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Advances in the treatment of metastatic melanoma have improved responses and survival. However, many patients continue to experience resistance or toxicity to treatment, highlighting a crucial need to identify biomarkers and understand mechanisms of response and toxicity. Neoadjuvant therapy for regional metastases might improve operability and clinical outcomes over upfront surgery and adjuvant therapy, and has become an established role for drug development and biomarker discovery in other cancers (including locally advanced breast cancer, head and neck squamous cell carcinomas, gastroesophageal cancer, and anal cancer). Patients with clinically detectable stage III melanoma are ideal candidates for neoadjuvant therapy, because they represent a high-risk patient population with poor outcomes when treated with upfront surgery alone. Neoadjuvant therapy is now an active area of research for melanoma with numerous completed and ongoing trials (since 2014) with disparate designs, endpoints, and analyses under investigation. We have, therefore, established the International Neoadjuvant Melanoma Consortium with experts in medical oncology, surgical oncology, pathology, radiation oncology, radiology, and translational research to develop recommendations for investigating neoadjuvant therapy in melanoma to align future trial designs and correlative analyses. Alignment and consistency of neoadjuvant trials will facilitate optimal data organisation for future regulatory review and strengthen translational research across the melanoma disease continuum.

Dacarbazine chemotherapy has been the mainstay of melanoma treatment for >30 years. In the early 2000s, carboplatin (with or without other agents, such as paclitaxel) was the most commonly used second-line therapy in the UK. The aim of the present study was to report a significant response rate to second-line carboplatin in patients from three UK institutions who had been previously treated and failed to respond to dacarbazine, and investigate whether sequential therapy may be more effective compared with combination therapy. A total of 104 patients were identified, the majority of whom were treated with carboplatin (area under the curve 5-6) every 3 weeks for a maximum of 6 cycles. A total of 102 patients were evaluable for response, among whom 11 patients had an objective response (1 complete response and 10 partial responses) and 15 had stable disease, giving an overall response rate of 11% and disease control rate of 26%. The median progression-free survival was 1.8 months (range, 0.2-36+ months) and the median overall survival was 4.6 months (range, 0.2-36+ months). Surprisingly, the majority of the patients who benefited from second-line carboplatin therapy were those with visceral metastases, the survival of whom would not be expected to exceed 6 months after first-line treatment.

Single-agent anti-PD1 antibodies are usually very well tolerated, but serious toxicity can still occur. Despite the PD-1 pathway seems to be relevant in the pathogenesis of immune-related myositis, anti-PD1-related myositis is generally a rare side effect of the treatment and usually not serious. However, its frequency is likely to increase as the use of immune checkpoint blockades. We present here a case of life-threatening polymyositis with associated spontaneous muscular hematoma in a patient treated with single-agent nivolumab in the adjuvant setting. Spontaneous hematoma is an extremely rare complication with unclear etiology of idiopathic myositis. Very few cases have been reported in the literature and their outcome has been often fatal. To our knowledge, this is the first case of autoimmune myositis and spontaneous heamatoma associated with the administration of single-agent checkpoint blockade. Anti-PD1 antibodies have changed the treatment landscape for a number of cancer entities in the past few years. When given as single agent they are usually very well tolerated, but serious rare toxicity can still occur. We present here a case of polymyositis with associated spontaneous muscular hematoma in a patient treated with single agent nivolumab.

Immune checkpoint blockades have recently emerged as a breakthrough treatment for solid tumors showing high response rates and long durability. In melanoma, the combination of ipilimumab with nivolumab showed high efficacy. However, still half the patients do not respond to this treatment. In order to increase the therapeutic ratio in melanoma and other cancers, different approaches are under evaluation. Three relevant questions are at the moment driving the research community: how to maximize benefit while minimizing toxicity; how to better identify patients who are more likely to benefit from immunotherapy; how to convert nonresponders into responders. In this review we summarize the most recent findings and we outline the most likely future challenges.

BACKGROUND: Despite the majority of patients do not gain any benefit from dendritic cells (DC) vaccines, this approach has occasionally given rise to dramatic responses in melanoma. Biomarkers are crucial to identify which patients are more likely to respond. We looked for correlations between pre- or post- vaccination biomarkers and clinical outcomes to DC therapy in a cohort of patients with stage IV melanoma receiving a vaccine with autologous ex-vivo expanded DCs pulsed with allogeneic tumor cell lysate. METHODS: Serial serum samples were collected at baseline, week 4 and 12 and they were analyzed for a panel of different inflammatory markers using cytometric bead array technology and ELISA. RESULTS: Twenty-one patients were evaluable for response. Patients were separated into responders and non-responders based on clinical benefit. Responders were defined as patients who achieved a complete response, partial response or stable disease the latter lasting for at least 6 months. Responders (N = 9) showed a significantly longer Progression-free Survival (PFS; HR 0.23; 95% CI 0.08–062; P < .001) and Overall Survival (OS; HR 0.22; 95% CI 0.08–0.59; P < .001). The clinical non-responder phenotype correlated with an elevated pre-vaccination level of cytokines associated with inflammation compared to clinical responders (Apolipoprotein C111; IL-12 p40; MiP1α; Stem Cell Factor and TNFα). Apolipoprotein E (ApoE) was also significantly elevated in the pre-vaccine sera of the clinically non-responding group and in addition it was found to correlate with outcomes. Patients with increased levels of ApoE had a significantly shorter PFS (HR 3.02; 95% CI 1.09–8.35; P = .015) and OS (HR 2.40; 95% CI 0.9–6.3; P = .034). CONCLUSION: Our findings support the notion that treating the inflammatory background may have an impact on clinical outcome for patients receiving immunotherapy. A larger study is needed to confirm the significance of ApoE as a predictive biomarker for response to DC vaccines.

Targeted therapies and immunotherapies have transformed melanoma care, extending median survival from ∼9 to over 25 months, but nevertheless most patients still die of their disease. The aim of precision medicine is to tailor care for individual patients and improve outcomes. To this end, we developed protocols to facilitate individualized treatment decisions for patients with advanced melanoma, analyzing 364 samples from 214 patients. Whole exome sequencing (WES) and targeted sequencing of circulating tumor DNA (ctDNA) allowed us to monitor responses to therapy and to identify and then follow mechanisms of resistance. WES of tumors revealed potential hypothesis-driven therapeutic strategies for BRAF wild-type and inhibitor-resistant BRAF-mutant tumors, which were then validated in patient-derived xenografts (PDX). We also developed circulating tumor cell–derived xenografts (CDX) as an alternative to PDXs when tumors were inaccessible or difficult to biopsy. Thus, we describe a powerful technology platform for precision medicine in patients with melanoma. Significance: Although recent developments have revolutionized melanoma care, most patients still die of their disease. To improve melanoma outcomes further, we developed a powerful precision medicine platform to monitor patient responses and to identify and validate hypothesis-driven therapies for patients who do not respond, or who develop resistance to current treatments. Cancer Discov; 6(3); 286–99. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 217

The treatment of malignant melanoma has changed beyond recognition in the last 7 years. Where previously single agent dacarbazine was often the only treatment used for advanced disease, now there are potentially multiple lines of treatment, based on immunotherapy and targeted treatment options, either as monotherapy or in combination. In this brave new world the question arises, does chemotherapy still have any relevance in the modern management of melanoma? In this review, we summarize the various chemotherapeutic options that have been trialled in melanoma to date, and discuss the role chemotherapy may still play in treating melanoma, potentially in combination with more novel agents, or in certain subtypes of melanoma.

Background\ud The use of checkpoint inhibitors (ipilimumab, pembrolizumab, nivolumab) has revolutionised the treatment of metastatic melanoma. However still more than the half the patients do not respond to single-agent immunotherapy. This has led to the development of combining these agents in an attempt to enhance the anti-cancer activity. More than 300 different studies with 15 different drug doses are currently ongoing. Combining different checkpoint inhibitors (CPIs) does indeed lead to an increase in response rate, but this is associated with significant toxicity. IMM-101 is a heat killed Mycobacterium preparation which induces marked immune modulation and little systemic toxicity. It has been reported as having activity in melanoma as single agent and in pancreatic cancer in combination with gemcitabine, the latter in a randomised study.\ud \ud Methods\ud Here we report the effect of adding CPIs to 3 patients who had previously been on IMM-101, either as a trial or a named patient programme and a patient who received the IMM-101 together with nivolumab.\ud \ud Results\ud All 4 patients had rapid and very good responses, three of them maintained over 18 months with no significant additional toxicity.\ud \ud Conclusions\ud The rapid and complete clinical responses seen in these patients may suggest that IMM-101 is activating a complementary pathway which is synergistic with CPI treatment.

Background The purpose of this study was to determine whether single nucleotide polymorphisms (SNPs) in AKT1, AKT2, FRAP1, PIK3CA, and PTEN were associated with treatment response and clinical outcome in patients with head and neck squamous cell carcinoma (HNSCC). Methods Genomic DNA was extracted from formalin-fixed tissue of 45 patients with recurrent or initially metastatic HNSCC, and SNPs were genotyped by means of real-time polymerase chain reaction (PCR) system or direct sequencing. Results The AKT2:rs8100018 and the PTEN:rs12569998 homozygous variants resulted as associated with an increased risk of progression (hazard ratio [HR], 4.83; 95% confidence interval [CI], 1.11–21.03; and HR, 2.36; 95% CI, 1.24–4.50, respectively). An additive effect on risk of progression was observed. The AKT2:rs8100018 homozygous variant was significantly associated with a higher risk of death (HR, 3.57; 95% CI, 1.06–12.00), whereas the presence of at least one variant allele of AKT1:rs3803304 was associated with a lower risk of death (HR, 0.51; 95% CI, 0.27–0.97). Conclusion We identified combined genotypes associated with outcome of HNSCC, which might have an impact for identification of a target population for cetuximab-docetaxel treatment. Results should be considered as an initial finding and warrant validation in larger clinical trials. © 2014 Wiley Periodicals, Inc. Head Neck 37: 471–478, 2015

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.

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I–IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab., The heterogeneity of colorectal cancer has important clinical and therapeutic implications. Here the authors analysed the responses of a large biobank of organoids and xenografts derived from colorectal patients to a panel of clinically relevant therapeutic agents to identify genes signatures associated with drug response.

Recent years have witnessed increased interest in the detection of circulating tumour cells (CTCs) for diagnosis, monitoring, and treatment decision making in patients with cancer. Factors that have led to accelerated research in this field include advances in technologies for examination of intact CTCs, personalised medicine with treatment selection according to molecular characteristics, and continued lack of understanding of the biology of treatment resistance and metastasis. CTCs offer promise as a surrogate for tissue where there is insufficient tissue for molecular analysis and where there is a requirement to serially monitor molecular changes in cancer cells through treatment or on progression. In patients with either small cell or non-small cell lung cancer (NSCLC), there is evidence that CTC number is prognostic and that CTCs counted before and after treatment mirror treatment response. In patients with molecularly defined subtypes of NSCLC, CTCs demonstrate the same molecular changes as the cancer cells of the tumour. However, CTCs are not quite ready for "primetime" in the lung cancer clinic. There are still more questions than answers with respect to the optimal technologies for their detection and analysis, their biological significance, and their clinical utility. Despite this the current pace of progress in CTC technology development seems set to make "liquid biopsies" a clinical reality within the next decade. For the everyday clinician and clinical trialist, it will be important to maintain knowledge of the strengths and weaknesses of the technologies and evolving evidence base for CTCs as a routinely used diagnostic tool.

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.

For breast cancer patients, treatment decisions based on the molecular profile of the primary tumor have been used for many years. In Her2-overexpressing tumors, trastuzumab is a key component of therapy. However, despite persistent expression of Her2, most tumors eventually become resistant to trastuzumab. When this happens, the patients benefit from a regime containing lapatinib, a dual EGFR and Her2 tyrosine kinase inhibitor. We report on a patient affected by chemo-refractory metastatic Her2-positive breast cancer enrolled in a translational research program for the detection and characterization of circulating tumor cells (CTCs). Depletion of the EGFR-positive CTC pool in the blood was associated with tumor response, whereas disease progression was related to a recurrence in CTCs, which were both EGFR and Her2 negative. Although a proof for the clinical significance of EGFR-positive circulating tumor cells is currently lacking, expression of EGFR may predict response to lapatinib-based treatments as in the case presented.

Summary Background There is continued interest in markers indicative of circulating melanoma cells. Nestin is a neuroepithelial intermediate filament protein that was found to be expressed in melanoma and in various cancer stem cells. Objectives We investigated expression of nestin in peripheral blood of patients with melanoma. Methods We analysed nestin expression by flow cytometry and by quantitative reverse transcription–polymerase chain reaction both in tissues (n = 23) and in blood samples (n = 102) from patients with American Joint Committee on Cancer stage III–IV melanoma. Forty-six negative controls were also added. Results Flow cytometry did not reveal nestin-expressing cells in peripheral blood of healthy volunteers. In patients with melanoma, however, nestin protein was expressed in a proportion of melanoma cells enriched from peripheral blood by immunomagnetic sorting. In melanoma tissue samples a significant correlation was found between mRNAs coding for nestin and tyrosinase (P = 0·001) and melan-A (P = 0·002), whereas in blood a significant correlation was observed only for tyrosinase (P = 0·015), but not for melan-A (P = 0·53). Nestin expression was higher in stage IV patients compared with stage III/IV with no evidence of disease, in patients with high tumour burden, and was positively correlated to expression of tyrosinase and melan-A. Conclusions Nestin was found to be an additional marker of interest for circulating melanoma cells. Prospective studies should investigate its potential added informative value in comparison with markers already in use for melanoma cell detection.

Antibodies against immune checkpoints including CTLA-4, PD-1 and PD-L1 are increasingly being used in lung cancer. They are associated with novel, immune related toxicities not previously encountered with established treatments for lung cancer including colitis, hepatitis, rashes, neuropathies and other rarer immune mediated toxicities. Although generally these are low grade, there is a potential to be life threatening if not managed promptly. Early recognition of toxicity and institution of management algorithms are key to ensuring patient safety. We review the common toxicities and provide recommendations on their management.

Prostate cancer represents one of the most important health problems in industrialized countries. It is the second leading cause of cancer-related death in the United States. Therapeutic options are different according to the stage of the disease at the diagnosis. Patients with localized disease may be treated with surgery or radiation, whereas the treatment for patients with a metastatic disease is purely palliative. Hormonal treatment represents the standard therapy for stage IV prostate cancer, but patients ultimately become unresponsive to androgen ablation and are classified as hormone-refractory prostate cancer patients. The molecular mechanisms involved in progression in hormone resistance are characterized by mutations, down and up-regulation in the androgen receptor gene, mutations in p53 and over-expression of Bcl2 and other alterations in genes and in gene expression. The important thing is that we understand these mechanisms to define potential therapeutic agents for the treatment of hormone-refractory prostate cancer patients. Conventional options for patients with hormone-refractory prostate cancer include secondary hormone therapy, radiotherapy and cytotoxic chemotherapy. The commonest antineoplastic agents are mitoxantrone, estramustine and taxanes. Despite an improvement In the palliative benefit, none of these agents has demonstrated a beneficial impact on the overall survival of patients. Therefore, there is no standard therapy for these patients, thus we need new approaches which should be studied in clinical trials. The evaluation and incorporation of new agents into current treatment regimens could have a role in the treatment of hormone-refractory prostate cancer, but their efficacy has not yet been demonstrated.

The combination of epirubicin, cisplatin, and protracted venous-infusion 5-fluorouracil is the standard treatment of advanced gastric carcinoma in many European countries, and it is also an active regimen in hepatobiliary tumors. Raltitrexed is a specific inhibitor of thymidylate synthase with clinical activity in gastrointestinal malignancies. The aim of the study was to evaluate the clinical activity and toxicity of the combination of epirubicin, cisplatin, and raltitrexed in patients with advanced gastric and hepatobiliary tumors. Twenty consecutive patients with gastric carcinoma, 7 with biliary-tract carcinoma, and 5 patients with hepatocarcinoma were treated with epirubicin (60 mg/m2), cisplatin (60 mg/m2) on day 1, and raltitrexed (1 mg/m2) on days 1 and 8, every 3 weeks. The median age was 63 years (range, 28-76). Eight patients had locally advanced disease and 24 had metastatic tumors. Seven of the 18 evaluable patients (39%) with gastric carcinoma and 2 of the 5 patients with hepatocarcinoma have a partial response; 1 minimal response and 4 stabilization of disease were documented in the 7 patients with biliary-tract carcinoma. The median time to progression of the entire group was 6.8 months, and the median survival was 9.0 months. The toxicity was mild and no toxic death occurred. The combination of epirubicin, cisplatin, and raltitrexed, using this schedule, is tolerable and has clinical activity in gastric and hepatobiliary tumors.

Purpose: The monocarboxylate transporter 1 (MCT1) inhibitor, AZD3965, is undergoing phase I evaluation in the United Kingdom. AZD3965 is proposed, via lactate transport modulation, to kill tumor cells reliant on glycolysis. We investigated the therapeutic potential of AZD3965 in small cell lung cancer (SCLC) seeking rationale for clinical testing in this disease and putative predictive biomarkers for trial use. Experimental Design: AZD3965 sensitivity was determined for seven SCLC cell lines, in normoxia and hypoxia, and for a tumor xenograft model. Proof of mechanism was sought via changes in intracellular/tumor lactate. Expression of MCT1 and related transporter MCT4 was assessed by Western blot analysis. Drug resistance was investigated via MCT4 siRNAi and overexpression. The expression and clinical significance of MCT1 and MCT4 were explored in a tissue microarray (TMA) from 78 patients with SCLC. Results: AZD3965 sensitivity varied in vitro and was highest in hypoxia. Resistance in hypoxia was associated with increased MCT4 expression. In vivo, AZD3965 reduced tumor growth and increased intratumor lactate. In the TMA, high MCT1 expression was associated with worse prognosis (P = 0.014). MCT1 and hypoxia marker CA IX expression in the absence of MCT4 was observed in 21% of SCLC tumors. Conclusions: This study provides a rationale to test AZD3965 in patients with SCLC. Our results suggest that patients with tumors expressing MCT1 and lacking in MCT4 are most likely to respond. Clin Cancer Res; 20(4); 926–37. ©2013 AACR.

Acral melanoma is a rare subtype of melanoma found on the non-hair bearing surfaces of the skin. It is associated with a worse prognosis than cutaneous melanoma, with higher proportions of patients developing metastatic disease. Combination treatments targeting the MAP kinase (MAPK) pathway and immune checkpoint inhibitors have improved overall survival in patients with stage IV disease. However, resistance to both modalities remains a challenge. This is exemplified by our comprehensive analysis of a patient presenting with acral melanoma that developed resistance to both immune and targeted therapy. The patient developed metastatic disease in multiple sites and we obtained fresh tumour tissue and generated cell lines from a skin metastasis at baseline prior to nivolumab. Following an initial complete response, the patient developed new mediastinal lymph node and brain metastases, which were also resected. On further relapse, the patient commenced dabrafenib with a partial response. Despite ongoing extra-cranial control, the brain lesion progressed and was therefore resected. To investigate resistance to immune therapy, we performed comprehensive genomic analysis and gene expression profiling on the lesions from the three sites, which identified common mutations in all three lesions and a low overall mutational burden consistent with acral melanoma. Heterogeneity of the MHC class I and II restricted antigen profile of the pre-treatment subcutaneous metastasis versus lymph node and brain progressions could not account for the development of resistance to immunotherapy. Gene expression profiling revealed up-regulation of known mechanisms of immune tolerance in both the lymph node and brain metastasis compared to the pre-treatment subcutaneous metastasis. To investigate resistance to targeted therapy, we compared patient-derived cell lines from brain lesions taken pre (DabS) and on progression on dabrafenib (DabR), and showed ongoing sensitivity to dabrafenib despite the patient having progressed in the brain. When cells from the DabR cell line were cultured in cerebrospinal fluid (CSF) in the presence of dabrafenib, we observed a reduction in cell death. Thus, extrinsic factors present in CSF may have resulted in the progression of the brain lesion in this patient. Using a combination of platforms to study patient derived tissues, we show that immune editing through selection of cells with an immune resistant phenotype may have resulted in the resistance of this patient's tumours to immune therapy whilst resistance to MAPK targeted therapy could have been mediated by extrinsic factors in the CSF. Citation Format: Rebecca J. Lee, Maria Romina Girotti, Amaya Viros, Franziska Baenke, Amit Mandal, Garima Khandelwal, John Bridgeman, Elena Galvani, Gabriela Gremel, Milena Kalaitsidou, Garry Ashton, Isabel Peset, Matthew Smith, Jacqueline Swan, Kang Zeng, Haoran Tang, Caron Abbey, Robert Hawkins, Alberto Fusi, Crispin Miller, David Gilham, Nathalie Dhomen, Paul Lorigan, Richard Marais. Mechanisms of resistance to immuno and targeted therapies in acral melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2400.

BRAF/MEK inhibitors and immunotherapies have revolutionized care for patients with advanced melanoma, improving expected median survival from 9 months to 25-30 months, but the majority of patients still die of their disease. Personalized medicine strives to individualize and improve patient care. To individualize treatment decisions in advanced melanoma we analyzed 364 samples from 214 patients. We performed whole exome sequencing (WES) and circulating tumor DNA (ctDNA) analysis, and we developed patient derived xenografts (PDX). WES and targeted sequencing of ctDNA allowed us to predict responses to therapy and to identify and monitor mechanisms of resistance. WES of tumors revealed new therapeutic strategies in BRAF V600 wild-type and BRAF inhibitor-resistant melanoma and we validated these in patient derived xenografts (PDX). Thus, we describe a powerful combination of techniques for personalized medicine to improve the management of melanoma patients. Citation Format: Maria R. Girotti, Gabriela Gremel, Rebecca Lee, Elena Galvani, Dominic Rothwell, Amaya Viros, Amit Kumar Mandal, Kok Haw Jonathan Lim, Grazia Saturno, Simon J Furney, Franziska Baenke, Malin Pedersen, Jane Rogan, Jacqueline Swan, Matthew Smith, Alberto Fusi, Deemesh Oudit, Nathalie Dhomen, Ged Brady, Caroline Dive, Richard Marais. Application of sequencing, liquid biopsies and patient derived xenografts for personalized medicine in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 470.

e23141Background: Cancer profiling is receiving increased attention, but comprehensive algorithms to guide targeted and immunotherapies for patients are scarce and complex biomarker data are not ea...

We analysed mRNA levels of interferon response genes (ISG15, STAT1, CXCL10) of inhibitors of the JAK/STAT pathway (STAT3, SOCS1, SOCS3) and of cytokines (TNF alpha, IL10, TGF1) in peripheral blood of 91 stage III melanoma patients enrolled in EORTC 18991 trial to find biomarkers indicative for disease stage and predictive for efficacy of pegylated interferon alpha-2b (PEG-IFN alpha-2b) therapy. mRNA levels were analysed at baseline and after 6 months. Univariate and multivariate analyses were performed to estimate the prognostic and predictive role of mRNA levels for distant metastasis-free survival (DMFS) and relapse-free survival (RFS). Compared to healthy controls, melanoma patients showed significantly higher TGF beta 1 mRNA levels. In a multivariate model, increasing SOCS1 and SOCS3 mRNA levels were associated with worse RFS (P = 0.02 and P = 0.04, respectively) and DMFS (P = 0.05 and P = 0.05, respectively) due to negative correlation between, respectively, SOCS1/SOCS3 mRNA levels and ulceration or Breslow thickness. No impact of PEG-IFN alpha-2b on mRNA levels was observed except for ISG15 mRNA levels, which decreased in the treatment arm (P = 0.001). It seems that patients with a decrease > 60 % of ISG15 mRNA levels during 6 months PEG-IFN alpha-2b had inferior outcome.

Background The study was performed to investigate the expression of chemokine receptors (CR) on circulating tumor cells (CTC), which may be of importance for organ-specific metastases and cancer treatment since CR are potential drug-targets. Methods Blood samples from patients with metastatic carcinoma (MC) or melanoma (MM) were enriched for CTC and expression of CR (CXCR4, CCR6, CCR7 and CCR9) was evaluated by flow cytometry. Results CTC were detected in 49 of 68 patients (72%) [28 MC; 21 MM] with a median number of 3 CTC (range: 1-94)/10 mL of blood. CXCR4 was expressed on CTC in 82% (40/49) of patients [median number 1 CTC/10 mL blood; range 1-14] and CCR6 in 29 patients (59%; median 1, range: 1-14). In MM patients, CCR7 was expressed on CTC in 6 (29%) samples and CCR9 in 12 (57%). A positive correlation between surface expression of CR and organ-specific metastatic pattern was not observed. Conclusions CR were expressed on CTC of patients with solid tumors. Along with our findings, the observation that CR could be involved in CTC proliferation and migration of tumor cells appoints CTC as potential CR-antagonist therapeutic target.

Background A limitation of positive selection strategies to enrich for circulating tumor cells (CTCs) is that there might be CTCs with insufficient expression of the surface target marker which may be missed by the procedure. We optimized a method for enrichment, subsequent detection and characterization of CTCs based on depletion of the leukocyte fraction. Methods The 2-step protocol was developed for processing 20 mL blood and based on red blood cell lysis followed by leukocyte depletion. The remaining material was stained with the epithelial markers EpCAM and cytokeratin (CK) 7/8 or for the melanoma marker HMW-MAA/MCSP. CTCs were detected by flow cytometry. CTCs enriched from blood of patients with carcinoma were defined as EpCAM+CK+CD45-. CTCs enriched from blood of patients with melanoma were defined as MCSP+CD45-. One-hundred-sixteen consecutive blood samples from 70 patients with metastatic carcinomas (n = 48) or metastatic melanoma (n = 22) were analyzed. Results CTCs were detected in 47 of 84 blood samples (56%) drawn from carcinoma patients, and in 17 of 32 samples (53%) from melanoma patients. CD45-EpCAM-CK+ was detected in pleural effusion specimens, as well as in peripheral blood samples of patients with NSCLC. EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin. This was confirmed by CGH. By defining CTCs in carcinoma patients as CD45-CK+ and/or EpCAM+, the detection rate increased to 73% (61/84). Conclusion Enriching CTCs using CD45 depletion allowed for detection of epithelial cancer cells not displaying the classical phenotype. This potentially leads to a more accurate estimation of the number of CTCs. If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined.

Background: The mechanisms regulating tumor cell dissemination in locally advanced squamous cell carcinoma of the head and neck region (SCCHN) are largely unresolved. We assessed the frequency of circulating tumor cells (CTCs), their association with clinicopathologic parameters and their kinetics during radiochemotherapy. Patients and methods: Peripheral blood samples from 42 patients with locally advanced SCCHN were included. CTCs were detected using flow cytometric analysis of CD452epithelial cell adhesion molecule+cytokeratin+ cells and results were validated by nested RT-PCR analysis of circulating epidermal growth factor receptor transcripts. The association between the presence of CTCs and T stage, tumor volume, N stage and human papillomavirus status was evaluated. The influence of radiochemotherapy on CTC numbers was determined. Results: CTCs were detected in 18 of 42 SCCHN patients (43%), with a mean 6 standard deviation of 1.7 6 0.9 CTCs per 3.75 ml blood. We observed no significant correlation between the presence of CTCs and T stage or tumor volume. However, a nodal stage of N2b or higher was associated with higher frequency of CTCs. Though concurrent radiochemotherapy reduced their frequency, CTCs persisted during treatment in 20% of cases. Conclusions: Detection of CTCs correlates with regional metastasis in inoperable SCCHN. Further follow-up is needed to evaluate the prognostic significance of CTC detection, in addition to clinical staging of lymph nodes, for regional or distant recurrence.

Aim: To evaluate the prognostic and predictive importance of detection of melanoma cells in peripheral blood using reverse transcriptase polymerase chain reaction (RT-PCR) in stage III cutaneous melanoma patients after sentinel or regional lymph node dissection. Patients and methods: Serial testing for tyrosinase and Mart-1/Melan-A transcripts in peripheral blood was performed every 6 months over a maximum period of 60 months in a subset of patients enrolled in EORTC 18991 phase 3 trial, comparing pegylated interferon-alpha-2b with observation. Univariate and multivariate analyses were performed to estimate the role of RT-PCR as prognostic and predictive factor for distant metastasis-free survival (DMFS). Results: Among 299 patients who underwent RT-PCR analyses, 109 (36.5%) had at least one positive sample, either at time of randomisation (N = 17) or subsequently (N = 92). The cumulative rate of positive results was similar in the two treatment groups, as the DMFS from first RT-PCR positivity. RT-PCR result, positive versus negative, at a given time point, had no prognostic impact on subsequent DMFS. Cox time-dependent analysis indicated a significantly higher risk of developing distant metastasis in patients with a positive sample as compared to those with a negative one: hazard ratio (HR) of 2.23 (95% confidence interval (CI), 1.40-3.55; p < .001). These results were comparable in the 2 treatment groups, indicating that RT-PCR assessment was not predictive for treatment outcome. Conclusion Detection of circulating tumour cells by RT-PCR for tyrosinase and Mart-1/Melan-A was a time-dependent moderate prognostic factor for subsequent development of distant metastasis in stage III melanoma patients. (C) 2009 Elsevier Ltd. All rights reserved.

Nowadays cisplatin doublets are considered the gold-standard treatment in advanced non-small cell lung cancer (NSCLC) patients, but are often associated to poor toxicity profile. In the last years different schedules have been developed in order to improve the tolerability of these regimens. Carboplatin is gradually replacing cisplatin in clinical practice as well as in clinical trials even if it is still unclear whether it has an equivalent efficacy compared to cisplatin. Oxaliplatin, the trans-l oxalato platinum compound, showed encouraging antineoplastic activity and favourable toxicity profile in NSCLC, but confirmatory randomized phase III trials are warranted. We performed a literature search in order to better understand the possible role of oxaliplatin-based combinations in advanced NSCLC treatment strategies.

Background: The addition of cytokines to chemotherapy (CT) has obtained encouraging but contradictory results in metastatic melanoma. In this phase III trial, we compared the effects of CT [cisplatin, vindesine and dacarbazine (CVD)] with those of concurrent biochemotherapy (bioCT) consisting of CVD plus interleukin-2 and interferon-a2b. Patients and methods: A total of 151 untreated metastatic melanoma patients were randomized, 75 on arm A (cisplatin 30 mg/m 2 on days 1–3, vindesine 2.5 mg/m 2 on day 1 and dacarbazine 250 mg/m 2 on days 1–3), and 76 on arm B (same CVD scheme plus interferon-a2b on days 1–5 and interleukin-2 on days 1–5 and 8–15, both administered subcutaneously), either recycled every 3 weeks. Response was assessed every two cycles. Results: Ten percent of the patients were alive at a median of 52 months from start of therapy. We observed a response rate (RR) of 21% on arm A versus 33% on arm B; three patients (4%) given bioCT had complete responses (CRs). Median time to progression (TTP) was identical; median overall survival (OS) time was 12 months on arm A and 11 months on arm B. Conclusions: BioCT is not better than CT alone; the trend in favor of the bioCT in terms of RR did not translate into better TTP or OS. Therefore, bioCT cannot be recommended as standard first-line therapy for metastatic melanoma.

Neuroendocrine tumors are rare neoplasms originating from cells belonging to a diffuse or confined neuroendocrine system and characterized by a significant histopatologic and biologic heterogeneity. Timely diagnosis is delayed because they are often clinically silent for their low differentiation grade and the absence of any symptom due to abnormal hormone release. For these reasons, many neuroendocrine tumor patients are not treated medically for metastatic or inoperable disease. Medical treatments include biotherapy, with interferon-α and somatostatin analogues, and chemotherapy. Somastostatin analogues are widely used in patients with symptoms and with carcinoids of low differentiation grade. Interferon-α is used alone or in combination with somatostatin analogues. Chemotherapy is active in patients with poorly differentiated neuroendocrine tumors. The therapeutic regimen commonly used is the combination of cisplatinum and etoposide. In conclusion, no standard treatment for NET has yet been identified, and the response criteria suggested by ITMO remain a reference point. The clinical aspect of the disease and biologic features suggest the identification of neuroendocrine tumors patients suitable for the appropriate therapies. On these bases, it is recommended that diagnosis and treatment of neuroendocrine tumors be carried out at specialized oncological centers involved in clinical trials.

e18010 Background: In NSCLC heterogeneity or switch to neuroendocrine phenotype and the expression of stem-cell related markers have been associated with higher tumor aggressiveness. We evaluated the presence of CTC bearing stem cell-related marker CD133+ and the neuroendocrine marker synaptophysin (SYP) in patients with metastatic non small cellular lung cancer (NSCLC). Methods: Forty-four blood samples (20 mL) were collected after informed consent from 16 consecutive patients (pts) with metastatic NSCLC before and during chemotherapy at our Institution. Blood was enriched for tumor cells by CD45 depletion using a magnetic bead separation technique and potential CTC were identified by flow cytometry. CTC were defined as EpCAM+ CK7/8 + CD45-CD53-. Multiparameter cytometry assessed surface expression of the combinations of CD133 and SYP. Results: CTC phenotype: Twenty-five of all 44 samples (57%) were found positive for presence of at least 1 CTC. The median number of CTC was 4 (range: 1-9) / 10mL blood. Twelve samples (27%) showed to be positive for the cancer putative stem cell marker CD133. The median number of CD133+ CTC was 2 (range: 1-7) / 10mL. Seventeen samples (38%) were positive for SYP with a median number of 2 SYN+ CTC (range: 1-6 ) /10ml. Eleven samples (25%) resulted double positive for CD133 and SYP. Tumor heterogeneity: Twelve of the 16 patients (75%) had at least one sample positive for CTC. In 3 pts (25%) CTC expressed only SYP, in 5 pts (42%) both the markers and in 4 pts (33%) neither CD133 nor SYP were expressed. Conclusions: A higher rate of CD133-SYP-double positive CTC compared to the single positive CTC was observed. This suggests that the switch to the neuroendocrine phenotype might occur concomitantly with a stem-cell like more resistant and aggressive phenotype. It needs to be clarified, whether the presence of stem-cell like and neuroendocrine CTC may be a predictive factor for treatment response and outcome in patients with metastatic NSCLC.

5523 Background: Lymph node metastasis frequently occurs in SCCHN and is generally associated with poor prognosis. The molecular mechanisms regulating tumor cell dissemination and thus the potentia...

e22056 Background: Within circulating tumor cells there may be a subset of cell with stem cell (tumor initiating) characteristics able to develop distant metastasis. Several markers including nestin and CD133 have been found to be possible candidates to identify such a kind of subpopulation in other experimental models. We evaluated the presence of melanoma cells bearing stem cell phenotype in the bloodstream of patients with cutaneous or uveal melanoma after depletion of the leukocytes fraction. Methods: Between 50 and 100 ml of peripheral blood were collected from 12 melanoma patients with various tumor burden as well as three healthy volunteers. Blood samples were enriched for tumour cells by CD45 depletion of the leukocyte fraction using magnetic beads separation (EasySep, Stem Cell Technologies. Inc.). The remaining material was stained with antibodies for the markers Melan-A/Mart-1 (Dako) and HMB45 (Dako), CD133 (Miltenyi Biotec) and nestin (R&D System) and analysed by flow cytometry (BD FACSCalibur). Ten ml of blood were further processed and CD133, nestin, Melan-A/Mart-1 transcripts were quantified by Real Time RT-PCR (LightCycler, Roche Diagnostic). Results: CD45-depleted fractions in healthy controls were negative for melanoma markers. Melan-A/Mart-1 and/or HMB45 positive cells were detectable in 11 out of 12 melanoma patients. The absolute number of melanoma cells identified ranged from 6 to 176 per 10 ml of blood. Nestin expressing cells were more represented compared to CD133 expressing cells (median 27.4%, range 0.3% to 65.1% vs. median 9.3%, range 0.1% to 16.8%) within the melanoma fraction of cells positive for Melan-A/Mart-1 and/or HMB45. In one patient two different melanoma cell populations were detectable. The population of cells with lower expression of the melanoma markers showed at the same time higher expression of nestin and CD133 (5.9% vs. 1.3% and 10.2% vs. 6.7% respectively). Nestin results were in good accordance to the FACS data (nestin: r=0.55; CD133: r=0.23; Pearson test). Conclusions: The novel negative separation technique allows reliable isolation of melanoma cells from peripheral blood of patients with metastatic disease. A significant fraction of melanoma cells in peripheral blood bears a stem cell phenotype. No significant financial relationships to disclose.

7118 Background: Platinum analogues have shown good activity and better toxicity profile than cisplatin/GEM regimens in advanced NSCLC, while sequential administration of two platinum/non-platinum ...

Introduction The ability of doublet therapy in the second-line setting in patients with platinum-refractory non-small cell lung cancer (NSCLC) has not yet been proven. In this setting, docetaxel (D) has shown efficacy and irinotecan (I) has only recently been introduced. This study was initiated to explore the activity and tolerability of three D + I regimens in platinum pretreated NSCLC patients. Methods From March 2003 to June 2006, 65 patients (age range, 39–71 years; 83% male) with relapsed stage III/IV NSCLC were randomly assigned to receive either I 160 mg/m 2 plus D 60 mg/m 2 on day 1 every 21 days (arm A), I 80 mg/m 2 on days 1,8 plus D 60 mg/m 2 on day 1 every 21 days (arm B), or I 60 mg/m 2 plus D 30 mg/m 2 on days 1, 8, 15, and 22 every 42 days (arm C), for a maximum of 18 weeks. Results Per protocol analysis (47 of 65) overall response rates were 5.6% (A), 6.7% (B), and 7.1% (C). Median times to progression were 3.4, 4.0, and 4.3 months, respectively. Overall survival was 8.9 (A), 8.3 (B), and 9.4 (C) months. G3/4 neutropenia was more frequent in arms A (42%) and B (55%) whereas G3/4 nonhematologic toxicity was similarly prevalent in all arms, although diarrhea occurred in 47% of arm C patients. Conclusions Single-agent treatment with D or the multitarget antifolate pemetrexed or erlotinib remain the best choices and investigational studies, following first-line therapy, are required.

In July 1988 a healthy 67-year-old man with a longsmoking history underwent a direct microlaryngoscopyfor dysphonia. A neoplastic lesion attached to the rightarytenoid was detected and subsequently resected endo-scopically. The histological diagnosis was argentophiliccarcinoid. Locoregional relapses were endoscopicallyresected in October 1991 and March 1999. Because thelast pathology report revealed an increase in cytologicalatypias, a right arytenoidectomy and lateral pharyngoto-my were performed in August 1999. Surgery was notcurative due to tumor involvement of the resection mar-gins. In April 2001 right laterocervical and retro-mandibular adenopathies in association with involve-ment of the right aryepiglottic fold and second-leveladenopathies were detected. Cervical lymph node dis-section along with laser therapy to the right aryepiglot-tic fold was performed. The histological findings wereneuroendocrine tumor relapsed to the right aryepiglotticfold with metastases to the right cervical lymph nodes.TNM stage was pT1, N2b, M0 R1.Because of the involvement of the resection margins,radiation therapy to the larynx (right supraglottic re-gion) and neck was administered with a total dose of50.4 Gy. Eight months later magnetic resonance imag-ing showed tumor relapse in the right hemilarynx. Fur-ther resection including the second and third lymphaticlevels and subcutaneous tissue was performed. Wide-spread subcutaneous metastatic involvement was ob-served without evidence of tumor cells in the lymphnodes. The patient started treatment with long-acting re-lease octreotide for two months, when positron emis-sion tomography (PET) showed metastatic involvementof mediastinal lymph nodes, soft tissues of the thigh,larynx and bone (left ischium and femur). The patient was referred to the Istituto Nazionale Tu-mori of Milan where a disease workup was performed(Figure 1). Physical examination revealed multiplepainful subcutaneous metastases on the scalp, rightshoulder and arm, thorax, left thigh and inguinal region,while bone metastases were excluded by further radio-logical examination. Circulating levels of chromograninA (CgA), vanillylmandelic acid and 5-hydroxyindoleacetic acid (5-HIAA) were within the normal limits,while the plasma concentration of neuron-specific eno-lase (NSE) was increased (32.26 ng/mL; normal value