Decreased differentiation capacity and altered expression of extracellular matrix components in irradiation-mediated senescent human breast adipose-derived stem cells.

Radiotherapy is widely used for the treatment of breast cancer. However, we have shown that ionizing radiation can provoke premature senescence in breast stromal cells. In particular, breast stromal fibroblasts can become senescent after irradiation both in vitro and in vivo and they express an inflammatory phenotype and an altered profile of extracellular matrix components, thus facilitating tumor progression. Adipose-derived stem cells (ASCs) represent another major component of the breast tissue stroma. They are multipotent cells and due to their ability to differentiate in multiple cell lineages they play an important role in tissue maintenance and repair in normal and pathologic conditions. Here, we investigated the characteristics of human breast ASCs that became senescent prematurely after their exposure to ionizing radiation. We found decreased expression levels of the specific mesenchymal cell surface markers CD105, CD73, CD44, and CD90. In parallel, we demonstrated a significantly reduced expression of transcription factors regulating osteogenic (i.e., RUNX2), adipogenic (i.e., PPARγ), and chondrogenic (i.e., SOX9) differentiation; this was followed by an analogous reduction in their differentiation capacity. Furthermore, they overexpress inflammatory markers, that is, IL-6, IL-8, and ICAM-1, and a catabolic phenotype, marked by the reduction of collagen type I and the increase of MMP-1 and MMP-13 expression. Finally, we detected changes in proteoglycan expression, for example, the upregulation of syndecan 1 and syndecan 4 and the downregulation of decorin. Notably, all these alterations, when observed in the breast stroma, represent poor prognostic factors for tumor development. In conclusion, we showed that ionizing radiation-mediated prematurely senescent human breast ASCs have a decreased differentiation potential and express specific changes adding to the formation of a permissive environment for tumor growth.

Down-Regulation of the Proteoglycan Decorin Fills in the Tumor-Promoting Phenotype of Ionizing Radiation-Induced Senescent Human Breast Stromal Fibroblasts.

Down-regulation of the small leucine-rich proteoglycan decorin in the stroma is considered a poor prognostic factor for breast cancer progression. Ionizing radiation, an established treatment for breast cancer, provokes the premature senescence of the adjacent to the tumor stromal fibroblasts. Here, we showed that senescent human breast stromal fibroblasts are characterized by the down-regulation of decorin at the mRNA and protein level, as well as by its decreased deposition in the pericellular extracellular matrix in vitro. Senescence-associated decorin down-regulation is a long-lasting process rather than an immediate response to γ-irradiation. Growth factors were demonstrated to participate in an autocrine manner in decorin down-regulation, with bFGF and VEGF being the critical mediators of the phenomenon. Autophagy inhibition by chloroquine reduced decorin mRNA levels, while autophagy activation using the mTOR inhibitor rapamycin enhanced decorin transcription. Interestingly, the secretome from a series of both untreated and irradiated human breast cancer cell lines with different molecular profiles inhibited decorin expression in young and senescent stromal fibroblasts, which was annulled by SU5402, a bFGF and VEGF inhibitor. The novel phenotypic trait of senescent human breast stromal fibroblasts revealed here is added to their already described cancer-promoting role via the formation of a tumor-permissive environment.

Ionizing radiation-mediated premature senescence and paracrine interactions with cancer cells enhance the expression of syndecan 1 in human breast stromal fibroblasts: the role of TGF-ß.

The cell surface proteoglycan syndecan 1 (SDC1) is overexpressed in the malignant breast stromal fibroblasts, creating a favorable milieu for tumor cell growth. In the present study, we found that ionizing radiation, a well-established treatment in human breast cancer, provokes premature senescence of human breast stromal fibroblasts in vitro, as well as in the breast tissue in vivo. These senescent cells were found to overexpress SDC1 both in vitro and in vivo. By using a series of specific inhibitors and siRNA approaches, we showed that this SDC1 overexpression in senescent cells is the result of an autocrine action of Transforming Growth Factor-ß (TGF-ß) through the Smad pathway and the transcription factor Sp1, while the classical senescence pathways of p53 or p38 MAPK - NF-kB are not involved. In addition, the highly invasive human breast cancer cells MDA-MB-231 (in contrast to the low-invasive MCF-7) can also enhance SDC1 expression, both in early-passage and senescent fibroblasts via a paracrine action of TGF-ß. The above suggest that radiation-mediated premature senescence and invasive tumor cells, alone or in combination, enhance SDC1 expression in breast stromal fibroblasts, a poor prognostic factor for cancer growth, and that TGF-ß plays a crucial role in this process.

Sonication assisted microbiological diagnosis of implant-related infection caused by Prevotella disiens and Staphylococcus epidermidis in a patient with cranioplasty.

BACKGROUND: Infections present a major complication of cranioplasty procedures and in many cases removal of the implant material becomes a necessity. Sonication of the artificial implant material has been used during the last years, in order to facilitate better diagnosis of these infections, nevertheless its use in cranial implant infections is still limited. CASE PRESENTATION: A case of a 63-year-old Caucasian male patient who underwent a decompressive craniectomy, due to intracranial hemorrhage, and a consequent cranioplasty using an autogenic bone flap fixed by titanium clamps, is reported. After three unsuccessful cranioplasty efforts to repair a persistent skin defect, removing the bone flap and the titanium clamps was a necessity. Tissue and bone cultures were unable to reveal any microorganism whilst sonication of the removed titanium clamps and consequent culture of the resulting sonication liquid yielded Prevotella disiens and Staphylococcus epidermidis. The patient was treated with daptomycin and metronidazole until discharge and the skin defect was successfully repaired. CONCLUSION: The present case report indicates that the use of the sonication procedure assisted the microbiological diagnosis. This is the first known neurosurgical case of the implementation of the sonication procedure.

A new mathematical model for the interpretation of translational research evaluating six CTLA-4 polymorphisms in high-risk melanoma patients receiving adjuvant interferon.

Adjuvant therapy of stage IIB/III melanoma with interferon reduces relapse and mortality by up to 33% but is accompanied by toxicity-related complications. Polymorphisms of the CTLA-4 gene associated with autoimmune diseases could help in identifying interferon treatment benefits. We previously genotyped 286 melanoma patients and 288 healthy (unrelated) individuals for six CTLA-4 polymorphisms (SNP). Previous analyses found no significant differences between the distributions of CTLA-4 polymorphisms in the melanoma population vs. controls, no significant difference in relapse free and overall survivals among patients and no correlation between autoimmunity and specific alleles. We report new analysis of these CTLA-4 genetic profiles, using Network Phenotyping Strategy (NPS). It is graph-theory based method, analyzing the SNP patterns. Application of NPS on CTLA-4 polymorphism captures allele relationship pattern for every patient into 6-partite mathematical graph P. Graphs P are combined into weighted 6-partite graph S, which subsequently decomposed into reference relationship profiles (RRP). Finally, every individual CTLA-4 genotype pattern is characterized by the graph distances of P from eight identified RRP's. RRP's are subgraphs of S, collecting equally frequent binary allele co-occurrences in all studied loci. If S topology represents the genetic "dominant model", the RRP's and their characteristic frequencies are identical to expectation-maximization derived haplotypes and maximal likelihood estimates of their frequencies. The graph-representation allows showing that patient CTLA-4 haplotypes are uniquely different from the controls by absence of specific SNP combinations. New function-related insight is derived when the 6-partite graph reflects allelic state of CTLA-4. We found that we can use differences between individual P and specific RRPs to identify patient subpopulations with clearly different polymorphic patterns relatively to controls as well as to identify patients with significantly different survival.

Evaluation of six CTLA-4 polymorphisms in high-risk melanoma patients receiving adjuvant interferon therapy in the He13A/98 multicenter trial.

PURPOSE: Interferon is approved for adjuvant treatment of patients with stage IIb/III melanoma. The toxicity and uncertainty regarding survival benefits of interferon have qualified its acceptance, despite significant durable relapse prevention in a fraction of patients. Predictive biomarkers that would enable selection of patients for therapy would have a large impact upon clinical practice. Specific CTLA-4 polymorphisms have previously shown an association with response to CTLA-4 blockade in patients with metastatic melanoma and the development of autoimmunity. EXPERIMENTAL DESIGN: 286 melanoma patients and 288 healthy controls were genotyped for six CTLA-4 polymorphisms previously suggested to be important (AG 49, CT 318, CT 60, JO 27, JO30 and JO 31). Specific allele frequencies were compared between the healthy and patient populations, as well as presence or absence of these in relation to recurrence. Alleles related to autoimmune disease were also investigated. RESULTS: No significant differences were found between the distributions of CTLA-4 polymorphisms in the melanoma population compared with healthy controls. Relapse free survival (RFS) and overall survival (OS) did not differ significantly between patients with the alleles represented by these polymorphisms. No correlation between autoimmunity and specific alleles was shown. The six polymorphisms evaluated where strongly associated (Fisher's exact p-values < 0.001 for all associations) and significant linkage disequilibrium among these was indicated. CONCLUSION: No polymorphisms of CTLA-4 defined by the SNPs studied were correlated with improved RFS, OS, or autoimmunity in this high-risk group of melanoma patients.

Randomized phase III study of 1 month versus 1 year of adjuvant high-dose interferon alfa-2b in patients with resected high-risk melanoma.

PURPOSE: A high-dose interferon alfa (IFN-alpha) regimen as reported in E1684 was unique for the incorporation of an induction phase of maximally tolerated dosages of intravenous (IV) therapy for the initial 4 weeks. This is the only trial that has shown prolongation of overall survival and relapse-free survival (RFS) in comparison with observation. Analysis of the hazard curves for RFS and overall survival (OS) in E1684 revealed separation of the high-dose and observation arms, suggesting that the induction phase may represent a critical component of this regimen, although this has not been tested prospectively. PATIENTS AND METHODS: We conducted a prospective randomized study of IV induction therapy versus a full year of high-dose IFN, with primary end points of RFS and OS for patients with stage IIB, IIC, and III melanoma, within 56 days of curative surgery. Patients were randomly assigned to receive IFN-alpha-2b 15 x 10(6) U/m2 IV x 5/7 days weekly x 4 weeks (arm A) versus the same regimen followed by IFN-alpha-2b 10 x 10(6) U (flat dose) administered subcutaneously three times a week for 48 weeks (arm B). RESULTS: Between 1998 and 2004, 364 patients were enrolled (353 eligible: arm A, n = 177; arm B, n = 176). At a median follow-up of 63 months (95% CI, 58.1 to 67.7), the median RFS was 24.1 months versus 27.9 months (P = .9) and the median OS was 64.4 months versus 65.3 months (P = .49). Patients in arm B had more grade 1 to 2 hepatotoxicity, nausea/vomiting, alopecia, and neurologic toxicity. CONCLUSION: There were no significant differences in OS and RFS between the regimens of 1 month and 1 year of treatment.

Prognostic significance of autoimmunity during treatment of melanoma with interferon.

BACKGROUND: Immunotherapy for advanced melanoma induces serologic and clinical manifestations of autoimmunity. We assessed the prognostic significance of autoimmunity in patients with stage IIB, IIC, or III melanoma who were treated with high-dose adjuvant interferon alfa-2b. METHODS: We enrolled 200 patients in a substudy of a larger, ongoing randomized trial. Blood was obtained before the initiation of intravenous interferon therapy, after 1 month of therapy, and at 3, 6, 9, and 12 months. Serum was tested for antithyroid, antinuclear, anti-DNA, and anticardiolipin autoantibodies, and patients were examined for vitiligo. RESULTS: The median duration of follow-up was 45.6 months. Relapse occurred in 115 patients, and 82 patients died. The median relapse-free survival was 28.0 months, and the median overall survival was 58.7 months. Autoantibodies and clinical manifestations of autoimmunity were detected in 52 patients (26 percent). The median relapse-free survival was 16.0 months among patients without autoimmunity (108 of 148 had a relapse) and was not reached among patients with autoimmunity (7 of 52 had a relapse). The median survival was 37.6 months among patients without autoimmunity (80 of 148 died) and was not reached among patients with autoimmunity (2 of 52 died). In univariate and multivariate regression analyses, autoimmunity was an independent prognostic marker for improved relapse-free survival and overall survival (P<0.001). CONCLUSIONS: The appearance of autoantibodies or clinical manifestations of autoimmunity during treatment with interferon alfa-2b is associated with statistically significant improvements in relapse-free survival and overall survival in patients with melanoma.

The effect of temozolomide-based chemotherapy in patients with cerebral metastases from melanoma.

Cerebral metastases from melanoma are correlated with a poor prognosis. Temozolomide is an oral alkylating agent that can cross the blood-brain barrier and in phase II and III trials, patients with advanced metastatic melanoma achieved overall response rates of 13 to 21%. The present study evaluated the efficacy and toxicity of temozolomide-based chemotherapy in patients with cerebral metastases from melanoma. Twenty-five patients (median age 48 years) with histologically confirmed stage IV melanoma and cerebral metastases treated with temozolomide-based chemotherapy. 10 patients received temozolomide plus docetaxel, nine patients temozolomide plus cisplatin and six patients temozolomide as single agent. Six patients achieved an objective response (24%). All responses were partial. The disease was stable in five patients (20%) and 13 patients progressed (52%). The median response duration was 6.9 months (range 1.8 to 16 months). The median time to progression (TTP) for all patients was 2 months, compared with a median TTP of 3.9 months, among responders and a median TTP of 1.8 months, for patients who remained stable or progressed (P<0.0001). The median survival time for the entire patient population was 4.7 months. The median survival for responders was 5.5 months and for non-responders was 3.6 months. The difference was statistically significant (P<0.05). The toxicity was mild. The most frequently reported adverse event were myelotoxicity and nausea and vomiting. Four patients developed grade 3/4 leukopenia, two grade 4 neutropenia, and one patient developed grade 3 thrombocytopenia. There was no treatment discontinuation caused by toxicity. Temozolomide-based chemotherapy may have a role in patients with cerebral metastases from melanoma. Further exploration is required. Toxicity was manageable.

Tolerability of adjuvant high-dose interferon alfa-2b: 1 month versus 1 year--a Hellenic Cooperative Oncology Group study.

BACKGROUND: High-dose interferon alfa-2b (IFN-alpha2b) as adjuvant therapy for melanoma is associated with substantial dose-limiting toxicity. It has been suggested that the 1-month intravenous (i.v.) induction regimen may be sufficient to reduce the risk of relapse and death. PATIENTS AND METHODS: The Hellenic Cooperative Oncology Group is conducting a multicenter, randomized trial of 1-month i.v. induction versus 1 year of adjuvant IFN-alpha2b therapy in patients with stage IIB/III melanoma. Adverse events reported by the first 200 patients to complete therapy are described. RESULTS: Both induction and maintenance regimens were well tolerated. The most common toxicities were flu-like and gastrointestinal symptoms, neutropenia, liver toxicity, and neurologic toxicity. The incidence of grade 3/4 toxicity was low and occurred mainly during the induction phase in both arms. Dose was reduced in 31% of patients during induction. Only 2% of patients discontinued. Dose was reduced in 8% of patients during maintenance and only 5% of patients discontinued. CONCLUSION: Intravenous induction with 15 MIU/m2/day IFN-alpha2b is well tolerated. Efficacy results from this trial are eagerly anticipated.