Lack of tumor recognition by hTERT peptide 540-548-specific CD8(+) T cells from melanoma patients reveals inefficient antigen processing.

Telomerase is a ribonucleoprotein complex responsible for the maintenance of the length of the telomeres during cell division, which is active in germ-line cells as well as in the vast majority of tumors but not in most normal tissues. The wide expression of the human telomerase catalytic subunit (hTERT) in tumors makes it an interesting candidate vaccine for cancer. hTERT-derived peptide 540-548 (hTERT(540)) has been recently shown to be recognized in an HLA-A*0201-restricted fashion by T cell lines derived from peptide-stimulated peripheral blood mononuclear cells (PBMC) from healthy donors. As a first step to the inclusion of this peptide in immunotherapy clinical trials, it is crucial to assess hTERT(540)-specific T cell reactivity in cancer patients as well as the ability of hTERT-specific CD8(+) T lymphocytes to recognize and lyse hTERT-expressing target cells. Here, we have analyzed the CD8(+) T cell response to peptide hTERT(540) in HLA-A*0201 melanoma patients by using fluorescent HLA-A*0201/hTERT(540) peptide tetramers. HLA-A*0201/hTERT(540) tetramer(+) CD8(+) T cells were readily detected in peptide-stimulated PBMC from a significant proportion of patients and could be isolated by tetramer-guided cell sorting. hTERT(540)-specific CD8(+) T cells were able to specifically recognize HLA-A*0201 cells either pulsed with peptide or transiently transfected with a minigene encoding the minimal epitope. In contrast, they failed to recognize hTERT-expressing HLA-A*0201(+) target cells. Furthermore, in vitro proteasome digestion studies revealed inadequate hTERT processing. Altogether, these results raise questions on the use of hTERT(540) peptide for cancer immunotherapy.

The results of surgery, with or without radiotherapy, for primary spinal myxopapillary ependymoma: a retrospective study from the rare cancer network.

PURPOSE: The aim of this study was to assess the outcome of patients with primary spinal myxopapillary ependymoma (MPE). MATERIALS AND METHODS: Data from a series of 85 (35 females, 50 males) patients with spinal MPE were collected in this retrospective multicenter study. Thirty-eight (45%) underwent surgery only and 47 (55%) received postoperative radiotherapy (RT). Median administered radiation dose was 50.4 Gy (range, 22.2-59.4). Median follow-up of the surviving patients was 60.0 months (range, 0.2-316.6). RESULTS: The 5-year progression-free survival (PFS) was 50.4% and 74.8% for surgery only and surgery with postoperative low- (<50.4 Gy) or high-dose (>or=50.4 Gy) RT, respectively. Treatment failure was observed in 24 (28%) patients. Fifteen patients presented treatment failure at the primary site only, whereas 2 and 1 patients presented with brain and distant spinal failure only. Three and 2 patients with local failure presented with concomitant spinal distant seeding and brain failure, respectively. One patient failed simultaneously in the brain and spine. Age greater than 36 years (p = 0.01), absence of neurologic symptoms at diagnosis (p = 0.01), tumor size >or=25 mm (p = 0.04), and postoperative high-dose RT (p = 0.05) were variables predictive of improved PFS on univariate analysis. In multivariate analysis, only postoperative high-dose RT was independent predictors of PFS (p = 0.04). CONCLUSIONS: The observed pattern of failure was mainly local, but one fifth of the patients presented with a concomitant spinal or brain component. Postoperative high-dose RT appears to significantly reduce the rate of tumor progression.

Long-term complete responses after 131I-tositumomab therapy for relapsed or refractory indolent non-Hodgkin's lymphoma.

We present the long-term results of 18 chemotherapy relapsed indolent (N = 12) or transformed (N = 6) NHL patients of a phase II anti-CD20 (131)I-tositumomab (Bexxar) therapy study. The biphasic therapy included two injections of 450 mg unlabelled antibody combined with (131)I-tositumomab once as dosimetric and once as therapeutic activity delivering 75 or 65 cGy whole-body radiation dose to patients with normal or reduced platelet counts, respectively. Two patients were not treated due to disease progression during dosimetry. The overall response rate was 81% in the 16 patients treated, including 50% CR/CRu and 31% PR. Median progression free survival of the 16 patients was 22.5 months. Median overall survival has not been reached after a median observation of 48 months. Median PFS of complete responders (CR/CRu) has not been reached and will be greater than 51 months. Short-term side effects were mainly haematological and transient. Among the relevant long-term side effects, one patient previously treated with CHOP chemotherapy died from secondary myelodysplasia. Four patients developed HAMA. In conclusion, (131)I-tositumomab RIT demonstrated durable responses especially in those patients who achieved a complete response. Six of eight CR/CRu are ongoing after 46-70 months.

Melan-A/MART-1-specific CD8 T cells: from thymus to tumor.

The self-antigen Melan-A/MART-1 is frequently involved in T-cell responses against malignant melanoma. The use of fluorescent tetramers incorporating the immunodominant Melan-A/MART-1 peptide has provided new insights into HLA-A2-restricted T-cell responses against this antigen in cancer patients and in healthy individuals. Direct evidence has been provided that a large Melan-A/MART-1-specific CD8 T-cell pool is generated during thymic selection. Although several other examples of naive self-peptide-specific T-cell repertoires are known, this is the only one directly accessible to analysis in healthy individuals

Lack of antitumour activity of human recombinant tumour necrosis factor-alpha, alone or in combination with melphalan in a nude mouse human melanoma xenograft system.

The most promising developments in the field of isolated limb perfusion have centred around the use of the recombinant cytokine tumour necrosis factor-alpha (rTNF-alpha) in combination with melphalan. While the results of clinical trials are impressive, the exact antitumour mechanisms of rTNF-alpha and its role in combination with melphalan remain unclear. Our aim was to study the antitumour activity of human rTNF-alpha with or without the combination of melphalan in a nude mouse human melanoma xenograft system. In a first attempt to define the maximal tolerated single dose of rTNF-alpha in this setting, 15 animals were exposed to increasing doses of rTNF-alpha (60-2500 microg/kg intraperitoneally). All but one animal survived and tumour growth was not influenced by these single dose applications of rTNF-alpha even at the very high doses. Anti-tumour activity of repeated application of melphalan (three times 9 mg/kg in group 2 and three times 6 mg/kg in group 3), of rTNF-alpha alone (nine doses of 50 microg/kg in group 4), and of rTNF-alpha in combination with melphalan (nine doses of 50 microg/kg rTNF-alpha and three times 6 mg/kg melphalan in group 5) was further compared with non-treated animals (group 1). Tumour growth was significantly inhibited in all animals treated with melphalan (group 2, 3 and 5), but was not decreased in animals treated with rTNF-alpha alone (group 4). Mean final tumour volumes and mean tumour weight were not different in group 2 (789 +/- 836 mm3, 0.38 +/- 0.20 g), group 3 (1173 +/- 591 mm3, 0.55 +/- 0.29 g) and group 5 (230 +/- 632 mm3, 0.37 +/- 0.29 g), but significant lower than group 1 (3156 +/- 1512 mm3, 2.35 +/- 0.90 g) and group 4 (3228 +/- 1990 mm3, 2.00 +/- 1.16 g). There were no significant differences between high and low dose melphalan treatment and between melphalan treatment in combination with rTNF-alpha. Histological examination did not show differences between treated and non-treated animals besides slightly inhibited mitotic activities of tumour cells in melphalan-treated animals. While tumour growth of human xenotransplanted melanoma in nude mice could be inhibited by melphalan, we failed to demonstrate any antitumour effect of rTNF-alpha. The combination of melphalan and rTNF-alpha did not enhance the antiproliferative effect of melphalan alone. Human xenotransplanted tumours on nude mice might not be the ideal experimental setting for studies of potential direct antineoplastic activity of rTNF-alpha, and these results support the concept that TNF-alpha exerts its antitumour activity indirectly, possibly by impairing the tumour vasculature and by activating the immune system.

Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis.

Background Following the discovery that mutant KRAS is associated with resistance to anti-epidermal growth factor receptor (EGFR) antibodies, the tumours of patients with metastatic colorectal cancer are now profiled for seven KRAS mutations before receiving cetuximab or panitumumab. However, most patients with KRAS wild-type tumours still do not respond. We studied the effect of other downstream mutations on the efficacy of cetuximab in, to our knowledge, the largest cohort to date of patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab plus chemotherapy in the pre-KRAS selection era. Methods 1022 tumour DNA samples (73 from fresh-frozen and 949 from formalin-fixed, paraffin-embedded tissue) from patients treated with cetuximab between 2001 and 2008 were gathered from 11 centres in seven European countries. 773 primary tumour samples had sufficient quality DNA and were included in mutation frequency analyses; mass spectrometry genotyping of tumour samples for KRAS, BRAF, NRAS, and PIK3CA was done centrally. We analysed objective response, progression-free survival (PFS), and overall survival in molecularly defined subgroups of the 649 chemotherapy-refractory patients treated with cetuximab plus chemotherapy. Findings 40.0% (299/747) of the tumours harboured a KRAS mutation, 14.5% (108/743) harboured a PIK3CA mutation (of which 68.5% [74/108] were located in exon 9 and 20.4% [22/108] in exon 20), 4.7% (36/761) harboured a BRAF mutation, and 2.6% (17/644) harboured an NRAS mutation. KRAS mutants did not derive benefit compared with wild types, with a response rate of 6.7% (17/253) versus 35.8% (126/352; odds ratio [OR] 0.13, 95% CI 0.07-0.22; p<0.0001), a median PFS of 12. weeks versus 24 weeks (hazard ratio [HR] 1 98, 1.66-2.36; p<0.0001), and a median overall survival of 32 weeks versus 50 weeks (1.75, 1.47-2.09; p<0.0001). In KRAS wild types, carriers of BRAF and NRAS mutations had a significantly lower response rate than did BRAF and NRAS wild types, with a response rate of 8.3% (2/24) in carriers of BRAF mutations versus 38.0% in BRAF wild types (124/326; OR 0.15, 95% CI 0.02-0.51; p=0.0012); and 7.7% (1/13) in carriers of NRAS mutations versus 38.1% in NRAS wild types (110/289; OR 0.14, 0.007-0.70; p=0.013). PIK3CA exon 9 mutations had no effect, whereas exon 20 mutations were associated with a worse outcome compared with wild types, with a response rate of 0.0% (0/9) versus 36.8% (121/329; OR 0.00,0.00-0.89; p=0.029), a median PFS of 11.5 weeks versus 24 weeks (HR 2.52, 1.33-4.78; p=0.013), and a median overall survival of 34 weeks versus 51 weeks (3.29, 1.60-6.74; p=0.0057). Multivariate analysis and conditional inference trees confirmed that, if KRAS is not mutated, assessing BRAF, NRAS, and PIK3CA exon 20 mutations (in that order) gives additional information about outcome. Objective response rates in our series were 24.4% in the unselected population, 36.3% in the KRAS wild-type selected population, and 41.2% in the KRAS, BRAF, NRAS, and PIK3CA exon 20 wild-type population. Interpretation While confirming the negative effect of KRAS mutations on outcome after cetuximab, we show that BRAF, NRAS, and PIK3CA,exon 20 mutations are significantly associated with a low response rate. Objective response rates could be improved by additional genotyping of BRAF, NRAS, and PIK3CA exon 20 mutations in a KRAS wild-type population.

Node-negative T1-T2 anal cancer: radiotherapy alone or concomitant chemoradiotherapy?

PURPOSE: To evaluate the influence of concomitant chemotherapy on loco-regional control (LRC) and cancer-specific survival (CSS) in patients with T1-T2 N0 M0 anal cancer treated conservatively by primary radiotherapy (RT). MATERIALS AND METHODS: Between 1976 and 2008, 146 patients with T1 (n=29) or T2 (n=117) N0 M0 anal cancer were treated curatively by RT alone (n=71) or by combined chemoradiotherapy (CRT) (n=75) consisting of mitomycin C±5-fluorouracil. Univariate and multivariate analyses were performed to assess patient-, tumor- and treatment-related factors influencing LRC and CSS. RESULTS: With a median follow-up of 62.5 months (interquartilerange, 26-113 months), 122 (84%) patients were locally controlled. The five-year actuarial LRC, CSS and overall survival for the population were 81.4%±3.6%, 91.9%±2.6%, and 75.4%±3.9%, respectively. The five-year LRC and CSS for patients treated with RT alone and with CRT were 75.5%±6.0% vs. 86.8%±4.1% (p=0.155) and 88.5%±4.5% vs. 94.9%±2.9% (p=0.161), respectively. In the multivariate analysis, no clinical or therapeutic factors were found to significantly influence the LRC and CSS, while the addition of chemotherapy was of borderline significance (p=0.065 and p=0.107, respectively). CONCLUSIONS: In the management of node negative T1-T2 anal cancer, LRC and CSS tend to be superior in patients treated by combined CRT, even though the difference was not significant. Randomized studies are warranted to assess definitively the role of combined treatment in early-stage anal carcinoma.

Information provision and information needs in adult survivors of childhood cancer.

BACKGROUND: Knowledge about their past medical history is central for childhood cancer survivors to ensure informed decisions in their health management. Knowledge about information provision and information needs in this population is still scarce. We thus aimed to assess: (1) the information survivors reported to have received on disease, treatment, follow-up, and late effects; (2) their information needs in these four domains and the format in which they would like it provided; (3) the association with psychological distress and quality of life (QoL). PROCEDURE: As part of the Follow-up survey of the Swiss Childhood Cancer Survivor Study, we sent a questionnaire to all survivors (≥18 years) who previously participated to the baseline survey, were diagnosed with cancer after 1990 at an age of <16 years. RESULTS: Most survivors had received oral information only (on illness: oral: 82%, written: 38%, treatment: oral: 79%, written: 36%; follow-up: oral: 77%, written: 23%; late effects: oral: 68%, written: 14%). Most survivors who had not previously received any information rated it as important, especially information on late effects (71%). A large proportion of survivors reported current information needs and would like to receive personalized information especially on late effects (44%). Survivors with higher information needs reported higher psychological distress and lower QoL. CONCLUSIONS: Survivors want to be more informed especially on possible late effects, and want to receive personalized information. Improving information provision, both qualitatively and quantitatively, will allow survivors to have better control of their health and to become better decision makers.

Chimioradiothérapie postopératoire des cancers des voies aérodigestives : vers un nouveau standard [Post-operative radiochemotherapy of the head and neck: Towards new standards?]

Head and neck squamous cell carcinomas are frequently diagnosed at an advanced stage. Their treatment remains controversial, and has to be multidisciplinary. External beam radiotherapy is a recognized treatment option after radical curative surgery in order to improve local control. Different adjuvant treatment options have been studied in order to improve the outcome of these patients. We review in this paper the different prognostic factors indicating an adjuvant treatment and the interest of treatment intensification in bad prognostic patients.

Differential regulation of vascular endothelial growth factor expression by peroxisome proliferator-activated receptors in bladder cancer cells.

The growth of any solid tumor depends on angiogenesis. Vascular endothelial growth factor (VEGF) plays a prominent role in vesical tumor angiogenesis regulation. Previous studies have shown that the peroxisome proliferator-activated receptor gamma (PPARgamma) was involved in the angiogenesis process. Here, we report for the first time that in two different human bladder cancer cell lines, RT4 (derived from grade I tumor) and T24 (derived from grade III tumor), VEGF (mRNA and protein) is differentially up-regulated by the three PPAR isotypes. Its expression is increased by PPARalpha, beta, and gamma in RT4 cells and only by PPARbeta in T24 cells via a transcriptional activation of the VEGF promoter through an indirect mechanism. This effect is potentiated by an RXR (retinoid-X-receptor), selective retinoid LG10068 providing support for a PPAR agonist-specific action on VEGF expression. While investigating the downstream signaling pathways involved in PPAR agonist-mediated up-regulation of VEGF, we found that only the MEK inhibitor PD98059 reduced PPAR ligand-induced expression of VEGF. These data contribute to a better understanding of the mechanisms by which PPARs regulate VEGF expression. They may lead to a new therapeutic approach to human bladder cancer in which excessive angiogenesis is a negative prognostic factor.

Testing for local adaptation in brown trout using reciprocal transplants.

ABSTRACT: BACKGROUND: Local adaptation can drive the divergence of populations but identification of the traits under selection remains a major challenge in evolutionary biology. Reciprocal transplant experiments are ideal tests of local adaptation, yet rarely used for higher vertebrates because of the mobility and potential invasiveness of non-native organisms. Here, we reciprocally transplanted 2500 brown trout (Salmo trutta) embryos from five populations to investigate local adaptation in early life history traits. Embryos were bred in a full-factorial design and raised in natural riverbeds until emergence. Customized egg capsules were used to simulate the natural redd environment and allowed tracking the fate of every individual until retrieval. We predicted that 1) within sites, native populations would outperform non-natives, and 2) across sites, populations would show higher performance at 'home' compared to 'away' sites. RESULTS: There was no evidence for local adaptation but we found large differences in survival and hatching rates between sites, indicative of considerable variation in habitat quality. Survival was generally high across all populations (55% +/- 3%), but ranged from 4% to 89% between sites. Average hatching rate was 25% +/- 3% across populations ranging from 0% to 62% between sites. CONCLUSION: This study provides rare empirical data on variation in early life history traits in a population network of a salmonid, and large-scale breeding and transplantation experiments like ours provide powerful tests for local adaptation. Despite the recently reported genetic and morphological differences between the populations in our study area, local adaptation at the embryo level is small, non-existent, or confined to ecological conditions that our experiment could not capture.

Localized fibrous tumours of the pleura: 15 new cases and review of the literature.

OBJECTIVE: To present a series of localized fibrous tumours of the pleura (LFTP), to define the clinical and histopathological diagnostic criteria of this tumour, and to determine the optimal treatment and follow-up. METHODS: Review of the charts of the patients with the diagnosis of LFTP (formerly called benign fibrous mesothelioma), as well as of all the histological sections, including immunohistochemical stains. Review of the literature with special emphasis on the clinical and histological criteria of malignancy. RESULTS: During the last 30 years, we found 15 patients with a complete clinical chart and histological material, particularly paraffin blocks of the tumour. The mean age was 57 years (range 27-79). Eight patients were asymptomatic, and the remaining seven presented with non-specific symptoms. All but one had complete resection of the tumour, including partial lung resection in two and partial chest wall resection in three. The diagnosis was confirmed by histological review in 15 cases. Immunohistochemical stainings showed positivity for vimentin in all cases, for CD 34 in 80%, but were consistently negative for cytokeratins. Nine tumours were histologically classified as malignant. Among them, five recurred, two of which were responsible for death. One benign tumour recurred after 1 year, and was treated successfully by repeat resection and radiotherapy. Overall, 13 patients (86%) were alive with no evidence of disease between 10 months and 27 years after the first resection. CONCLUSIONS: LFTP is a rare tumour which has a benign clinical course in over 80% of the cases, and is asymptomatic in half the patients. The diagnosis is difficult to establish before operation. Treatment consists of complete resection including adjacent structures if necessary. The clinical behaviour of LFTP cannot be predicted on the basis of histological aspects only. If histologically malignant tumours are more prone to recurrence and poor outcome, broad-based and locally invasive tumours bear a higher risk of recurrence. Long term follow-up is therefore mandatory in all cases in order to perform early re-resection when recurrence occurs.

Cyclic AMP induces differentiation in vitro of human melanoma cells.

Treating human melanoma lines with dibutyryl adenosine 3':5'-cyclic monophosphate (dbc AMP) resulted in morphologic changes associated with the altered expression of cell surface antigens. After treatment, cells developed long cellular projections characteristic of mature melanocytes and showed the presence of an increased number of Stage II premelanosomes. In addition, induction of melanin synthesis, detected as brown perinuclear pigmentation, was observed. The AMP further drastically reduced the growth rate of the five melanoma cell lines that were tested. The influence of dbc AMP was completely reversible 3 days after the agent was removed from the culture medium. The antigenic phenotype of the melanoma lines was compared before and after dbc AMP treatment. This was done with four monoclonal antibodies directed against major histocompatibility complex (MHC) Class I and II antigens and 11 monoclonal antibodies defining eight different melanoma-associated antigenic systems. Treatment with dbc AMP reduced the expression of human leukocyte antigen (HLA)-ABC antigens and beta-2-microglobulin in five of five melanoma lines. In the two HLA-DR-positive cell lines dbc AMP reduced the expression of this antigen in one line and enhanced it in the other. No induction of HLA-DR or HLA-DC antigens was observed in the Class II negative cell lines. Furthermore, dbc-AMP modulated the expression of the majority of the melanoma antigenic systems tested. The expression of a 90-kilodalton (KD) antigen, which has been found to be upregulated by interferon-gamma, was markedly decreased in all the five cell lines. A similar decrease in the expression of the high molecular weight proteoglycan-associated antigen (220-240 KD) was observed. The reduced expression of Class I and II MHC antigens as well as the altered expression of the melanoma-associated antigens studied were shown to be reversible after dbc AMP was removed. Our results collectively show that the monoclonal antibody-defined melanoma-associated molecules are linked to differentiation. They could provide useful tools for monitoring the maturation of melanomas in vivo induced by chemical agents or natural components favoring differentiation.

Younger age as a bad prognostic factor in patients with carcinoma of the cervix.

OBJECTIVE: To verify the influence of age on the prognosis of cervix carcinoma. STUDY DESIGN: Five hundred and sixty eight patients treated for a FIGO stage IB-IVA with radical irradiation in the Centre Hospitalier Universitaire Vaudois of Lausanne were subdivided according to the following age categories: < or = 45, 46-60, 61-69 and >70 years. Taking the 46-60 years age group as the reference, the hazard ratios (HR) of death and corresponding 95% confidence intervals (95% CI) were estimated by means of a Cox multivariate analysis. RESULTS: The 5-year survival rates were, respectively, 57%, 67%, 60% and 45%. For the youngest women the risk of death was significantly increased (HR = 2.00, 95% CI [1.32-3.00]) and was even more accentuated in advanced stages. CONCLUSION: Age under 45 years is a bad prognostic factor in carcinoma of the cervix.

Combining MHC tetramer and intracellular cytokine staining for CD8(+) T cells to reveal antigenic epitopes naturally presented on tumor cells.

As more tumor antigens are discovered and as computer-guided T cell epitope prediction programs become more sophisticated, many potential T cell epitopes are synthesized and demonstrated to be antigenic in vitro. However, it is estimated that about 50% of such tumor antigen-specific T cells have not been demonstrated to recognize the naturally presented epitopes due to either technical difficulties, such as T cell cloning which is still challenging for many laboratories; or the predicted T cell epitopes are not generated or not generated in sufficient amounts by the antigen processing machinery. However, to potentially identify clinically relevant vaccine candidate epitopes, it is essential to demonstrate natural antigen presentation. Here we combine the advantages of MHC tetramer and intracellular cytokine staining to sensitively detect natural antigen presentation by tumor cells for epitopes of interest. The novel method does not require T cell cloning or long-term T cell culture. Because the antigen-specific T cells are positively identified, this method is much less influenced by IFNgamma producing cells with unknown specificities and should be widely applicable.