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Purpose This phase III, randomized, double-blind, placebo-controlled study was conducted to evaluate the efficacy and safety of sorafenib with carboplatin and paclitaxel (CP) in patients with advanced melanoma who had progressed on a dacarbazine- or temozolomide-containing regimen. Patients and Methods A total of 270 patients were randomly assigned to receive intravenous paclitaxel 225 mg/m2 plus intravenous carboplatin at area under curve 6 (AUC 6) on day 1 of a 21-day cycle followed by either placebo (n = 135) or oral sorafenib 400 mg (n = 135) twice daily on days 2 to 19. The primary efficacy end point was progression-free survival (PFS); secondary and tertiary end points included overall survival and incidence of best response, respectively. Results The median PFS was 17.9 weeks for the placebo plus CP arm and 17.4 weeks for the sorafenib plus CP arm (hazard ratio, 0.91; 99% CI, 0.63 to 1.31; two-sided log-rank test P = .49). Response rate was 11% with placebo versus 12% with sorafenib. Dermatologic events, grade 3 thrombocytopenia, diarrhea, and fatigue were more common in patients treated with sorafenib plus CP versus placebo plus CP. Conclusion In this study, the addition of sorafenib to CP did not improve any of the end points over placebo plus CP and cannot be recommended in the second-line setting for patients with advanced melanoma. Both regimens had clinically acceptable toxicity profiles with no unexpected adverse events. A trial of similar design for the first-line treatment of patients with advanced melanoma (intergroup trial E2603) is currently ongoing.

Approximately 20% of skin tumors occur in the head and neck region. The therapeutic procedure is laid down in the current interdisciplinary guidelines of the EBM on skin tumors (squamous epithelial carcinoma, melanoma, Merkel cell carcinoma and basal cell carcinoma). For mucous membrane tumors primary R0 surgery is recommended. Approximately 57% of Merkel cell carcinomas occur in the head and neck region. Bilateral selective neck dissection and adjuvant radiation therapy is recommended for every stage. Operative therapy with histological confirmation is the standard procedure for treatment of basal cell carcinoma. The therapeutic procedure for squamous epithelial carcinoma depends on the extent and the histological subtype of the tumor. Complete surgical excision with topographically assigned histopathological control of the incision edges is the first choice treatment as long as this is medically acceptable. Malignant growths in the region of the nasal and paranasal cavities form approximately 4% of head and neck tumors. The general therapeutic principles depend on the possibility of complete resection of the tumor. Postoperative radiation and chemotherapy should be carried out depending on experience with other head and neck tumors in a pT3/4 situation. A dilemma with rare soft tissue sarcomas with many histological subtypes is that at present there is no appropriate classification for this group of tumors. In addition there are no guidelines or evidence with respect to indications for resection of residues after primary chemotherapy of pediatric rhabdomyosarcoma-like tumors. It is therefore recommended that these tumors should be treated in interdisciplinary oncology centers. Small cell carcinomas are highly malignant and rapidly form metastases. The therapy of small cell carcinomas in the head and neck region depend on the localization, tumor spread, lymph node status and the presence of distant metastases.

Dieser Beitrag gibt einen Uberblick zu folgenden „seltenen“ Kopf-Hals-Tumoren: den Speicheldrusenkarzinomen, Plattenepithelkarzinomen, Nasopharynxkarzinomen und Paragangliomen. Aufgrund der Seltenheit der Tumoren liegen in vielen Fallen keine prospektiv-randomisierten Studien vor. Die Empfehlungen zur Therapie beruhen daher uberwiegend auf retrospektiven Studien oder auf den Erfahrungen der Autoren. Der Artikel befasst sich ausschlieslich mit malignen Tumoren. Die Prognose der Speicheldrusenkarzinome richtet sich neben dem histologischen Subtyp auch nach dem Differenzierungsgrad. Die Therapie ist primar chirurgisch und orientiert sich an den bildmorphologischen Kriterien der „sinnvollen“ Resektablilitat. Die Indikation zur adjuvanten Radiotherapie von Speicheldrusenkarzinomen besteht beim Vorliegen von Risikofaktoren, die postoperativ die lokoregionare Rezidivwahrscheinlichkeit erhohen. Plattenepithelkarzinome sind als auserst maligne anzusehen, stellen sich aber vergleichsweise als radio- und chemosensibel dar. Bei Nasopharynxkarzinomen in den fruheren Stadien I–IIA ist die alleinige Radiotherapie, im fortgeschrittenen Stadium III/IV die simultane cisplatinhaltige Radiochemotherapie Standard. Die Wirksamkeit der Chemotherapie ist bei dieser Entitat besonders hoch. Paragangliome sind primar chirurgisch anzugehen. Sie sind grundsatzlich strahlensensibel, aber kaum chemosensibel.

Chemokine receptors are thought to be involved in the process of cancer metastases. When investigating cell lines and tissues from colorectal cancer (CRC), the CCR7 protein unexpectedly was confined to the cytoplasm and not present on the cell surface. This study investigated at the DNA, mRNA and protein level, the mechanism and the consequences of the failure of CCR7 to localize to the cell membrane. In all 15 CRC cell lines tested, no surface CCR7 was detected and no chemotactic response was elicited upon in-vitro exposure to CCR7 chemokine ligands (CCL) 19 and CCL21. Integrity of CCR7 DNA and mRNA was examined with respect to signal peptide expression in cell lines and CRC tissues by real-time RT-PCR and sequencing. Nine of 15 CRC cell lines and 8 of 14 CRC tissues revealed a truncated CCR7 mRNA species containing various incomplete signal peptide encoding sequences, while the corresponding DNA was intact. These results indicate in CRC frequent alternative splicing or post-transcriptional mRNA modification resulting in a CCR7 molecule lacking an intact signal peptide prohibiting membrane translocation. Further studies would be necessary to identify a potential intracellular role of the truncated CCR7, abundantly present in the cytoplasm.

A novel approach for cancer immunotherapy is to augment T-cell-mediated immunity by blocking inhibitory signals that suppress T-cell function. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a key negative regulator of T-cell activation. CTLA-4 blockade using anti-CTLA-4 monoclonal antibodies (mAbs) potentiates the T-cell response against tumors, and preliminary data on these agents demonstrate good efficacy and tolerability in the treatment of patients with metastatic melanoma and other cancers. This paper will review data from studies with anti-CTLA-4 mAbs to date, discuss some of the key clinical considerations emerging from early clinical trials with this therapeutic strategy, and provide an overview of ongoing and planned clinical trials for anti-CTLA-4 mAb therapy in metastatic melanoma and other cancers.

CCR6 is expressed in various tumors and has been implicated in the process of tumor progression and metastasis. Its chemokine ligand, CCL20, is present in different tissues including lymph nodes, but also the normal prostate. This study was performed to investigate a potential relationship between CCR6 and CCL20 expression and features of human prostate cancer (PCA) at time of primary treatment. Immunohistochemistry was used to detect CCR6 and CCL20 expression in archival tissue blocks of 80 PCA cases of various tumor grades and stages. Evaluation was semiquantitatively by visual scoring and quantitatively by digital image analysis (DIA). CCR6 and CCL20 expression was compared with Gleason score, stage, perineural invasion, nodal metastasis, age, and preoperative serum prostate-specific antigen (PSA) level by univariate and multivariate analyses. Staining intensity of CCR6 in tumor cells varied considerably, with it being: weak in 21 tumors (26.2%), intermediate in 44 (55.0%), and strong in 15 (18.8%), with 3.6-log differences in DIA measurements. CCL20 expression was absent in eight tumors (10.0%), weak in 41 (51.2%), intermediate in 23 (28.8%), and strong in eight (10.0%). CCR6 and CCL20 expression did not correlate. CCR6 expression was associated with T-category (P

Systemic medical treatment of melanoma is administered in the adjuvant and palliative setting. Adjuvant therapy may be considered in patients with primary melanoma with more than 1.5 mm tumor thickness and with regional node metastasis. Presently no indication for systemic adjuvant chemotherapy or for adjuvant therapy with nonspecific immune-stimulatory agents outside controlled studies is seen. Interferon-alpha is the first substance in the adjuvant therapy of melanoma, which has shown to present a significant advantage to the patients in some prospective randomized studies. Good arguments for using adjuvant interferon-alpha therapy in high-risk melanoma patients exist. Both high-dose and low-dose interferon-alpha show promise. The major indications for systemic chemotherapy and chemoimmunotherapy are inoperable recurrent tumors, inoperable regional metastases and distant metastases (stage IV). As treatment in such situations is primarily palliative, the effect of any regimen on the quality of life must be carefully weighed. As a first line treatment, single agent therapy is recommended, as polychemotherapy or biochemotherapy did not show significant advantages for prolongation of survival; hence they are more toxic. An urgent need for development of new treatment modalities is necessary and general principles of experimental immunotherapy are outlined.

Recombinant antibody fusion constructs with heterologous functional domains are a promising approach to new therapeutic targeting strategies. However, expression of such constructs is mostly limited to cost and labor-intensive mammalian expression systems. Here we report on the employment of Pichia pastoris for the expression of heterologous antibody fusion constructs with green fluorescent protein, A33scFv::GFP, or with cytosine deaminase, A33scFv::CDy, their production in a biofermenter and a modified purification strategy. Combined, these approaches improved production yields by about thirty times over established standard protocols, with extracellular secretion of the fusion construct reaching 12.0 mg/l. Bifunctional activity of the fusion proteins was demonstrated by flow cytometry and an in-vitro cytotoxicity assay. With equal amounts of purified protein, the modified purification method lead to higher functional results. Our results demonstrate the suitability of methylotrophic Pichia expression systems and laboratory-scale bioreactors for the production of high quantities of bifunctionally active heterologous single-chain fusion proteins.

Purpose: In a previous immunohistochemical study of dendritic cells (DC) in sentinel lymph nodes (SLN) draining regressing melanomas, we found that the accumulation of mature DC-LAMP+ DCs in SLNs was associated with local expansion of antigen-specific memory effector CTLs and the absence of metastasis in downstream lymph nodes. The aim of this study was to investigate the prognostic importance of the maximal density of mature DCs in SLNs.Experimental Design: A total of 458 consecutive patients with micrometastatic melanoma within SLNs were eligible for analysis. The maximal density of mature DC-LAMP+ DCs was evaluated by three independent observers and categorized into three classes (Results: There was excellent interobserver reproducibility for maximum density of mature DC-LAMP+ DC scores (κ score = 0.82). There were differences in the maximal density scores and staining intensity according to the treating melanoma center (P < 0.001). The higher the mature DC density in the SLN is, the longer is the duration of survival [P = 0.047; hazard ratio, 0.70; 95% confidence interval, 0.50-1.00]. Adjusted by thickness and ulceration, the prognostic importance of DC density was lower (P = 0.36).Conclusion: This study is the first to report the prognostic value of DC-LAMP+ DC counts in SLNs containing metastatic melanoma. Patients with a high density of mature DCs (≥200/mm2) have the lowest risk of death. It also provides evidence that a lack of maturation in the SLNs is important in biological facilitation of melanoma progression.

In recent years, reduced-intensity conditioning (RIC) regimens before allogeneic stem cell transplantation (SCT) are increasingly used in patients not eligible for conventional conditioning. We did a retrospective, multicenter analysis to assess the feasibility of conditioning with fludarabine and treosulfan before allogeneic SCT in multiple myeloma patients. Thirty-four patients with a median age of 51.5 years were included in the analysis. All patients underwent myeloablation after conditioning followed by stable engraftment, and 29 of 31 evaluable patients (94%) showed early complete hematopoietic chimerism. Non-hematological toxicities were limited and encompassed mainly fever in neutropenia and infections. Grade II-IV acute and chronic graft-versus-host disease was observed in 33 and 39%, respectively. With a median follow-up of 708 days (range 60-1729 days), the median progression-free survival was 180 days. The treatment-related mortality was 10% on day 100 and 25% after 1 year. The median overall survival has not yet been reached. Our data indicate that conditioning with fludarabine and treosulfan before allogeneic SCT is feasible in intensively pretreated multiple myeloma patients and leads to stable engraftment and complete hematopoietic chimerism. Randomized trials are warranted to determine if this approach might be incorporated in an algorithm of multiple myeloma treatment.

This paper covers highlights from the International Society for Biological Therapy of Cancer (iSBTc) 21st Annual Meeting, including the associated iSBTc Primer on Tumor Immunology and Biological Therapy, and the iSBTc Mini-Symposium on the Biologic Effects of Targeted Therapies. The programs ran from 26 – 29 October 2006 and were held at the Hyatt Regency Century Plaza in Los Angeles, CA, USA. Focussed on the varying aspects of biological cancer therapies and tumor immunology, the iSBTc 21st Annual Meeting featured 131 scientific posters, 29 oral abstract presentations, 20 invited speaker lectures and 2 keynote addresses. The 21st Annual Meeting, Primer and Mini-Symposium were attended by > 400 clinicians and basic scientists from academia, industry and government. The iSBTc was founded in 1984 as the Society for Biological Therapy with 40 charter members. At present, the Society has > 500 members consisting primarily of MDs, PhDs, RNs and corporate representatives. Scientific and business meetings have b...

Die palliative medikamentose Systemtherapie von Patienten mit Plattenepithelkarzinomen der Kopf-Hals-Region ist ein komplexes Feld, zu dem nur wenige Daten vorliegen und das von zahlreichen Einzelfallentscheidungen gepragt ist. Eine Reihe zytostatischer Substanzen ist fur die Behandlung zugelassen, z. B Platinsalze, 5-Fluorouracil, Methotrexat. Die Taxane gewinnen derzeit zunehmend an Bedeutung. Zusatzlich besteht die Moglichkeit der EGF-Rezeptorblockade mittels Antikorpern und Tyrosinkinaseinhibitoren. Der Stellenwert der Behandlung mit angiogeneseinhibierenden Substanzen ist noch unklar. Da eine Kombinationsbehandlung in der palliativen Therapie keinen eindeutigen Vorteil zeigt, werden die verfugbaren Medikamente in der Regel sequenziell eingesetzt, immer unter Beachtung der bei dieser spezifischen Patientengruppe haufigen Komorbiditaten.

Organerhaltende Therapieansatze bei Karzinomen des Larynx und Hypopharynx als Alternative zur kompletten Kehlkopfentfernung (Laryngektomie) wurden in den letzten 2 Jahrzehnten signifikant weiterentwickelt und in der klinischen Betrachtung etabliert. Insbesondere die transorale Laserchirurgie hat sich einen festen Stellenwert in der Behandlung mikrolaryngoskopisch gut ubersehbarer Karzinome (bis Tumorgrose T3) erarbeitet, wobei je nach Erfahrung des Operateurs in Einzelfallen auch offene Teilresektionen des Kehlkopfs zur Anwendung kommen. Ubereinkunft herrscht bezuglich der nach wie vor gultigen Notwendigkeit der Laryngektomie, die bei fortgeschrittenen Tumoren des Larynx und Hypopharynx oft die rehabilitativ gunstigste und onkologisch sauberste Therapie der Wahl darstellt. Hat das Karzinom eine Ausbreitung, die durch Teilresektionen nicht mehr sinnvoll behandelbar ist, konnen multimodale Therapieansatze zum Einsatz kommen, die in den letzten Jahren im Rahmen klinischer Studien diversifiziert wurden. Fruh konnte gezeigt werden, dass solche Organerhaltungsprogramme in onkologischer Hinsicht gleichwertig mit der Laryngektomie zu setzen sind und langfristig funktionell zufrieden stellende Organerhaltungsraten bis zu 60% erreichen konnen. Der aktuelle Fokus richtet sich derzeit auf den Stellenwert der Induktionschemotherapie, dem zusatzlichen Einsatz von Biologicals (insb. EGFR-Blockade) und die Reduktion der Spattoxizitat.

This study was performed to investigate hematotoxicity and occurrence of PPE in patients with high-grade non-Hodgkins lymphoma treated with a modified CHOP regimen (CLAOP), where doxorubicin had been substituted by a noncardiotoxic pegliposomal doxorubicin. An open-label phase I/IIa study was performed evaluating CLAOP21/20 with 20 mg/m(2) of pegliposomal doxorubicin every 21 days (12 patients), and a dose-dense filgrastim supported CLAOP14 regimen every 14 days with escalating doses of pegliposomal doxorubicin (24 patients) in elderly high-grade lymphoma patients or patients with comorbidity interfering with cardiac function. CLAOP21/20 was well tolerated. Hematotoxicity was similar to that reported with regular CHOP. In the CLAOP14 cohort, a pegliposomal doxorubicin dose of 25 mg/m(2) was associated with dose-limiting hematotoxicity, febrile episodes, and PPE. Both regimens were active with an overall response rate of 60% and 77%, respectively. The recommended dose of pegliposomal doxorubicin in the biweekly regimen for Phase II/III testing is 20 mg/m(2).

The purpose of this study was to assess the efficacy and safety of pegylated liposomal doxorubicin in combination with cyclophosphamide and dexamethasone (CLAD). In this prospective open-label phase II study, 60 patients with advanced multiple myeloma (MM) received three weekly cycles of CLAD, consisting of cyclophosphamide 200 mg/m2 i.v. d1-4, pegylated liposomal doxorubicin 20 mg/m2 i.v. d1 and dexamethasone 40 mg p.o. d1-4 for a maximum of six cycles in absence of disease progression. Efficacy and toxicity was compared to our immediate historical cohort of 46 patients treated with cyclophosphamide, dexamethasone and conventional doxorubicin (CAD). A total of 239 cycles of CLAD and 209 cycles of CAD, respectively, were given. The objective response rate was 71% (CLAD) and 74% (CAD). Non-cumulative hematological toxicity was predominant in both regimens. It was found that CLAD is an active and well-tolerated treatment regimen for MM. Response rate is comparable to other anthracycline containing regimens like CAD with an advantage in hematological toxicity and lower infectious complications, and a presumed advantage of lower cardiotoxicity.

Background Cyclin A1 is essential for male gametopoiesis. In acute myeloid leukemia, it acts as a leukemia-associated antigen. Cyclin A1 expression has been reported in several epithelial malignancies, including testicular, endometrial, and epithelial ovarian cancer (EOC). We analyzed Cyclin A1 expression in EOC and its correlation with clinical features to evaluate Cyclin A1 as a T-cell target in EOC. Methods Cyclin A1 mRNA expression in EOC and healthy tissues was quantified by microarray analysis and quantitative real-time PCR (qRT-PCR). Protein expression in clinical samples was assessed by immunohistochemistry (IHC) and was correlated to clinical features. Results Cyclin A1 protein was homogeneously expressed in 43 of 62 grade 3 tumor samples and in 1 of 10 grade 2 specimens (p

Squamous cell cancer of the head and neck (SCCHN) is a frequent aggressive malignancy with limited therapeutic options. Increasing evidence suggests that mammalian target of rapamycin (mTOR)-inhibitors might be effective in advanced SCCHN. However, non-invasive biomarkers for early prediction of treatment efficacy are not established in SCCHN. Highly proliferating tumours are characterised by enhanced cell turnover which is associated with enhanced apoptosis. During apoptosis of epithelial cells caspases cleave cytokeratin (CK)-18 can be detected in the blood. In this study we analysed sera from patients with relapsed or metastatic SCCHN patients who have been treated with temsirolimus for caspase-cleaved and total (caspase-cleaved and uncleaved) CK-18 by enzyme-linked immunosorbent assays (ELISAs). In addition, caspase-3 activity was detected by luminometric substrate assay. SCCHN patients revealed higher serum levels of those biomarkers compared to healthy controls. Importantly, patients with short progression-free survival (PFS) showed higher serum levels of caspase-3 activity compared to patients with longer PFS (⩾ 2months). Caspase-3 activity is inversely correlated with PFS. A cut-off value for caspase-3 activity was determined that correctly predicted PFS2months with a sensitivity of 86% and a specificity of 67%. These data demonstrate that detection of serum caspase-3 activity might be a useful non-invasive biomarker for early identification of SCCHN patients not responding to treatment with novel targeted therapies such as mTOR-inhibitors.

Summary Background Patients with recurrent or metastatic squamous-cell carcinoma of the head and neck (HNSCC) progressing after first-line platinum regimens have a poor prognosis and few treatment options. Afatinib, an irreversible ERBB family blocker, has shown efficacy in a phase 2 study in this setting. We aimed to assess the efficacy and safety of afatinib compared with methotrexate as second-line treatment in patients with recurrent or metastatic HNSCC progressing on or after platinum-based therapy. Methods In this open-label, phase 3, randomised controlled trial conducted in 101 centres in 19 countries, we enrolled patients aged 18 years or older with histologically or cytologically confirmed HNSCC that was recurrent, metastatic, or both who had progressed on or after first-line platinum-based therapy, were not amenable for salvage surgery or radiotherapy, and who had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Previous treatment with more than one systemic regimen in this setting was not allowed; previous treatment with EGFR-targeted antibody therapy (but not EGFR-targeted tyrosine-kinase inhibitors) was allowed. We randomly assigned eligible patients in a 2:1 ratio to receive oral afatinib (40 mg/day) or intravenous methotrexate (40 mg/m 2 per week), stratified by ECOG performance status and previous EGFR-targeted antibody therapy for recurrent or metastatic disease. Randomisation was done centrally with an interactive voice or web-based response system. Clinicians and patients were not masked to treatment allocation; independent review of tumour response was done in a blinded manner. The primary endpoint was progression-free survival as assessed by an independent, central imaging review committee. Efficacy analyses were done in the intention-to-treat population and safety analyses were done in patients who received at least one dose of study drug. This ongoing study is registered with ClinicalTrials.gov, number NCT01345682. Findings Between Jan 10, 2012, and Dec 12, 2013, we enrolled 483 patients and randomly assigned 322 to afatinib and 161 to methotrexate. After a median follow-up of 6·7 months (IQR 3·1–9·0), progression-free survival was longer in the afatinib group than in the methotrexate group (median 2·6 months [95% CI 2·0–2·7] for the afatinib group vs 1·7 months [1·5–2·4] for the methotrexate group; hazard ratio [HR] 0·80 [95% CI 0·65–0·98], p=0·030). The most frequent grade 3 or 4 drug-related adverse events were rash or acne (31 [10%] of 320 patients in the afatinib group vs none of 160 patients in the methotrexate group), diarrhoea (30 [9%] vs three [2%]), stomatitis (20 [6%] vs 13 [8%]), fatigue (18 [6%] vs five [3%]), and neutropenia (1 [ vs 11 [7%]); serious adverse events occurred in 44 (14%) of afatinib-treated patients and 18 (11%) of methotrexate-treated patients. Interpretation Afatinib was associated with significant improvements in progression-free survival and had a manageable safety profile. These findings provide important new insights into the treatment of this patient population and support further investigations with irreversible ERBB family blockers in HNSCC. Funding Boehringer Ingelheim.

Aim The detection of circulating tumour cells in the bloodstream before and after surgical manipulation, and the qualitative detection of potential shedding of tumour cells during surgical manipulation of patients with uveal melanoma. Methods 202 patients treated for a newly diagnosed uveal melanoma were included in the study. Blood samples were acquired 24 h before and 30 min after the basic surgical steps. Detection of potential circulating melanoma cells was extrapolated from the presence of tyrosinase and MelanA/Mart1 transcripts by reverse transcription PCR. Results Based on the measurement of tyrosinase transcripts, as a result of the first and second surgical manipulation there were three and zero transitions from negative to positive respectively, while there were two and one transitions from positive to negative, respectively. According to MelanA/Mart1 transcripts, there were 19 and 5 transitions from negative to positive respectively, and 15 and 2 transitions from positive to negative, respectively. No statistically significant differences were documented, concerning the presence of circulating tumour cells in the blood samples acquired before and after the first surgical manipulation or the second one. Conclusion The change in the percentage of patients with detected tumour cells in their bloodstream was not statistically significant. The frequent shifts from negative to positive samples as well as from positive to negative samples comparing preoperative to postoperative samples indicates discontinuous shedding or variation due to measurements close to the threshold of detection. As a conclusion, the surgical manipulation does not seem to have a measurable contribution to the spread of melanoma cells in the bloodstream.

Purpose Mutations in the gene coding for the kinase B-Raf are associated with tumour growth in conjunctival melanoma. The purpose of this study is to explore effects of pharmacological B-Raf inhibition in conjunctival melanoma cell lines. Methods The B-Raf genotypes were assessed by PCR and subsequent sequencing. Cytotoxicity, cell viability, proliferation, apoptosis rate and phosphorylation rate of ERK and Akt were analysed in three different conjunctival melanoma cell lines under the influence of the B-Raf inhibitor PLX 4720 at various concentrations. Results The cell lines CRMM-1 and CM2005.1 showed the B-Raf V600E mutation, whereas CRMM-2 expressed a B-Raf wild type. CM2005.1 was highly sensitive to PLX 4720, showing a complete cytotoxic effect for >1 µM, as well as a significant concentration-dependent reduction of the proliferation rate and viability rate. Even though CRMM-1 also carries the B-Raf V600E mutation, it did not react as sensitive to PLX 4720 inhibition as CM2005.1, but showed a significant concentration-dependent reduction regarding proliferation and viability. PLX 4720 had only slight impact on CRMM-2 in high concentrations (10 µM) regarding cytotoxicity, proliferation and viability. Fluorescence-activated cell sorting analysis revealed that PLX 4720 acted predominantly antiproliferative and not via an induction of apoptosis. The phosphorylation rate of ERK was significantly reduced in CRMM-1 and CM2005.1, while it remained unchanged in CRMM-2. The phosphorylation rate of Akt was significantly elevated in CRMM-2. Conclusions Proliferation inhibition of conjunctival melanoma cells by PLX 4720 depends on their B-Raf genotype. Therefore, therapeutic application of B-Raf inhibitors should take into account the specific B-Raf genotype.

Background: Several trials demonstrated efficacy of the gemcitabine/treosulfan (GeT) combination in metastatic uveal melamoma. This randomized phase II trial compared the GeT combination versus treosulfan alone (T) in this rare disease. Patients and methods: Chemotherapy-naive patients with proven metastatic uveal melanoma were randomly assigned to receive 1000 mg/m 2 of gemcitabine plus 3500 mg/m 2 of treosulfan (GeT) or 3500 mg/m 2 of T. Chemotherapy was administered on days 1 and 8 in both arms, cycles were repeated on day 29. Primary end point was rate of responses and disease stabilizations. Results: Forty-eight patients were randomized. Seven confirmed stable diseases (SDs) and one partial remission (PR) were observed in 24 patients treated with the GeT regimen, whereas no PR and only three SDs were observed in the T arm (P = 0.08). Median progression-free survival (PFS) was 3 months (95% CI 1.1–4.9) and 2 months (95% CI 1.7–2.3) in the GeT and T arm (P = 0.008, log-rank). Six and 12 months PFS was 34.8% and 17.9% and 16.7% and 0% always favoring the GeT arm. Conclusions: This first randomized trial in metastatic uveal melanoma showed a superior PFS and a trend for a higher response/stabilization rate of the GeT combination over T.

Purpose The liver is the primary organ of metastasis in colorectal cancer (CRC). Chemokine receptor CCR6 is expressed on a subset of T cells and is associated with their migration into the liver. This study was performed to analyze a possible association between CCR6 expressed by primary CRC and liver metastases. Patients and Methods CCR6 expression levels were evaluated by immunohistology in 64 CRC primary tumor specimens. Twenty-four of 64 patients had synchronous liver metastases. Evaluation of immunostaining was performed semiquantitatively by visual assessment and quantitatively by digital image analysis (DIA). Multiple logistic regression analysis was performed to assess relevant parameters for liver metastases. Results CCR6 expression was verified in all 64 primary tumor specimens with considerable variations in intensity; 21 tumors (33%) demonstrated weak CCR6 staining, 32 (50%) demonstrated intermediate staining, and 11 (17%) demonstrated strong staining. Quantitative assessment by DIA showed an up to 5-log difference in CCR6 values. CCR6 staining was significantly stronger in tumor cells compared with adjacent colon epithelial cells (P < .0005). Multiple logistic regression analysis, controlling for age, sex, tumor stage, nodal status, pathologic grade, and preoperative carcinoembryonic antigen levels, revealed that CCR6 staining in the primary tumor was independently associated with the presence of liver metastases (odds ratio = 2.1; P = .002). Conclusion The association between expression level of CCR6 in primary CRC and synchronous liver metastases suggests that CCR6 and its ligand may be involved in the metastatic spread to the liver. Therefore, CCR6 may be a potential target for specific therapeutic interventions.

Monitoring cellular immune responses is one prerequisite for rational development of cancer vaccines. The primary objective of immune monitoring is to determine the efficacy of a vaccine to induce or augment a specific T-cell response. Further questions relate to the prevalence and functional relevance of spontaneous tumor-directed immune responses, the functional characteristics of T-cell responses, and, finally and most importantly, the relationship between immune monitoring assay results and clinical end points. The issue of T-cell monitoring has become more complex as different types and generations of assays have been adopted during the past decade and both standardization and validation of assays have often been insufficient. Because the development of assays parallel the clinical development of cancer vaccines, technical advances have been achieved simultaneously with broadening understanding of cancer immunity. Suitable animal models for immune monitoring are, however, lacking, because preclinical vaccine development in rodents does not allow serial immune monitoring of the peripheral blood, as is commonly used in patients. The current situation is characterized by a lack of universal standards for T-cell assessment, uncertainty about the association between immune monitoring assay results and clinical antitumor end points, and lack of knowledge regarding the contribution of different aspects of T-cell function to clinical efficacy. It is acknowledged that T-cell monitoring will have to be validated in large trials with clinically effective vaccines, but this necessity should not discourage the current application of novel assays within clinical trials of all stages.

Innovations and Challenges in Melanoma, chaired by Michael Atkins and cochaired by Ulrich Keilholz, John Kirkwood, and Jeffrey Sosman, was held July 15 to 16, 2005, in Cambridge, Massachusetts. The conference brought together leading experts in the fields of cancer research, medical oncology, surgical oncology, anatomic pathology, dermatology, and immunotherapy who wished to advance the field of melanoma treatment by exchanging information and perspectives regarding recent advances and recommendations for further study. The conference proceedings published in this educational supplement to Clinical Cancer Research are intended to provide timely information and recommendations on how genetics, biology, and data information can enhance our understanding of melanoma biology and help inform the use of therapies for this disease.

Das Aderhautmelanom unterscheidet sich in der Tumorbiologie und im Metastasierungsverhalten vom kutanen malignen Melanom. Das metastasierte Aderhautmelanom ist eine seltene Erkrankung mit schlechter Prognose. Zu den therapeutischen Moglichkeiten zahlen operative Verfahren, die nur fur sehr wenige Patienten sinnvoll sind, die systemische Chemotherapie und lokale Behandlungsverfahren wie die Chemoembolisation und die lokoregionare Chemotherapie uber die A. hepatica. Die wenigen vorliegenden klinischen Studien haben nur eine geringe Zahl von Patienten eingeschlossen. Sie belegen eine Wirksamkeit der Chemoembolisation und der lokoregionaren Chemotherapie bei Patienten mit alleiniger Metastasierung in die Leber. Bei Patienten mit Metastasierung in mehrere Organe werden zurzeit Zytostatikakombinationen aus Gemcitabin und Treosulfan in klinischen Studien untersucht. Fortemustin, eine moderne Nitroseharnstoffverbindung, stellt ebenfalls eine therapeutische Option dar.

Item does not contain fulltext BACKGROUND: Chemoimmunotherapy for patients with metastatic melanoma is associated with high toxicity, and only a subset of patients will benefit. This randomised phase II study was performed with the primary objective of exploring whether two cycles of dacarbazine monotherapy could select the subset of patients that would benefit most from more intensive chemoimmunotherapy. PATIENTS AND METHODS: Patients with metastatic melanoma were randomised to either receive chemoimmunotherapy with dacarbazine, cisplatin, interferon-alpha and interleukin-2 (arm A) or initial treatment with two cycles of dacarbazine monotherapy followed irrespective of response by the same 4-drug regimen of chemoimmunotherapy (arm B). Chemoimmunotherapy was continued in the absence of disease progression for a maximum of four cycles. Primary end-point was the disease stabilisation rate. RESULTS: A total of 93 patients were randomised, and 89 patients were eligible. Disease stabilisation (complete/partial response or stable disease) was achieved in 19 patients (42.2%) in arm A and 9 patients (20.5%) in arm B. In arm B 32 of the 44 patients continued chemoimmunotherapy after two cycles of dacarbazine. Of 20 patients with progressive disease (PD) after two cycles of dacarbazine in arm B, only 2 patients achieved an objective response. Median overall survival (OS) in arms A and B was 10.5 months and 9.5 months, respectively. CONCLUSIONS: Despite a lower initial stabilisation rate, the strategy of starting with 2 courses of DTIC prior to a 4-drug regimen led to comparable median overall survival. Only few transient responses were achieved with the 4-drug regimen in patients with disease progression on DTIC, suggesting frequent cross resistance. Two cycles of dacarbazine monotherapy cannot be recommended to select patients for more intensive chemoimmunotherapy.

Temsirolimus showed clinical efficacy in refractory squamous cell carcinoma of the head and neck (SCCHN), who failed platinum and epidermal growth factor receptor inhibition. Tumor shrinkage in 39% and a progression-free survival rate of 40% at 12 weeks warrants future studies in SCCHN, which may define a novel treatment paradigm in SCCHN. However, no predictive molecular marker was identified. Background Squamous cell carcinoma of the head and neck (SCCHN) is a common disease, which has a poor prognosis after failure of therapy. Activation of the PI3K–AKT–mTOR axis is commonly detected in recurrent or metastatic SCCHN, and provided the rationale for the clinical phase II trial in pretreated SCCHN. Patients and methods The primary end point was the progression-free survival rate (PFR) at 12 weeks. Forty eligible patients have been recruited after failure of platinum chemotherapy and cetuximab. A preplanned futility analysis was successfully passed after ≥1 success was detected in 20 patients. Secondary objectives consisted of progression-free survival (PFS), disease control rate (DCR), overall survival (OS), safety and tolerability, and predictive biomarkers for KRAS, BRAF, PIK3CA mutations, and HPV status. Archived tumor tissue was analyzed for DNA sequence. Results A total of 40 patients were eligible. The PFR at 12 weeks was 40% (95% CI 25.0–54.6). The median PFS and OS were 56 days (95% CI 36–113 days) and 152 days (76–256 days), respectively. In 33 assessable patients, disease stabilization occurred in 57.6%, with tumor shrinkage in 13 patients (39.4%). Overall, the treatment was well tolerated. Fatigue (47.5%), anemia (25.0%), nausea (20.0%), and pneumonia (20.0%) were the most common adverse events. Neither PIK3CA mutations, nor HPV status were predictive for success with temsirolimus treatment. No mutations were found for KRAS or BRAF. Conclusion Tumor shrinkage and efficacy parameter indicate that inhibition of the PI3K–AKT–mTOR axis was a putative novel treatment paradigm for SCCHN. We could not identify parameters predictive for treatment success of temsirolimus, which underscores the need for refinement of the molecular analysis in future studies. Clinical trials number NCT01172769.

Increases in incidence of oropharyngeal squamous cell carcinoma (OPSCC) in countries with falling tobacco use have been attributed to a growing role of human papilloma virus (HPV) in the carcinogenesis. Trends of HPV prevalence in populations with persistently high portions of smokers are poorly characterised.Registry data from East Germany were used to determine incidence trends between 1998 and 2011. Data from patients treated at the Charité University Medicine Berlin between 2004 and 2013 (cohort 1, N=436) were used for estimation of trends in HPV prevalence, smoking and survival. HPV prevalence was prospectively confirmed in cohort 2 (N=213) comprising all primary HNSCC cases at the Charité in 2013.Between 1998 and 2011 incidence of both OPSCC and non-OPSCC increased. An increase in HPV prevalence (% of HPV+ cases in 2004-2006 versus 2012-2013: 27% versus 59%, P=0.0004) accompanied by a moderate decrease in the portion of current smokers was observed in OPSCC but not in non-OPSCC. The change in disease epidemiology in OPSCC was associated with significant improvement in overall survival. Increased HPV prevalence in OPSCC (48%) compared to non-OPSCC (11%) was confirmed in cohort 2.Despite clear differences to the United States in terms of tobacco use, the increase in OPSCC incidence in a European population was also mainly attributed to HPV, and the HPV status significantly affected prognosis. For clinical trial design it is important to consider the large group of smokers within HPV-induced OPSCC.

Background Based on phase II trial results, chemoimmunotherapy combinations have become the preferred treatment for patients with metastatic melanoma in many institutions. This study was performed to determine whether interleukin-2 (IL-2) as a component of chemoimmunotherapy influences survival of patients with metastatic melanoma. Patients and Methods Patients with advanced metastatic melanoma were randomly assigned to receive dacarbazine 250 mg/m2 and cisplatin 30 mg/m2 on days 1 to 3 combined with interferon-alfa-2b 10 × 106 U/m2 subcutaneously on days 1 through 5 without (arm A) or with (arm B) a high-dose intravenous decrescendo regimen of IL-2 on days 5 through 10 (18 × 106 U/m2/6 hours, 18 × 106 U/m2/12 hours, 18 × 106 U/m2/24 hours, and 4.5 × 106 U/m2 for 3 × 24 hours). Treatment cycles were repeated in the absence of disease progression every 28 days to a maximum of four cycles. Results Three hundred sixty-three patients with advanced metastatic melanoma were accrued. The median survival was 9 months in both arms, with a 2-year survival rate of 12.9% and 17.6% in arms A and B, respectively (P = .32; hazard ratio, 0.90; 95% CI, 0.72 to 1.11). There was also no statistically significant difference regarding progression-free survival (median, 3.0 v 3.9 months) and response rate (22.8% v 20.8%). Conclusion Despite its activity in melanoma as a single agent or in combination with interferon-alfa-2b, the chosen schedule of IL-2 added to the chemoimmunotherapy combination had no clinically relevant activity.

Contains fulltext : 49192.pdf (Publisher’s version ) (Closed access) BACKGROUND: Individuals affected by melanoma with thick primary tumours or regional node involvement have a poor outlook, with only 30-50% alive at 5 years. High-dose and low-dose interferon alfa have been assessed for the treatment of these patients, with the former having considerable toxicity and a consistent effect on disease free survival, but not on overall survival, and the latter no consistent effect on either. Our aim was, therefore, to assess the effect of two regimens of interferon of intermediate dose versus observation alone on distant metastasis-free interval (DMFI) and overall survival in such patients. METHODS: We did a randomised controlled trial in 1388 patients who had had a thick primary tumour (thickness > or = 4 mm) resected (stage IIb) or regional lymph node metastases dissected (stage III) and had been assigned to 13-months (n=553) or 25 months (n=556) of treatment with subcutaneous interferon alfa 2b, or observation (n=279). Treatment comprised 4 weeks of 10 million units (MU) of interferon alfa (5 days per week) followed by either 10 MU three times a week for 1 year or 5 MU three times a week for 2 years, to a total dose of 1760 MU. Our primary endpoint was DMFI. Analyses were by intent to treat. FINDINGS: After a median follow-up of 4.65 years, we had recorded 760 distant metastases and 681 deaths. At 4.5 years, the 25-month interferon group showed a 7.2% increase in rate of DMFI (hazard ratio 0.83, 97.5% CI 0.66-1.03) and a 5.4% improvement in overall survival. The 13-month interferon group showed a 3.2% increase in rate of DMFI at 4.5 years (0.93, 0.75-1.16) and no extension of overall survival. Toxicity was acceptable, with 18% (195 of 1076) of patients going off study because of toxicity or as a result of refusal of treatment because of side-effects. INTERPRETATION: Interferon alfa as used in the regimens studied does not improve outcome for patients with stage IIb/III melanomas, and cannot be recommended. With respect to efficacy of the drug, duration of treatment seemed more important than dose, and should be assessed in future trials.

Contains fulltext : 47941.pdf (Publisher’s version ) (Closed access) The transcription factor Wilms' tumour gene 1 (WT1) is important as a prognostic marker as well as in the detection and monitoring of minimal residual disease in leukaemia and myelodysplastic syndromes. Evidence has accumulated over the past decade to show that WT1 is a key molecule for tumour proliferation in a large number of human neoplasms most prominent in acute leukaemias, making it a suitable target for therapeutic strategies. Based on animal results, showing safety and efficacy of immunization with WT1 peptides and protein, early clinical trials in leukaemia have recently been initiated. The First International Conference on WT1 in Human Neoplasia was held in Berlin, March 11--12, 2004. This report reviews the current knowledge on the role of WT1 in tumour promotion and as a diagnostic and therapeutic target, and summarizes the data presented and discussed in this meeting.

Purpose: Preclinical investigations suggest that histamine dihydrochloride (HDC) protects T cells and natural killer cells from inhibition by monocyte-derived reactive oxygen metabolites and synergizes with interleukin (IL) 2 in inducing T-cell activation. Here, we investigate whether this mechanism is operational in patients with melanoma treated with HDC as an adjunct to IL-2. Experimental Design: Melanoma patients having liver metastases were treated with IL-2 with or without HDC within a randomized, multicenter, phase III trial. The effect of HDC on type 1 and type 2 T-cell cytokine production was investigated in peripheral blood samples from 19 patients with the use of intracellular cytokine flow cytometry. Melanoma-specific T-cell responses were analyzed in eight HLA-A2–positive patients. Results: Frequencies of CD3+ T cells producing IFN-γ (type 1 T cells) in response to phorbol myristate acetate/ionomycin increased (median, 1.8-fold) in patients receiving IL-2 plus HDC but not in those receiving IL-2 alone (P < 0.01 for comparison between arms). In contrast, the number of IL-13-producing type 2 T cells that increased in patients after treatment with IL-2 was not modulated by HDC. Melanoma- and tyrosinase-specific IFN-γ and IL-13-producing T cells were detected in two of four HLA-A2–positive patients with melanoma following treatment with HDC + IL-2. Conclusions: Treatment of patients with stage IV melanoma with HDC in combination with IL-2 increases type 1 T-cell responses and may promote induction of melanoma-specific T cells. These effects are of relevance for tumor immunotherapy and provide a potential mechanism for the clinical efficacy of HDC added to IL-2.

Uveal melanoma is the most frequent primary malignant neoplasm of the eye and has a poor prognosis in metastatic stage. Fotemustine or a combination of gemcitabine and treosulfan has demonstrated some efficacy in metastatic disease. We conducted a phase II trial to assess the second-line activity and toxicity of bendamustine hydrochloride, a nucleoside analogue with alkylating activity. Inclusion criteria were a Karnofsky performance status of > or = 60% and progressive disease during or after first-line chemotherapy. Bendamustine was administered at a dose of 120 mg/m2 on days 1 and 2. Cycles were repeated on day 22. The primary endpoint of the study was the determination of the number of patients achieving an objective response or stable disease. The secondary endpoint was toxicity. Eleven patients were enrolled into the trial. Grade III and IV toxicity consisted of anaemia, thrombocytopenia and leucocytopenia in two, one and two patients, respectively. No grade III or IV non-haematological toxicity was observed. According to Response Evaluation Criteria in Solid Tumours (RECIST), all patients showed progressive disease. We conclude that bendamustine is ineffective as second-line chemotherapy for metastatic uveal melanoma.

Superiority of irinotecan (CPT-11) plus cisplatin over etoposide plus cisplatin in small cell lung cancer (SCLC) has recently been demonstrated. This study determines dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of escalating doses of carboplatin to a fixed dose of irinotecan in Caucasians with small cell lung cancer.Patients with extensive small cell lung cancer received 50 mg/m(2) irinotecan on day 1, 8, and 15. Dose escalation of carboplatin on day 1 started in dose level 1 at an AUC of 5 mg x min/ml and was escalated to AUC 6 in level 2. Cycles were repeated on day 29. Dose escalation was evaluated after 3 consecutive patients. If no grade IV neutropenia lasting foror =7 days or thrombopenia or non-hematologic toxicityor = grade III occurred, treatment was continued in the next dose level.16 patients were treated. DLT was reached in dose level 2 with 2 grade IV neutropenias and 1 grade IV thrombopenia and diarrhea. Toxicity was further investigated at dose level 1 in a total of 10 patients, which revealed 2 grade III neutropenias and 1 grade III diarrhea. Of 15 evaluable patients, 10 had a partial response, 3 had disease stabilization and 2 progressed.Dose level 1 was found to be MTD, this dose is currently compared to the combination of etoposide plus carboplatin within a randomised phase II/III trial.

Antigen-specific vaccination is a promising emerging treatment option for cancer patients. Results from early clinical vaccination trials with tumour peptides in patients with metastatic disease have shown tumour regressions in few patients usually with limited disease. Current clinical studies focus on the development of more potent vaccination strategies and on the vaccination of patients with occult or small volume metastatic disease. The novel generation of sensitive T-cell assays allowing direct quantitation and characterisation of specific T cells provide an essential tool for further systematic clinical development of vaccine protocols. There is accumulating evidence from clinical cancer vaccination trials of a relation between the induction of specific T cells and clinical efficacy.

The detection of disseminated tumor cells in peripheral blood from colorectal cancer patients by RT-PCR could be an attractive method for selecting patients for adjuvant therapy. We here report on real-time RT-PCR assays (LightCycler) to quantitate potential mRNA markers. We investigated specimens from colon carcinoma and normal colon mucosa tissues, cell lines, blood samples from 129 patients with colorectal cancer (all stages) and 58 reference blood samples (healthy donors, persons suffering from inflammatory bowel or infectious diseases). The expression profile in tissues showed high values for CEA and CK20, whereas in cell lines ProtM was predominant. All markers were detected in reference and patient blood samples (ProtM, 22, 17%; CEA, 84, 86%; CK20, 85, 88%). After quantitative analysis, the definition of cutoff values for each marker and the combination of markers, 13% of patients were judged to have elevated marker concentrations in their blood, from which only 6 had values significantly differing from cutoff value. There were no differences between stages of disease. In the case of 19 patients, investigated prior to and 1 week after surgery, 2 samples revealed a significant postoperative increase in CEA or CK20 mRNA concentration. In spite of high expression levels in tissues and cell lines, we were not able to differentiate satisfyingly mRNA markers originating from tumor cells and those from illegitimate transcription in hematopoetic cells in blood. We conclude that either copy numbers of analyzed markers in circulating tumor cells are not sufficient for detection or, more probably, peripheral blood is not a suitable compartment for detection of tumor cells in colorectal cancer. © 2003 Wiley-Liss, Inc.

There is increasing evidence that tumors elicit specific T-cell responses in a substantial proportion of patients. Recently, we have shown that in patients with colorectal cancer specific T cells against the tumor-associated antigens (TAA) Ep-CAM, her-2/neu or CEA can be detected in peripheral blood using IFNγ-ELISPOT assay. In our study, we have analyzed T-cell responses against HLA-A*0201-restricted epitopes of these TAA in peripheral blood of patients with breast cancer and colorectal cancer. Surprisingly, a complete absence of ex vivo T-cell responses against these TAA was found in 20 patients with breast cancer. In contrast, specific T cells were detectable in 12 of 49 patients with colorectal cancer against at least 1 of these TAA, confirming our previous results. T-cell responses against influenza-derived peptides were similar in both malignancies. The results of our study indicate a difference either of tumor immunogenicity or of the migratory pattern of tumor-specific T cells between breast cancer and colorectal cancer patients. The findings reported here have implications for the development of antigen-specific T-cell therapies. © 2003 Wiley-Liss, Inc.

This study analyses the frequency and therapy of brain metastases in 94 stage IV melanoma patients after treatment with high-dose interleukin-2 (IL-2) and interferon-α (IFN-α) within three subsequent trials between 1990 and 1995. Central nervous system (CNS) metastases occurred in 28 patients (30%) during the potential follow-up period of 6 years. Time to occurence of brain metastases varied between 1 and 53 months, with a median of 10 months. Of 28 patients, 19 had

The aim of this study was to define prognostic factors for survival, and especially for long-term survival in a mature data-set of patients with stage IV melanoma treated within a randomised trial of cytokine-based protocols. Long-term follow-up data on patients enrolled into a European Organization for Research and Treatment of Cancer (EORTC) trial comparing interferon-alpha (IFNalpha) plus interleukin-2 (IL-2) with or without cisplatin were collected. Univariate and multivariate Cox regression analyses were performed to define prognostic factors for survival. The characteristics of patients alive at 2 and 5 years after randomisation were compared with the entire cohort using the chi(2) test. The minimum potential follow-up of the 131 evaluable patients was 5 years. 18 patients (14%) were alive 2 years after randomisation, and 11 (8%) 5 years after randomisation. Pretreatment performance status (PS), serum lactate dehydrogenase (LDH) and tumour mass were significant predictors for survival, whereas site of metastases and number of sites were non-significant. PS and LDH were the only independent prognostic factors. All except 1 patient alive at 2 and 5 years had a pretreatment PS of 100%, and only three long-term survivors had elevated pretreatment LDH. There was no association between the site of metastases and long-term survival. Response to treatment was a major predictor for long-term survival, whereas addition of cisplatin did not impact upon overall survival probability or on long-term survival. The probability of long-term survival in stage IV melanoma patients after IL-2-based treatments is governed by pretreatment PS, serum LDH and response to treatment. Site of metastases, the basis for the M-subcategories of the new AJCC staging system, was not informative in this study.

Die Behandlungsmodalitat des primaren Aderhautmelanoms hat bisher keinen direkten Einfluss auf die Uberlebensprognose des Patienten. Durch die Identifizierung signifikanter Prognoseparameter bezuglich des Risikos, Metastasen eines Aderhautmelanoms zu entwickeln, werden neue Behandlungsstrategien erarbeitet mit dem Ziel, die Entstehung von Metastasen zu verhindern. Dabei spielt die Immuntherapie durch die Entwicklung geeigneter Impfstoffe gegen melanomspezifische Tumorantigene eine zunehmende Rolle. Beim bereits metastasierten Aderhautmelanom betrug bis dato die mittlere Uberlebenszeit ca. 5 Monate. In der letzten Zeit konnte durch die intrahepatische Infusion von Fotemustin die mittlere Uberlebenszeit auf 14 Monate bei selektierten Patienten verlangert werden. Neue Therapieprotokolle werden aufgrund von Chemosensitivitatsuntersuchungen aktuell erarbeitet. Des Weiteren werden immuntherapeutische Modalitaten auch in der Behandlung von Metastasen von Aderhautmelanomen untersucht.

The EORTC Melanoma Group (MG) was founded in 1969 by both clinicians and scientists from various disciplines and fields of research with a common interest in malignant melanoma. This collaborative approach has always been the foundation of the groups strength. With an interest in tumour biology and especially the immunological aspects of the disease, the group has always pursued a scientific approach to treatment development in malignant melanoma. Over the years, the group has performed many clinical trials, epidemiological studies, histopathological studies defining standards and guidelines, translational research regarding prognostic factors and various metastatic and immunological aspects of melanoma, and developed quality assurance programmes for immunological and molecular biological assays in laboratory networks. At present, the EORTC MG runs the worldwide largest clinical trial programme in stages II, III and IV melanoma involving some 140 cancer centres in and outside Europe. Each trial is associated with the appropriate translational research programmes.

Background : Quantification of tumor markers expressed by occult-circulating tumor cells may be of prognostic value in a variety of neoplasms and disease stages. Here, we report on a quantitative nested real-time polymerase chain reaction (PCR) assay to quantify rare transcripts using the Light Cycler system. Tyrosinase mRNA, only expressed by melanocytes and melanoma cells, was used as the model tumor marker. Methods : For the establishment and sensitivity testing of this novel real-time PCR assay, 10 ml EDTA blood from healthy volunteers was spiked with 10 1 –10 5 MKR cells (the melanoma cell line). Following RNA extraction and cDNA synthesis, nested and single round PCRs were performed. Subsequently, 35 blood samples of 22 melanoma patients were analyzed. Results : Nested PCRs provided quantitative data with a quantitative range of five orders of magnitude of tyrosinase mRNA equivalent to 1–10 4 MKR cells/ml of blood. Nested PCR with 35 pre-amplification cycles displayed the quantitative data for nine tyrosinase transcript-positive samples out of 35 blood samples, whereas nested PCR with 20 pre-amplification cycles or single round PCR were less sensitive. Conclusions : These experiments indicate that nested PCR, which is much more sensitive than single round PCR, is a useful tool even for the quantification of rare transcripts—a result with important implications for the study of minimal residual disease (MRD) in melanoma as well as in other malignancies.

Integrins are cell surface molecules that mediate cell adhesion, but are also important regulators of tumor cell interactions with their microenvironment, tumor cell survival and growth. In addition, the αvβ3-integrins appear to be critical for microvessel formation in tumor-induced neoangiogenesis. The present study is the first to investigate the effects of therapeutic αvβ3-integrin inhibition in a chemically induced tumor model that largely resembles human colon carcinomas. Tumor induction was performed in 47 male Sprague-Dawley rats using 1,2 dimethylhydrazin (21 mg/kg) twice a week. After 20 weeks of tumor induction, 100% of the animals developed adenocarcinomas with a median of 13.5 macroscopic tumor nodules (range 12–17), but no distant metastases. During further tumor induction for an additional 10 weeks, rats were treated three times/week with (a) 15 mg/kg RGDfV-peptide that can block vitronectin and fibronectin receptors; (b) an equimolar amount of an ineffective cyclic control peptide; or (c) with equimolar amounts of a linear RGDS-peptide. At the end of this treatment period, rats were sacrificed, and tumor load was quantified macroscopically and confirmed by histological examination. For investigation of the involvement of tumor-induced neoangiogenesis microvessel, density was determined using CD31-immunostaining. After 30 weeks, control animals (group B) had 5–18 tumors (median 14.5). If rats were treated with RGDfV-peptide (group A), the number of tumor nodules was significantly reduced (P < 0.005) to a median of seven macroscopic tumors (range 2–10 tumors), which also represented a significant reduction (P < 0.005) compared with prior to treatment. Application of noncylic RGDS-peptides (group C) did not affect the number of tumor nodules (median 18; range 10–30 tumors). The diameters of tumor nodules were comparable (3.2–6.1 mm) in animals of all groups. In addition, microvessel density was significantly (P < 0.05) reduced in tumors in group A compared to control rats. The major side effect in the treatment group was increased susceptibility to respiratory infections. Our results demonstrate that αvβ3-integrin-receptor inhibition appears to be a therapeutic strategy for colorectal cancer. In our therapeutic model, late onset of treatment with integrin-blocking peptides resulted in an inhibition of tumor growth and a reduced tumor load which appeared to be mediated, at least in part, by inhibition of neoangiogenesis.

6059 Background: Recent next-generation sequencing (NGS) studies revealed a wide mutational spectrum in SCCHN. However, little is known about spatial intratumoral heterogeneity and temporal clonal evolution. Precise understanding of the genomic architecture of primary and recurrent/metastatic (R/M) tumors will be crucial for the development of personalized treatment and molecular biomarkers. Methods: In this pilot study, paired tumor samples (primary, R/M lesions) from 10 patients with locally advanced SCCHN who progressed after concurrent chemoradiation (CTRX) were included. Mutational profiling was performed by NGS targeting the exonic regions of 327 genes. Only somatic mutant variants with a difference of ≥0.15 in allele frequency (AF) between primary and R/M tumors were considered for further analysis. Results: Median time to progression was 6.2 months (range: 2.5-30.2). Overall, the difference in mutational patterns of primary and R/M tissue was very small. On average, one mutant variant (range: 0-2) was selectively detected in only one of the paired samples or differed in AF for ≥0.15. Nonetheless, clonal selection of mutant variants previously linked to disease progression was observed in 8 of 10 cases. In line with their gain of function, an increase in AF of TP53 missense mutations (R175H, R248Q) in the recurrent tumor - suggestive of the selection of treatment-resistant mutant TP53 subclones - was observed in two patients. Further variants with increased mutant AF in recurrent tumors were found in ADCY2, CDKN2A, FGFR3, MET, NOTCH1, PIK3CA and TGFBR2. Conclusions: We here provide first evidence that treatment-induced clonal selection after CRTX frequently occurs in SCCHN but is limited to only few gene alterations associated with an aggressive phenotype. This result is surprising given CRTX being a DNA-damaging regimen with inherent risk of de-novo mutagenesis and abundant time for clonal tumor evolution. Further investigations of spatial intratumoral heterogeneity and clonal evolution in larger patient cohorts are required for improving our understanding of treatment resistance and disease progression in SCCHN.

e14613 Background: Immune checkpoints are an emerging treatment target in head and neck squamous cell carcinoma (HNSCC). Expression of PD-L1 and an IFNG immune signature have been shown as biomarkers of response. In this study we analyzed mutational signatures to identify recurrent mutational patterns associated with inflammation. Methods: HNSCC mutation and gene expression data were downloaded (n = 496, TCGA). Inflamed tumors were identified through upregulation of a predefined 6-gene IFNG immune signature. Mutational load, base exchange motifs and a BRCAness signature were analyzed in inflamed and non-inflamed tumors. Results were validated in independent datasets of cervical, urothelial and lung cancer (TCGA). Results: Total mutational load did not correlate with tumor inflammation (r = -0.03, p = 0.5). An APOBEC-associated tCw (thymine – cytosine – adenine/thymine) mutational signature was significantly associated with inflammation (r = 0.26, p = 2.923e-09). The tCw signature was furthermore correlated with the expression of PD-L1 and other immune checkpoints (r = 0.19, p = 1.723e-05). The TCW mutational signature correlated with the expression of APOBEC3 genes. Expression of APOBEC3 genes also correlated with tumor inflammation (r = 0.62, p < 2.2e-16). As a second mutational signature, indels and an independent BRCAness signature were significantly enriched in non-inflamed tumors (p = 6.464e-14). Enrichment of an APOBEC mutational signature in inflamed tumors could be validated in cervical and urothelial carcinoma but not NSCLC. Conclusions: In HNSCC, overall mutational load is not associated with inflammation. BRCAness is significantly associated with immune-desertion whereas expression of APOBEC genes and an APOBEC mutational signature significantly correlates with the expression of PD-L1 and a T-cell inflamed signature. These findings suggest that a specific mutational pattern might serve as a biomarker for immune checkpoint inhibition in cancer.