic strategy based on mature dendritic cells (DCs) loaded with autologous tumor-cell lysates in 15 patients affected by recurrent GB. The median progression-free survival (PFS) of this patient cohort was 4.4 mo, and the median overall survival (OS) was 8.0 mo. Patients with small tumors at the time of the first vaccination (< 20 cm(3); n = 8) had significantly longer PFS and OS than the other patients (6.0 vs. 3.0 mo, p = 0.01; and 16.5 vs. 7.0 mo, p = 0.003, respectively). CD8(+) T cells, CD56(+) natural killer (NK) cells and other immune parameters, such as the levels of transforming growth factor ?, vascular endothelial growth factor, interleukin-12 and interferon ? (IFN?), were measured in the peripheral blood and serum of patients before and after immunization, which enabled us to obtain a vaccination/baseline ratio (V/B ratio). An increased V/B ratio for NK cells, but not CD8(+) T cells, was significantly associated with prolonged PFS and OS. Patients exhibiting NK-cell responses were characterized by high levels of circulating IFN? and E4BP4, an NK-cell transcription factor. Furthermore, the NK cell V/B ratio was inversely correlated with the TGF?2 and VEGF V/B ratios. These results suggest that tumor-loaded DCs may increase the survival rate of patients with recurrent GB after effective tumor debulking, and emphasize the role of the NK-cell response in this therapeutic setting.
Abstract Chromosomal translocations involving the immunoglobulin loci represent frequent oncogenic events in B-cell lymphoma development. Although IRF8 (ICSBP-1) protein expression has been demonstrated in germinal center B-cells and related lymphomas in a single report, the IRF8 gene was not described as an immunoglobulin heavy chain (IGH) translocation partner. In a discovery-driven approach we searched for new translocation partners of IGH in diffuse large B-cell lymphoma (DLBCL) by long distance inverse polymerase chain reaction (LDI-PCR) and Sanger sequencing. A t(14;16)(q32.33;q24.1) IGH/IRF8 was detected in a CD5+de novo DLBCL, confirmed by translocation specific PCR and fluorescence in situ hybridization (FISH) analysis. No further IRF8 aberration could be identified either by LDI-PCR in an additional five CD5+DLBCLs or by FISH on 78 formalin-fixed paraffin-embedded biopsies. Subsequent screening for IRF8 by immunohistochemistry revealed IRF8 expression in 18/78 (23%), correlating with a germinal center B-cell-like (GCB) type of DLBCL. This hitherto unknown translocation t(14;16)(q32.33;q24.1) is likely to represent the initiator of a multistep lymphomagenesis in a CD5+de novo DLBCL.
Non-invasive diagnostic tools are effective in the histomorphological study of melanocytic lesions. The role of melanoma susceptibility genes on melanocytic nevi histopathological features is not clear. The current study aimed to correlate genetic alterations and histomorphological features of melanocytic nevi. Clinical, dermoscopic and confocal features of 34 multiple melanoma patients and 34 controls were compared. Among patients with melanoma, carriers of CDKN2A mutations and/or MC1R variants, and wild-type genes were also compared. In patients with melanoma, a lighter phototype (P = 0.051), a higher number of nevi (P < 0.01) and clinically atypical nevi (P < 0.01) were observed. At dermoscopy, these nevi showed a complex pattern (P = 0.011), atypical network (P = 0.018) and irregular pigmentation (P = 0.037); at confocal, an irregular meshwork pattern (P = 0.026) with atypical nests (P = 0.016) and an inflammatory infiltrate (P = 0.048) were observed. Among patients with melanoma genetically tested, CDKN2A G101W mutation carriers were more frequently younger (P = 0.023), with clinically atypical nevi (P = 0.050), with cytological atypia (P = 0.033) at confocal. G101W mutation and MC1R variants carriers showed hypopigmented nevi (P = 0.002) and, at confocal, roundish cells infiltrating the junction (P = 0.019). These data suggest an influence of CDKN2A mutation and MC1R variants in the development of dysplastic melanocytic lesions. Non-invasive histomorphological evaluation, together with genetic studies, improves melanoma risk identification and early diagnosis, for a patient-tailored management.
PLX4032/vemurafenib is a first-in-class small-molecule BRAF(V600E) inhibitor with clinical activity in patients with BRAF mutant melanoma. Nevertheless, drug resistance develops in treated patients, and strategies to overcome primary and acquired resistance are required. To explore the molecular mechanisms involved in primary resistance to PLX4032, we investigated its effects on cell proliferation and signaling in a panel of 27 genetically characterized patient-derived melanoma cell lines. Cell sensitivity to PLX4032 was dependent on BRAF(V600E) and independent from other gene alterations that commonly occur in melanoma such as PTEN loss, BRAF, and MITF gene amplification. Two cell lines lacking sensitivity to PLX4032 and harboring a different set of genetic alterations were studied as models of primary resistance. Treatment with the MEK inhibitor UO126 but not with PLX4032 inhibited cell growth and ERK activation. Resistance to PLX4032 was maintained after CRAF down-regulation by siRNA indicating alternative activation of MEK-ERK signaling. Genetic characterization by multiplex ligation-dependent probe amplification and analysis of phosphotyrosine signaling by MALDI-TOF mass spectrometry analysis revealed the activation of MET and SRC signaling, associated with the amplification of MET and of CTNNB1 and CCND1 genes, respectively. The combination of PLX4032 with drugs or siRNA targeting MET was effective in inhibiting cell growth and reducing cell invasion and migration in melanoma cells with MET amplification; similar effects were observed after targeting SRC in the other cell line, indicating a role for MET and SRC signaling in primary resistance to PLX4032. Our results support the development of classification of melanoma in molecular subtypes for more effective therapies.
CHEK2 gene mutations occur in a subset of patients with familial breast cancer, acting as moderate/low penetrance cancer susceptibility alleles. Although CHEK2 is no longer recognized as a major determinant of the Li-Fraumeni syndrome, a hereditary condition predisposing to cancer at multiple sites, it cannot be ruled out that mutations of this gene play a role in malignancies arising in peculiar multi-cancer families. To assess the contribution of CHEK2 to the breast cancer/sarcoma phenotype, we screened for germ-line sequence variations of the gene among 12 probands from hereditary breast/ovarian cancer families with one case of sarcoma that tested wild-type for mutations in the BRCA1, BRCA2, and TP53 genes. Two cases harbored previously unreported mutations in CHEK2, the c.507delT and c.38A>G, leading to protein truncation (p.Phe169LeufsX2) and amino acid substitution (p.His13Arg), respectively. These mutations were not considered common polymorphic variants, as they were undetected in 230 healthy controls of the same ethnic origin. While the c.38A>G encodes a mutant protein that behaves in biochemical assays as the wild-type form, the c.507delT is a loss-of-function mutation. The identification of two previously unreported CHEK2 variants, including a truncating mutation leading to constitutional haploinsufficiency, in individuals belonging to families selected for breast cancer/sarcoma phenotype, supports the hypothesis that the CHEK2 gene may act as a factor contributing to individual tumor development in peculiar familial backgrounds.
Gliomas are very invasive brain tumors with poor prognosis and therefore any attempt to limit tumor cell dissemination in the brain is expected to improve glioma treatment. The recent deorphanization of CXCR7 as additional receptor for CXCL12 and CXCL11 has raised key issues on its interaction with the CXCL12/CXCR4 axis as a mechanism to modulate glioma cell migration. In this work we investigated protein and mRNA expression of the two chemokines CXCL12 and CXCL11, together with their receptors CXCR4 and CXCR7 in human glioma specimens and cell lines by immunohistochemistry, flow cytometry and quantitative real-time PCR. The main purpose of this study was to find out whether and at what extent CXCR4 and CXCR7 are differentially expressed in glioma cells. In human glioma specimens the levels of CXCL11 and CXCR4 mRNA were significantly higher in glioblastomas compared to non-tumor controls or low grade gliomas, whilst no difference was found for CXCL12 and CXCR7 mRNA expression. In cell lines, flow cytometry and immunocytochemical experiments showed CXCR4 was mainly expressed irrespective of its membrane or intracellular localization. In contrast, a predominant intracellular localization together with a negligible membrane expression of CXCR7 was found in all cells examined. In in vitro experiments CXCR4 and CXCR7 antagonists and the silencing of CXCR4 showed complete inhibition of glioma proliferation. Our findings, in agreement with previous data, suggest that in human glioma cells the prevalent intracellular localization of CXCR7 might modulate the functionality of CXCL11/12 either acting as a scavenger for these chemokines or interfering with the signaling pathways activated by the stimulation of CXCR4.
Until now, the evaluation of the effectiveness of guideline implementation in nursing and allied health professions has received relatively little attention. The aims of this study were (i) to describe the development process of guidelines concerning the management of peripheral venous catheters (PVCs) implemented in an Italian hospital; and (ii) to evaluate the effectiveness of guideline dissemination in terms of both clinical outcomes (signs of infection) and process outcomes (measures of appropriateness of PVC management).
The biological behavior of chronic lymphocytic leukemia and small lymphocytic lymphoma is unpredictable. Nonetheless, non-mutated IgV(H) gene rearrangement, ATM (11q22-23) and p53 (17p13) deletion are recognized as unfavorable prognosticators in chronic lymphocytic leukemia. The mRNA expression of activation-induced cytidine deaminase (AID), an enzyme indispensable for somatic hypermutation processes, was claimed to be predictive of non-mutated chronic lymphocytic leukemia cells in blood. Here, we evaluated AID protein expression compared with known molecular and immunohistochemical prognostic indicators in 71 chronic lymphocytic leukemia/small lymphocytic lymphoma patients using a tissue microarray approach. We found AID heterogeneously expressed in tumor cells as shown by colocalization analysis for CD5 and CD23. Ki-67 positive paraimmunoblasts of the proliferation centers displayed the highest expression. This observation is reflected by a significant association of AID positivity with a high proliferation rate (P=0.012). ATM deletion was detected in 10% (6/63) of patients and p53 deletion in 19% (13/67) of patients. Moreover, both ATM (P=0.002) and p53 deletion (P=0.004) were significantly associated with AID. IgV(H) gene mutation was seen in 45% (27/60) of patients. Twenty-five percent (17/69) of patients with AID-positive chronic lymphocytic leukemia/small lymphocytic lymphoma displayed a shorter survival than AID-negative chronic lymphocytic leukemia/small lymphocytic lymphoma patients (61 vs 130 months, P=0.001). Although there was a trend, we could not show an association with the IgV(H) gene mutation status. Taken together, our study shows that AID expression is an indicator of an unfavorable prognosis in chronic lymphocytic leukemia/small lymphocytic lymphoma patients, although it is not a surrogate marker for the IgV(H) status. Furthermore, the microenvironment of proliferation centers seems to influence AID regulation and might be an initiating factor in its transformation.
To evaluate molecular and immunohistochemical markers to develop a molecular grading of urothelial bladder cancer and to test these markers in voided urine samples. Experimental Design: 255 consecutive biopsies from primary bladder cancer patients were evaluated on a tissue microarray. The clinical parameters gender, age, adjacent carcinoma in situ, and multifocality were collected. UroVysion fluorescence in situ hybridization (FISH) was done. Expression of cytokeratin 20, MIB1, and TP53 was analyzed by immunohistochemistry. Fibroblast growth factor receptor 3 (FGFR3) status was studied by SNaPshot mutation detection. Results were correlated with clinical outcome by Cox regression analysis. To assess the predictive power of different predictor subsets to detect high grade and tumor invasion, logistic regression models were learned. Additionally, voided urine samples of 119 patients were investigated. After cytologic examination, urine samples were matched with their biopsies and analyzed for loss of heterozygosity (LOH), FGFR3 mutation, polysomy, and p16 deletion using UroVysion FISH. Receiver operator characteristic curves for various predictor subsets were plotted.
Childhood cutaneous melanoma is a rare disease with increasing incidence. It is not clear whether it differs from adult melanoma in etiology and clinical evolution. To genetically characterize childhood melanoma, 21 pediatric patients were studied by germ-line analysis of CDKN2A, CDK4, and MC1R genes. In addition, alterations in CDKN2A, c-Kit, BRAF, and NRAS genes were evaluated at the somatic level by direct gene sequencing, fluorescence in situ hybridization analysis, and immunohistochemistry. As a control group of susceptible patients, we studied patients from 23 melanoma-prone families. At the germ-line level, CDKN2A and MC1R gene variants were detected in 2/21 and 12/21 pediatric patients and in 9/23 and 19/22 in familial patients. At the somatic level, most lesions (9/14) from pediatric patients showed CDKN2A locus homozygous deletions and a null p16 immunophenotype, whereas most lesions (5/8) from familial patients were disomic and immunoreactive. A c-Kit low-polysomy profile seems to parallel CDKN2A homozygous deletions in pediatric melanoma whereas the single activating mutation observed segregates with familial patients. Loss of KIT protein expression was frequent (7/14) in pediatric melanomas, where metastatic cases were prevalent. BRAF(V600E) mutation occurred at a similar rate (approximately 50%) in lesions from pediatric and familial patients, whereas no NRAS mutations were detected.
Immune-based treatments represent a promising new class of therapy designed to boost the immune system to specifically eradicate malignant cells. Immunotherapy may generate specific anti-tumor immune responses, and dendritic cells (DC), professional antigen-presenting cells, are widely used in experimental cancer immunotherapy. Several reports describe methods for the generation of mature, antigen-pulsed DC for clinical use. Improved quality and standardization are desirable to obtain GMP-compliant protocols. In this study we describe the generation of DC from 31 Glioblastoma (GB) patients starting from their monocytes isolated by immunomagnetic CD14 selection using the CliniMACS® device. Upon differentiation of CD14+ with IL-4 and GM-CSF, DC were induced to maturation with TNF-?, PGE(2), IL-1?, and IL-6. Whole tumor lysate was obtained, for the first time, in a closed system using the semi-automated dissociator GentleMACS®. The yield of proteins improved by 130% compared to the manual dissociation method. Interestingly the Mean Fluorescence Intensity for CD83 increased significantly in DC pulsed with "new method" lysate compared to DC pulsed with "classical method" lysate. Our results indicate that immunomagnetic isolation of CD14(+) monocytes using the CliniMACS® device and their pulsing with whole tumor lysate proteins is a suitable method for clinical-scale generation of high quality, functional DC under GMP-grade conditions.
To evaluate chromosomal instability at 9p21-22 with p16 protein expression in organ transplant recipients (OTRs) compared with immunocompetent patients with squamous cell carcinoma (SCC).
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