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The detection and characterization of cell-free DNA (cfDNA) in peripheral blood from neuroblastoma patients may serve as a minimally invasive approach to liquid biopsy. Major challenges in the analysis of cfDNA purified from blood samples are small sample volumes and low cfDNA concentrations. Droplet digital PCR (ddPCR) is a technology suitable for analyzing low levels of cfDNA. Reported here are two quadruplexed ddPCR assay protocols that reliably quantify MYCN and ALK copy numbers in a single reaction together with the two reference genes, NAGK and AFF3, and accurately estimate ALKF1174L (exon 23 position 3522, C>A) and ALKR1275Q (exon 25 position 3824, G>A) mutant allele fractions using cfDNA as input. The separation of positive and negative droplets was optimized for detecting two targets in each ddPCR fluorescence channel by the adjustment of the probe and primer concentrations of each target molecule. The quadruplexed assays were validated using a panel of 10 neuroblastoma cell lines and paired blood plasma and primary neuroblastoma samples from nine patients. Accuracy and sensitivity thresholds in quadruplexed assays corresponded well with those from the respective duplexed assays. Presented are two robust quadruplexed ddPCR protocols applicable in the routine clinical setting and that require only minimal plasma volumes for the assessment of MYCN and ALK oncogene status.

Precision cancer medicine (PCM) holds great promises to offer more effective therapies to patients based on molecular profiling of their individual tumours. Although the PCM approach seems intuitive, multiple conceptional and structural challenges interfere with the broad implementation of PCM into clinical practice. Accordingly, concerted national and international efforts are needed to guide the further development and broad adoption of PCM in Germany. With support of the 'German Cancer Aid' (Deutsche Krebshilfe [DKH]) a task force 'Molecular Diagnostics and Therapy' was implemented. In two workshops supported by the DKH, delegates from the fourteen comprehensive cancer centresidentified key topics essential to implement quality-guided, harmonized and adaptable PCM. Based on an online questionnaire and using a modified Delphi approach, nine statements were drafted and evaluated within the group. These statements could serve as a basis to define a collaborative strategy for PCM in the future with the aim to sustain and further improve its quality.

Background: After promising results from TPEx phase II trial in first line recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC), this trial compared the TPEx regimen (docetaxel-platinum-cetuximab) to the standard EXTREME regimen. Methods: Patients with R/M HNSCC unsuitable for curative treatment were randomized between TPEx (four cycles docetaxel-cisplatin-cetuximab followed by cetuximab maintenance 500 mg/m² every two weeks) and EXTREME (six cycles 5FU-cisplatin-cetuximab followed by weekly cetuximab 250 mg/m² maintenance). Main inclusion criteria were patients fit for cisplatin, performance status (PS) ≤1, creatinine clearance >60ml/min, and age

Summary: Colon cancer is a heterogeneous tumor driven by a subpopulation of cancer stem cells (CSCs). To study CSCs in colon cancer, we used limiting dilution spheroid and serial xenotransplantation assays to functionally define the frequency of CSCs in a panel of patient-derived cancer organoids. These studies demonstrated cancer organoids to be enriched for CSCs, which varied in frequency between tumors. Whole-transcriptome analysis identified WNT and Hedgehog signaling components to be enhanced in CSC-enriched tumors and in aldehyde dehydrogenase (ALDH)-positive CSCs. Canonical GLI-dependent Hedgehog signaling is a negative regulator of WNT signaling in normal intestine and intestinal tumors. Here, we show that Hedgehog signaling in colon CSCs is autocrine SHH-dependent, non-canonical PTCH1 dependent, and GLI independent. In addition, using small-molecule inhibitors and RNAi against SHH-palmitoylating Hedgehog acyltransferase (HHAT), we demonstrate that non-canonical Hedgehog signaling is a positive regulator of WNT signaling and required for colon CSC survival. : Colon cancer is a heterogeneous tumor driven by a subpopulation(s) of therapy-resistant cancer stem cells (CSCs). Regan et al. use 3D culture models to demonstrate that CSC survival is regulated by non-canonical, SHH-dependent, PTCH1-dependent Hedgehog signaling, which acts as a positive regulator of WNT signaling to block CSC differentiation. Keywords: WNT pathway, non-canonical Hedgehog signaling, cancer stem cell, colon cancer, cancer organoid, PTCH1, HHAT, SHH

In patients with advanced squamous cell carcinoma of the head and neck (SCCHN), aggressive treatment is effective, but can significantly impact their health-related quality of life (HRQoL), particularly if there is damage to the structures involved in swallowing, breathing, speech or physical appearance. Long-term psychosocial support may be required for patients whose basic functioning and social interactions are affected. In randomised clinical trials in SCCHN, HRQoL has not been routinely reported and, with a number of different HRQoL tools available, data for many of the parameters that patients consider important in their day-to-day lives are not widely available to clinicians. In addition, the assessment of HRQoL outside of the clinical trial setting is even scarcer. In an era of personalised medicine, an individualised approach to assessing QoL (iQoL) is of paramount importance when treating and monitoring patients with SCCHN; by responding to QoL concerns, appropriate treatment regimens that result in meaningful improvements in clinical outcomes and quality of survival (QoS) for patients can be adopted. In this review we describe the tools available for assessing HRQoL in cancer patients and provide an overview of the HRQoL data currently available in head and neck cancer. How this information can be used to individualise treatment to optimise clinical outcomes, maximise the patient's day-to-day living and minimise healthcare spending in SCCHN is discussed.

Circulating tumour cells (CTCs) are cells of solid tumour origin detectable in the peripheral blood. Their occurrence is considered a prerequisite step for establishing distant metastases. Metastatic melanoma was the first malignancy in which CTCs were detected and numerous studies have been published on CTC detection in melanoma at various stages of disease. In spite of this, there is no general consensus as to the clinical utility of CTCs in melanoma, largely due to conflicting results from heterogeneous studies and discrepancies in methods of detection between studies. In this review, we examine the possible clinical significance of CTCs in cutaneous, mucosal and ocular melanoma, focusing on detection methods and prognostic value of CTC detection.

Background The prognostic role of persistence of circulating tumor cells (CTC) after upfront tumor surgery for outcome of adjuvant (chemo)radiation in locally advanced squamous cell carcinoma of the head and neck (LASCCHN) was evaluated. Patients and methods In this prospective study, peripheral blood samples from 144 patients with LASCCHN presenting after tumor resection for adjuvant treatment were analyzed for CTC. Their detection was correlated with tumor site, clinical risk factors, disease-free (DFS) and overall survival (OS). Results CTC were detected in 42 of 144 patients (29%). CTC detection was higher in cases with nodal involvement and in carcinomas located at the tonsil or base of tongue but was not influenced by age, smoking history, T stage, extracapsular lymph node extension, surgical margins or the human papillomavirus status. Overall, the presence of CTC was not predictive for OS or DFS. However, while in oropharyngeal carcinomas (OPC, n = 63), the detection of CTC was associated per trend with improved DFS [CTC+ versus CTC- (% of patients without evidence of disease at 2 years): 100% versus 79%; log rank: P = 0.059]; the reverse was observed for carcinomas from other sites (non-OPC, n = 81; CTC+ versus CTC-: 29% versus 75%; P = 0.001). In multivariate analysis, CTC remained an independent prognostic marker for DFS [hazard ratio (HR) 4.3, 95% confidence interval (CI) 1.7–10.9, P = 0.002] and OS (HR 2.7, 95% CI 1.2–6.3, P = 0.016) in non-OPC. Conclusions Assessment of CTC in non-OPC should prove useful for identification of patients who benefit from treatment intensification. The basis for the good prognostic value of CTC in OPC has to be elucidated in future studies.

Background Therapeutic options for neuroendocrine neoplasms (NEN) are limited, and only few NEN patients have been treated according to their individual genomic profile. We report the molecular and clinical characteristics of a large NEN cohort that was studied by prospective whole-genome (WGS), whole-exome (WES), and transcriptome sequencing (TS) within the NCT/DKTK MASTER precision oncology program. Methods Between 2013 and 2018, a total of 115 patients with advanced, heavily pretreated NEN were enrolled. Primary sites were gastrointestinal (n = 30), pancreatic/hepatic (n = 33), pulmonary (n = 20), genitourinary (n = 13), head/neck (n = 6), and other regions (n = 11). Grading according to the 2017 and 2019 WHO classification was neuroendocrine tumor (NET) G1, n = 12; NET G2, n = 31; NET G3, n = 12; neuroendocrine carcinoma, n = 36; mixed neuroendocrine-non-neuroendocrine neoplasm, n = 12; and other, n = 11. Results Of WES (n = 72) or WGS (n = 44) and TS (n = 92) were discussed in a multidisciplinary molecular tumor board. Recommendations were given in 103 cases (94%) for the following potential treatment alternatives: tyrosine or serine/threonine kinase inhibition (n = 67), PARP inhibition (n = 41), immunotherapy (n = 33), mTOR inhibition (n = 30), CDK4/6 inhibition (n = 19), MEK inhibition (n = 12), platinum-based chemotherapy (n = 7), BET inhibition (n = 7), anti-claudin18.2 (n = 5) or anti-DLL3 antibody treatment (n = 8). Somatic hypermutation (>10 mutations/megabase) was seen in 8.6% of patients. Treatment options could be implemented in 35 patients, of which 31 were evaluable for response: partial response, n = 10; mixed response, n = 1; stable disease, n = 11; progressive disease, n = 9; resulting in an overall response rate of 32% and a disease control rate of 71%. Conclusions Comprehensive genomic and transcriptomic characterization provides new insight into the molecular pathogenesis of NEN and creates therapeutic opportunities in a significant proportion of NEN patients who have exhausted standard treatment. Legal entity responsible for the study NCT Heidelberg & German Cancer Consortium. Funding German Cancer Research Center. Disclosure S. Kreutzfeldt: Research grant / Funding (institution), Travel / Accommodation / Expenses: PharmaMar; Honoraria (self): Novartis; Research grant / Funding (institution): Roche; Travel / Accommodation / Expenses: Pfizer. C.E. Heilig: Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: PharmaMar; Honoraria (institution), Travel / Accommodation / Expenses: Lilly; Travel / Accommodation / Expenses: Novartis; Travel / Accommodation / Expenses: Celgene; Travel / Accommodation / Expenses: Teva; Travel / Accommodation / Expenses: BMS; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Pfizer. All other authors have declared no conflicts of interest.

Background The development of new therapeutic options for cancer patients due to a better understanding of tumor biology has profound effects on the design and conduct of modern clinical cancer research especially with respect to clinical trial design. Until recently, cancer drugs have been investigated in clinical trials for a definitive histological tumor type but this approach is slowly changing and recently drugs have been approved rather histology agnostic based on genomic alterations in various histological tumor types. Here we investigated the changes in the landscape of clinical cancer trials in Germany from 2000-2016. Methods Clinical trials gov database was queried in 2017 for interventional clinical cancer trials running in Germany with study start between 01/01/2000 and 31/12/2016. Data extracted from the database included the conditions, interventions, study phase, Sponsor/Collaborators, funder type, study design, outcome measures and number enrolled. Results Overall, 2977 trials matching the criteria were extracted. Number of studies heavily increased within the first decade from 32 studies in 2000 to 214 studies in 2010, remained thereafter relatively stable (with 260 in 2011, 243 in 2012, 236 in 2013, 252 in 2014, 274 in 2015 to 224 in 2016). There was a clear change in the study design over the years towards more trials with a placebo. Furthermore, the number of entities investigated within one clinical trial (from 1 tumor entity, 2-5 tumor entities, 6-10 tumor entities and >10 tumor entities (including clinical trials targeting events from various entities) increased over the years. With respect to sponsorship, there was an increase of industry-alone sponsorship over the years. Table: 1638PD . 2000 - 2004 2005 - 2009 2010 - 2014 2015 - 2016 Masking open label 78% 75% 73% 77% single blind 1% 3% 3% 4% double blind 12% 20% 18% 12% quadruple 1% 2% 6% 6% Missing 9% 0% 0% 1% Placebo Placebo - no 95% 82% 80% 85% Placebo - yes 5% 18% 20% 15% Number of entities 1 88% 91% 89% 85% 2 - 5 6% 3% 4% 5% 6 - 10 0% 0% 0% 1% > 10 6% 5% 7% 10% Sponsorship Industrie alone 43% 68% 65% 64% other 57% 32% 35% 36% Conclusions Cancer clinical trials have changed with respect to trial design and sponsorship during the last decade to cope with recent development of new cancer therapeutics. Legal entity responsible for the study Charite Universitatsmedizin Berlin. Funding Has not received any funding. Disclosure T. Fureder: Honoraria (self), Honoraria (institution), Research grant / Funding (institution): Norvartis; Honoraria (self), Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: BMS; Honoraria (self), Honoraria (institution), Advisory / Consultancy, Research grant / Funding (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: MSD; Honoraria (self), Honoraria (institution), Advisory / Consultancy, Research grant / Funding (self), Research grant / Funding (institution): Merck Darmstadt; Honoraria (self), Honoraria (institution), Research grant / Funding (institution): AstraZeneca; Honoraria (self), Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Honoraria (self), Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Boehringer Ingelheim; Honoraria (self), Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Pfizer; Honoraria (self), Honoraria (institution), Research grant / Funding (institution): Sanofi; Honoraria (self), Honoraria (institution), Research grant / Funding (institution): Accord; Honoraria (institution), Research grant / Funding (institution): Celegene; Honoraria (institution), Research grant / Funding (institution): Ipsen; Honoraria (institution), Research grant / Funding (institution): Astellas; Non-remunerated activity/ies: President elect Austrian Head and Neck cancer society; Full / Part-time employment: Medical University of Vienna; Honoraria (institution): Johnson and Johnson ; Research grant / Funding (institution): Amgen. U. Keilholz: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Bristol-Myers Squibb; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Merck; Advisory / Consultancy: Serono; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: MSD Oncology; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Pfizer; Honoraria (self), Speaker Bureau / Expert testimony: Novartis; Honoraria (self), Speaker Bureau / Expert testimony: Glycotope GmbH. All other authors have declared no conflicts of interest.

Assessment of patients with synchronous primary cancers and metastases is challenging, as it can be difficult to assign the metastases to the correct primary due to low differentiation, high similarity on histology or inaccessibility of tumour tissue. Systemic treatment for metastatic disease, however, needs to be directed at the leading histology or cover multiple tumour types with the same regimen. Considering the additional obstacles in cancer management, including tumour heterogeneity and clonal evolution, blood-based genomic profiling (‘liquid biopsy’) is suggested to be a useful tool to provide accessible tumour-derived biomarkers. We herein report a case of a patient with independent primary tumours of the colon and pancreas, as well as liver metastases. All lesions were resected and genotyped revealing KRAS mutations G12C and G12D in the primary tumours, respectively. The G12D mutation detected in the pancreatic tumour was retrieved in the metastasis, thus confirming the pancreatic cancer to be the origin of the liver lesions. The prevalence of the pancreatic tumour was additionally verified by the detection of the G12D variant in circulating cell-free DNA (cfDNA). This case demonstrates the utility of liquid biopsy to identify the predominant tumour burden in patients with multiple primary cancers, based on the detection of the tumour-associated gene mutation in the plasma. Serial monitoring through liquid biopsies might allow disease surveillance to guide cancer management. The review of the literature highlights the importance of liquid biopsies in personalised oncology, even though only one case report refers to the benefit of cfDNA analysis in a patient affected by synchronous primary tumours.

Purpose Characterisation of circulating melanoma cells (CMC) for BRAF status can provide a strategy for non-invasive serial genotype monitoring in patients receiving BRAF inhibitors. We aimed to establish a method for BRAF mutation analysis at CMC level and we applied it in a cohort of CMC-positive patients. Methods We established a sensitive method for detection of BRAF mutations at codon 600 in whole blood samples of patients with melanoma, positive for presence of CMC. The method based on selective cleavage of wild-type sequences by taking the advantage of the presence of a TspR1 enzyme restriction site located at the site of mutation. Results In a serial dilution experiment spiking BRAF mutated cDNA into BRAF wild-type cDNA the method allowed to detect mutated cDNA till a dilution of 1:10 4 . Peripheral blood (PB) samples resulting positive for CMC and matched tissue specimens from 21 different AJCC stage IV melanoma patients were analysed. A 91% (19/21) correspondence between BRAF status in tissue and PB specimens was observed. In a patient (whose melanoma showed to bear the BRAF V600E mutation in blood, but not at tissue level) our analysis showed that blood samples with PCR evidence for CMC were heterogeneous for BRAF status under limiting-dilution conditions, suggestive of heterogeneity of CMC. Conclusion The method reported here represents a rapid approach for determination of BRAF status in the blood of patients with CMC.

Background Tyrosine kinase inhibitors (TKI) such as sorafenib have substantially improved the prognosis of metastatic renal cell carcinoma (mRCC) patients, but long-term remissions have only been reached with immunotherapy. Sequencing or combining TKI treatment with immunotherapy may represent an attractive therapeutic concept. However, in vitro data have shown that TKI may not only affect tumour cells, but also inhibit signalling in immune effector cells. Therefore, we asked whether sorafenib had an influence on peripheral immune effector cells in a cohort of 35 mRCC patients receiving sorafenib treatment. Methods Peripheral blood (pB) samples were analysed at baseline and after 8 weeks of treatment. IL-10 and TGF-s mRNA levels were quantified by RT-PCR; regulatory T cell (Treg) counts and intracellular cytokine responses (TNF-α, IFN-γ, IL-10 and TGF-s) of mononuclear cell subsets were determined by flow cytometry after in vitro stimulation with PMA/ionomycin. Results Sorafenib did not alter the elevated TGF-s and IL-10 mRNA levels or elevated frequencies of IL-10 and TGF-s producing monocytes and had no influence on type 1 cytokine responses in pB. CD4+CD25high FOXP3+/CD3+ T cells, likely representing Treg cells, decreased during sorafenib therapy. Conclusions In vivo, sorafenib treatment was associated with a decrease in frequency of Treg cells without influencing the function of peripheral immune effector cells. Therefore, although sorafenib did not convert the immunosuppressive phenotype associated with mRCC, it seemed to be a possible candidate for combination with immunotherapy.

Different molecular markers have been identified for melanoma-initiating cells including CD133 and nestin. Assuming that metastasis requires a dissemination of tumor-initiating cells, presence of circulating tumor-initiating cells should be associated with worse patient outcome. In this study, 20 ml blood was collected from 32 consecutive patients affected by metastatic melanoma and blood was enriched for circulating melanoma cells (CMCs) by CD45 depletion of the non-melanoma cell fraction. Multiparameter cytometry was carried out to co-stain with combinations of CD133 and nestin (NES). Six tissue samples from metastatic lesions of six different patients were stained with the same antibodies by immunohistochemistry. Percentage of NES-positive CMCs correlated with tumor burden and number of metastatic sites. Cox regression analysis revealed levels of lactate dehydrogenase (LDH; hazard ratio: 12.8 (1.35-121.5); P=0.02), number of metastatic sites (hazard ratio 3.87 (1.66-9.03); P=0.02), tumor burden (hazard ratio 5.72 (1.57-20.9); P=0.01), and percentage of NES-expressing CMCs ≥ 35% (hazard ratio 5.73 (1.66-19.7); P=0.006) to be factors related to shorter overall survival. CD133- and NES-expression profiles on CMCs were similar to matched metastatic tissue. These findings show that CMCs expressed stem cell-associated markers NES and CD133. Higher expression of NES on CMCs might represent an index of poor prognosis.

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.

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.

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.

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.

Introduction Next-generation sequencing (NGS) techniques render genetic investigations of tumor material more effective and will help to understand tumor biology and overcome mechanisms of treatment resistance. Targeted NGS which is feasible also for archival FFPE tumor material represents an attractive approach for the development of personalized cancer treatment. We describe here the establishment of an HNSCC-specific cancer gene panel for targeted NGS. Methods NGS data available at the cBioPortal reporting on whole exome sequencing of 412 HNSCC samples from three independent patient cohorts (Broad Institute, Science 2011; John Hopkins University, Science 2011; TCGA, provisional) were used. For the definition of relevant genes to be included in a specific HNSCC gene panel, selection criteria such as the relative mutational frequency in HNSCC, the presence of hotspot mutations within the coding region or the occurrence of mutations at recurrent positions were used. In order to consider the biological function of particular genes, genes were mapped to pathways using different databases. Results Overall, 1710 genes with genetic alterations fulfilling the selection criteria were identified in the HNSCC datasets. Of these, less than 10% of affected genes were altered in more than 5% of patients, speaking for a huge genetic heterogeneity of head and neck cancer. By our selection criteria, a gene set of 901 genes was identified, covering a coding region of 2.94 Mb in total. Of these genes, only 652 could be mapped to specific pathways, resulting in a final coding region length of the selected genes of 2.15 Mb to be targeted by panel NGS. The gene list covered a wide spectrum of already known but also novel unexpected genetic lesions. Conclusion Application of NGS for comprehensive molecular characterization of HNSCC revealed new insights into HNSCC biology and is expected to lead to the identification of new druggable targets also for this tumor entity. The gene list obtained by our approach can be used for the establishment of an HNSCC-specific panel for targeted NGS. Such a gene panel will help in the assessment the intratumor genetic heterogeneity and the characterization of spontaneous and treatment-induced clonal evolution of tumors and will thereby contribute to the development of personalized medicine in HNSCC.

A proliferation assay to detect a specific T cell response against HLA class I-binding peptides is reported. To establish the specificity and sensitivity of the assay we used a synthetic peptide derived from the CMV glycoprotein B containing the HLA-A2.1 ligand motif. Lymphocyte proliferation was measured following culture of PBMC in the presence or absence of peptide by BrdU uptake. Differing culture conditions were compared (cell number, time in culture, peptide concentration, +/- IL-2). A peptide-specific response was detected in 11 of 23 HLA-A2-positive and CMV IgG-positive donors (47.8%), and in 4 of 36 HLA-A2-positive and CMV IgG-negative donors (11.1%, p = 0.019) tested under optimized conditions. None of 22 HLA-A2-negative individuals tested showed a peptide-specific response and the reproducibility was high. Peptide-specific IFN-gamma-secreting T cells could be demonstrated in responding donors with the ELISPOT assay. This proliferation assay may be suitable for monitoring induction of a specific T cell response against known HLA class I-binding peptides following vaccination with tumor or viral antigens.

Background Prognosis of patients with failure of cisplatin-based 1st line chemotherapy for recurrent or metastatic SCCHN remains poor. We therefore evaluated temsirolimus after failure of cisplatin and cetuximab in SCCHN. Methods Patients with progressive SCCHN and failure of platinum-based chemotherapy and cetuximab were eligible. Patients with loco-regional recurrence as the sole lesion had to have a progression free survival (PFS) of at least 6 months from last therapy. 18 years of age, ECOG 0-2, life expectancy of at least 3 months, and adequate organ function are key inclusion criteria. Brain metastases were allowed, provided local therapy has been completed successfully. Tumor assessment was performed every 6 weeks and assessed according to RECIST 1.0. Primary endpoint was the progression free survival rate (PFR) at 12 weeks >20%. Secondary endpoints were time to progression (TTP), objective response rate (ORR), overall survival and toxicity (according CTCAE 3.0). 25 mg temsirolimus was given i.v. weekly until progression or toxicity prevail. PFS, PFR, and OS estimates were calculated by Kaplan-Meier-Curves. Results A total of 42 patients entered the trial, of whom 40 were eligible. ECOG performance status of 0/1/2 was found in 7/29/5 pts and missing in 1 patient. Mean age was 61.6y (range: 42-79y) with male predominance (31 pts; 74%). Progression free survival rate at 12 weeks was 40% (CI95% 25-55%), TTP was 56 d (CI95% 36-113d), and OS 152d (CI95% 76-256d). Stable disease (SD) was obtained in 19 pts. (56%), progressive disease (PD) in 10 pts. (29%), 1 pts. was not evaluable for response, and 10 pts. had missing scans. Treatment with temsirolimus was well tolerated. Safety data will be presented at the meeting. Conclusions Temsirolimus reaches its prespecified endpoint with a PFR at 12weeks of 40%. Efficacy data is encouraging and compare to single agent cetuximab activity in platinum-refractory SCCHN. Further studies with this drug in SCCHN are warranted. Disclosure V. Grunwald: research funding: Pfizer Advisory Board: Merck KG U. Keilholz: Research support and honorarium Whyeth / Pfizer. T.C. Gauler: ADVISORY BOARD: MERCK KG, Pfizer HONORARIA: Pfizer All other authors have declared no conflicts of interest.

A phase lb trial of a novel regional approach to adoptive immunotherapy is reported. Patients with liver metastases received continuous high-dose infusion of interleukin-2 (IL-2) into the splenic artery or intravenous infusion with subsequent transfer of lymphokine-activated killer (LAK) cells into the portal vein or the hepatic artery. Trafficking studies revealed homogeneous distribution of the LAK cells within the liver. The usual side-effects of IL-2 and LAK cells occurred without limiting liver toxicity. One partial (7+ months) and two complete responses (36 and 26+ months) were observed in 9 patients with metastases from cutaneous melanoma. None of 6 patients with metastases from ocular melanoma responded.

Aim: Patients with R/M HNSCC who progress after first-line platinum-based therapy have a dismal prognosis and no well-defined treatment. Afatinib, an irreversible ErbB family blocker, showed anti-tumor activity in a phase II trial in this setting. Methods: In this phase III trial, patients with R/M HNSCC after progression on/after platinum-based therapy were randomised 2:1 to 40 mg/day oral afatinib (n = 322) or 40 mg/m2/week intravenous MTX (n = 161), stratified by ECOG performance status (0/1) and prior use of anti-EGFR antibody therapy (Yes/No) in the R/M setting. The primary endpoint was progression-free survival (PFS); secondary endpoints included overall survival (OS), objective response rate (ORR), patient-reported outcomes (PROs) and safety. Results: Afatinib significantly improved the primary endpoint of PFS vs MTX (median 2.6 vs 1.7 months; HR = 0.80 [95% CI 0.65–0.98; p = 0.03]), but did not significantly improve OS vs MTX (median 6.8 vs 6.2 months; HR = 0.94 [0.75–1.18]). Disease control rate was higher with afatinib vs MTX (49.1% vs 38.5%; p = 0.04); ORR was 10.2% vs 5.6% (p = 0.10). Tumour shrinkage from baseline was observed in 34.8% of afatinib-treated patients compared to 22.4% of MTX-treated patients. Afatinib delayed deterioration of global health status, pain and swallowing vs MTX (all p ≤ 0.03) and provided improvement in pain (p = 0.03). The most frequent grade 3/4 drug-related adverse events were rash/acne (9.7%) and diarrhoea (9.4%) with afatinib, and leukopenia (15.6%) and stomatitis (8.1%) with MTX. Fewer treatment-related dose reductions, discontinuations and fatal events were seen with afatinib. Additional optional biomarker analyses, including p16 status, will be presented. Conclusions: In this phase III trial, second-line afatinib significantly improved the primary endpoint of PFS and delayed deterioration of PROs vs MTX, with a manageable safety profile, in patients with R/M HNSCC after failure of platinum-based therapy. Machiels JPH, Haddad RI and Fayette J contributed equally to this work. Disclosure: J-P. Machiels: Advisory boards for Boehringer Ingelheim, Novartis; R.I. Haddad: Advisory boards for Celgene, Bayer Corporate sponsored research for Boehringer Ingelheim; L.F. Licitra: Advisory boards for Eisai, Bristol-Myers Squibb, GlaxoSmithKline, Merk-Serono, Amgen, Boehringer Ingelheim, Debiopharm, VentiRX, Sobi, F. Hoffmann-La Roche, Celgene; M. Tahara: Advisory boards for Eisai, Boehringer Ingelheim, Otsuka Pharmaceutical Corporate sponsored research for Eisai, Boehringer Ingelheim Other substantive relationships with Merck Serono, Bristol-Myers Squibb; J.B. Vermorken: Advisory boards for Merck Serono, Genentech Other substantive relationships with Steering CIE LUX-HN P.M. Clement: Advisory boards for Merck Serono, Leo Pharma, Grunenthal Corporate sponsored research for Merck Sharp & Dohme, Grunenthal; T. Gauler: Stock ownership with Bayer AG Advisory boards for Boehringer Ingelheim (BI), Merck Serono, Novartis Corporate sponsored research for BI (ITS), Merck Serono (ITS) Other substantive relationships with BI, Merck Serono; G. De Castro Jr: Advisory boards for Boehringer Ingelheim, Bayer, F. Hoffmann-La Roche, Novartis; X.J. Cong, L. Svensson and E. Ehrnrooth: Other substantive relationships with Employee, Boehringer Ingelheim. All other authors have declared no conflicts of interest.

Aim: Platinum-based regimens are the current standard of care for patients (pts) with metastatic non-small cell lung cancer (NSCLC). Veliparib (V) is a potent, orally bioavailable PARP inhibitor that (1) enhances the efficacy of platinum-containing DNA damaging therapies in preclinical models, and (2) has been safely combined with full dose carboplatin (C) and paclitaxel (P) in Ph 1 trials. Methods: Pts with advanced or metastatic NSCLC were randomized 2:1 to V at 120 mg BID or placebo plus CP. Pts were stratified by histology and smoking history. The primary endpoint was progression-free survival (PFS, with 80% power and a = 0.05, assuming log-rank HR of 0.51). All data analyses were performed at the 78th PFS event except overall survival (OS, 90th event). In the case of a positive signal, continuation of the program to a phase 3 trial was planned. Results: 158 pts were randomized; 64% of pts were male; 49% had squamous NSCLC; 60% smoked within 1 year of study entry. Adverse events (AE) in 20% or more pts were alopecia (39% VCP/42% CP), anemia (31/42), neutropenia (36/29), nausea (28/25) and peripheral neuropathy (24/25). Leukopenia was seen in 11% VCP vs. 1% CP. Grade 3/4 AEs in 10% or more pts were neutropenia (23/19) and anemia (10/10). Mean chemo cycles were 4.5 C/4.5 P with CP and 4.5 C/4.3 P with VCP. Efficacy (ITT Population) CP n = 53 VCP n = 105 HR: VCP/CP PFS (mo, 95% CI) 4.2 (3.1-5.6) 5.8 (4.2-6.1) 0.71 (0.50-1.13) Squamous 4.1 (2.8-NA) 6.1 (5.8-8.3) 0.50 (0.24-1.04) Non-squamous 5.0 (2.8-5.6) 4.3 (2.8-6.0) 0.94 (0.52-1.71) OS (mo, 95% CI) 9.1 (5.4-12.3) 11.1 (8.8-13.4) 0.77 (0.50-1.18) Squamous 8.5 (5.0-11.4) 10.3 (8.3-13.2) 0.75 (0.41-1.38) Non-squamous 11.1 (4.8-NA) 12.6 (8.0-NA) 0.79 (0.43-1.47) Overall Response Rate (%, 95% CI) 28 (17-42) 31 (22-40) - Duration of Response (mo, 95% CI) 3.3 (2.7-4.3) 6.9 (4.4-7) 0.11 (0.03-0.50) Conclusions: Estimated HR for progression and death from NSCLC favored VCP over CP, but results were not statistically significant. VCP was delivered without excessive toxicity. Based on results in the squamous histology subgroup, a Phase 3 pivotal trial has been initiated for patient with squamous cell cancer (M11-089). Disclosure: S.S. Ramalingam: Has served as a consultant on scientific advisory board meetings for AbbVie and has received honorarium; Q. Qin: AbbVie employee and stock owner; J. Qian: AbbVie employee and stock owner; C. Nickner: AbbVie employee and stock owner; J. Dziubinski: AbbVie employee and stock owner; M.D. McKee: AbbVie employee and stock owner; V.L. Giranda: AbbVie employee and stock owner. All other authors have declared no conflicts of interest.

Aim: Analysis of a trial investigating, whether TPFC would be feasible and superior to PFC in patients with RM-HNSCC without limiting comorbidities, based on the findings, that D in addition to PF in induction treatment and C in addition to PF in RM disease both improved outcome in HNSCC. The first analysis was presented at ASCO 2014; here we present the final analysis. Methods: 180 patients were assigned 1:1 to receive either (arm A) P 40 mg/sqm, D 40 mg/sqm, F 2000 mg/sqm days 1 + 8, C 400/250 mg/sqm days 1, 8, 15 q3w or (arm B) standard PFC (P 100 mg/sqm day 1, F 1000 mg/sqm days 1-4, C 400/250 mg/sqm days 1, 8, 15) q3w. Chemotherapy was continued for a maximum of 6 cycles in absence of disease progression or limiting toxicity, followed by C maintenance (500 mg/sqm q2w). Because of excessive toxicity (gastrointestinal and infections) in arm A, P was reduced to 30 and F to 1000 mg/sqm after 20 patients/arm. Results: Median follow-up was 2 years. Toxicity was similar in both arms, with a rate of grade 4 toxicities of 21.3% vs. 30.8% of patients in arms A vs. B. 11.2% vs. 6.6% potentially treatment related deaths were observed in arms A vs. B. Median PFS A vs. B was 5.4 vs. 5.0 mo (HR 0.79, p = 0.375), median OS was 9.8 vs. 9.7 mo (HR 1.39, p = 0.993) and response rates were 38.2% vs. 31.9%, respectively. Conclusions: Despite of the previously described advantage of both components D and C in treatment of HNSCC, the four-drug regimen was not associated with improved median PFS or OS. The efficacy data of the CeFCiD trial are comparable with the results in the EXTREME trial. Disclosure: M. Knoedler: Financial support for conducting this trial by Merck GmbH and Sanofi Aventis for conducting this trial; T. Gauler, A. Dietz, V. Grunwald, J. Stoehlmacher-Williams, O. Guntinas-Lichius and G. Maschmeyer: Merck Serono; U. Keilholz: Merck Serono and Sanofi Aventis. All other authors have declared no conflicts of interest.