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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.

9554 Background: IMM-101 is a suspension of heat-killed whole cell Mycobacterium obuense (NCTC 13365), which enhances the innate immune response and dendritic cell maturation. In animal models, it increases antigen specific responses and number of CD8+ CTL and CD4 + Th1 cells. The clinical studies with IMM-101 have shown promising efficacy signals in pancreatic cancer when combined with gemcitabine and in melanoma as adjunctive or single agent. Methods: IMM-101-015 is an open-label Phase 2a study to investigate the safety and efficacy of IMM-101 in combination with checkpoint inhibitors (CPIs) in patients (pts) with advanced melanoma who were either treatment-naive (cohort A), or whose disease had progressed during PD-1 blockade (cohort B). Pts with evaluable lesions, adequate performance status and organ function were eligible. Pts received 1.0 mg of IMM-101 every 2 weeks for the first 3 doses followed by a rest period of 4 weeks, then every 2 weeks for the next 3 doses, and thereafter every 4 weeks. Nivolumab was given every 2 or 4 weeks (dependent on Investigator choice). Pts in cohort B had the option to change to ipilimumab and IMM-101 if their disease continued to progress. Biopsies and blood samples were obtained at baseline and during treatment for assessment of tumor biomarkers and immune correlatives. The primary objectives of the study were to evaluate the overall response rate (ORR) after a maximum of 18 months of treatment by RECIST 1.1 and to assess the safety and tolerability of the combination of IMM-101 + CPIs. Results: Sixteen pts (11 Cohort A and 5 Cohort B) were treated between October 2018 and May 2021. The median age was 68.5 yrs (range 36-92) and 11 (69%) were male. The ECOG ps was 0 in 9 (56%) and 1 in 7 (44%) pts. Four (25%) had unresectable stage III melanoma and 12 (75%) stage IV. In Cohort A (3 stage III, 5 stage IV M1b and 3 M1c) 3 pts (27%) had an elevated baseline LDH, 6 (55%) a positive PD-L1 status and 3 (27%) a BRAF mutation. Pts in cohort A were on study for a median time of 8.5 months (range 1.5 - 19.1) and those in cohort B for 3.0 months (range 1.5 - 7.4). All pts were evaluated for response. The ORR was 73% (95% CI 39.03, 93.98) in cohort A whereas all pts in cohort B reported progressive disease. With respect to cohort A, 2 (18%) pts had CR, 6 (55%) PR and 1 (9%) SD. The median progression-free survival time was 10.2 months (95% CI 2.50, NE) with 41% of the pts progression-free at 18 months. The most frequent treatment emergent adverse events (TEAEs) were injection site reaction (63%), pruritus (44%), fatigue (38%), skin rash (25%), hypothyroidism (25%) and diarrhoea (19%). There were no grade 4 TEAEs. Grade 3 TEAEs occured in 10 patients (63%), mostly skin toxicity (19%) and lab abnormalities (13%). Conclusions: IMM-101 in combination with nivolumab is safe and shows encouraging antitumor activity in treatment-naive patients with advanced melanoma. Clinical trial information: NCT03711188.

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.

Summary BRAF and MEK inhibitors are effective in BRAF mutant melanoma, but most patients eventually relapse with acquired resistance, and others present intrinsic resistance to these drugs. Resistance is often mediated by pathway reactivation through receptor tyrosine kinase (RTK)/SRC-family kinase (SFK) signaling or mutant NRAS, which drive paradoxical reactivation of the pathway. We describe pan-RAF inhibitors (CCT196969, CCT241161) that also inhibit SFKs. These compounds do not drive paradoxical pathway activation and inhibit MEK/ERK in BRAF and NRAS mutant melanoma. They inhibit melanoma cells and patient-derived xenografts that are resistant to BRAF and BRAF/MEK inhibitors. Thus, paradox-breaking pan-RAF inhibitors that also inhibit SFKs could provide first-line treatment for BRAF and NRAS mutant melanomas and second-line treatment for patients who develop resistance., Graphical Abstract, Highlights • pan-RAF inhibitors also inhibit SRC family kinases • The compounds do not induce paradoxical activation of ERK in RAS mutant cells • The compounds are active in BRAF and NRAS mutant melanomas • The compounds are active in PDXs resistant to BRAF or BRAF plus MEK inhibitors, Girotti et al. describe two pan-RAF inhibitors that also inhibit SRC-family kinases. These compounds do not drive paradoxical MEK/ERK activation and can inhibit MEK in NRAS mutant cells. Moreover, the agents can overcome resistance to clinical BRAF or combination BRAF/MEK inhibitors in patient-derived xenografts.

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.

T cell based immunotherapy has been investigated in a variety of malignancies and analyses have been mostly founded on in vitro data with tumor cell monolayers. However, three-dimensional (3D) culture models might mimic more closely the 'in vivo' conditions than 2D monolayers. Therefore, we analyzed the expression of tumor-associated antigens (TAA) and of molecules involved in antigen processing and presentation (APM) in tumor spheres, which served as an in vitro model for micrometastasis which might be enriched in tumor propagating cancer stem cells. For enrichment of sphere cells 12 human solid tumor cell lines were cultured in serum-free medium. Expression of a variety of TAA and APM were analyzed by RT-PCR and/or flow cytometry and compared to expression in corresponding adherent bulk cells grown in regular growth medium. Compared to adherent cells, spheres showed equal or higher mRNA expression levels of LMP2, LMP7 and MECL-1, of TAP1 and TAP2 transporters and, surprisingly, also of TAA including differentiation antigens. However, downregulation or loss of HLA-I and HLA-II molecules in spheres was observed in 8 of 10 and 1 of 2 cell lines, respectively, and was unresponsive to stimulation with IFN-γ. Although tumor spheres express TAA and molecules of intracellular antigen processing, they are defective in antigen presentation due to downregulation of HLA surface expression which may lead to immune evasion.

Clinically effective T-cell responses can be elicited by single peptide vaccination with Wilms' tumor 1 (WT1) epitope 126-134 in patients with acute myeloid leukemia (AML). We recently showed that a predominant T-cell receptor (TCR) β chain was associated with vaccine-induced complete remission in an AML patient (patient 1). In this study, we address the question of whether this predominant clone or the accompanying Vβ11 restriction could be found in other AML patients vaccinated with the same WT1 peptide.For assessment of Vβ usage, cytotoxic T lymphocytes (CTLs) from four vaccinated patients were divided into specific and non-specific by epitope-specific enrichment. Vβ families were quantified in both fractions using reverse transcribed quantitative PCR. Vβ11-positive 'complementary determining region 3' (CDR3) sequences were amplified from these samples, from bone marrow samples of 17 other vaccination patients, and from peripheral blood of six healthy controls, cloned and sequenced.We observed a clear bias towards Vβ11 usage of the WT1-specific CTL populations in all four patients. The predominant CDR3β amino acid (AA) sequence of patient 1 was detected in two other patients. CDR3β loops with closely related AA sequences were only found in patient 1. There were no CDR3β AA sequences with side chains of identical chemical properties detected in any patient.We provide the first data addressing TCR Vβ chain usage in WT1-specific T-cell populations after HLA A*0201-restricted single peptide vaccination. We demonstrate both shared Vβ restriction and the sharing of a TCR β transcript with proven clinical impact in one patient.

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.

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.

Approximately 50% of melanomas have mutations in the gene encoding BRAF. In recent years, new targeted therapies have transformed the landscape of metastatic melanoma treatment. Dabrafenib, a potent kinase inhibitor of mutated BRAF, has been showed to have high response rates with a rapid onset of response, as well as improved overall and progression-free survival when compared with chemotherapy. Dabrafenib in combination with trametinib, a MEK inhibitor, has demonstrated higher responses and improved clinical efficacy compared with monotherapy. Toxicity is distinct compared with chemotherapy but manageable. This article summarizes the pharmacology, key clinical trial data as well as practical experience with dabrafenib in clinical practice, and future directions.

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.

The protein kinase BRAF is mutated ∼40% of human melanomas. BRAF is a component of the RAS/RAF/MEK/ERK pathway and BRAF or MEK inhibitors increase progression-free and overall survival in melanoma patients with BRAF mutations. However, most patients relapse with acquired resistance and ∼20% of patients present intrinsic resistance and do not respond to these drugs. We describe here two novel compounds that target mutant BRAF and wild-type CRAF. Our compounds inhibited the growth of melanoma cells that were resistant to BRAF-selective inhibitors. ERK pathway reactivation is responsible for resistance to BRAF targeted therapies in ∼60% of the patients and in ∼25% of patients resistance is driven by acquisition of mutations in NRAS. We show that our compounds inhibited the growth of melanoma cells that were resistant to BRAF-selective inhibitors due to pathway reactivation mediated by different mechanisms. We show that the drugs were active against patient derived xenografts (PDXs) from patients with acquired or intrinsic resistance to BRAF-selective inhibitors and in whose tumors resistance was associated with ERK pathway reactivation. Further, our compounds are active in a PDX from a patient whose tumor developed acquired resistance to a combination of a BRAF-selective plus a MEK inhibitor and associated with acquisition of an NRAS mutation. Thus, our panRAF inhibitors can inhibit melanomas with different mechanisms of acquired or intrinsic resistance to BRAF-selective and BRAF-selective/MEK inhibitor combinations, potentially providing first-line treatment for naïve patients and second-line treatments for a range of relapsed patients. Citation Format: Maria R. Girotti, Filipa Lopes, Natasha Preece, Dan Niculescu-Duvaz, Alfonso Zambon, Lawrence Davies, Steven Whittaker, Grazia Saturno, Amaya Viros, Malin Pedersen, Bart MJM Suijkerbuijk, Delphine Menard, Robert Mcleary, Louise Johnson, Laura Fish, Sarah Ejiama, Berta Sanchez-Laorden, Neil Carragher, Kenneth Macleod, Garry Ashton, Anna Marusiak, Alberto Fusi, John Brognard, Margaret Frame, Paul Lorigan, Caroline J. Springer, Richard Marais. Novel panRAF inhibitors active in melanomas that are resistant to BRAF-selective, or BRAF-selective/MEK inhibitor combinations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3704. doi:10.1158/1538-7445.AM2014-3704

Data regarding the expression of epidermal growth factor receptor (EGFR) in melanoma and its role in the tumor biology are conflicting. In BRAF V600-mutant melanomas, the expression of EGFR has been associated with acquired resistance to BRAF inhibitors. In this study, we assessed EGFR expression and downstream signaling activity in a panel of melanoma cell lines and we investigated the effects of the BRAF inhibitor vemurafenib on expression of EGFR and its downstream effectors in a subgroup of BRAF-mutant melanoma cells. Three out of 10 melanoma cell lines expressed EGFR. Downstream signaling via ERK and AKT was responsive to either stimulation by EGF or inhibition by erlotinib. Constitutive activation of ERK occurred in all the cell lines investigated whereas constitutive activation of AKT only in three cell lines. Constitutive activation of ERK and AKT was independent from EGFR expression. Vemurafenib did not affect EGFR expression in general, but it increased EGFR phosphorylation in the cell line SkMel5. Induced EGFR phosphorylation was sensitive to treatment with erlotinib. Vemurafenib efficiently blocked ERK activation in all the BRAF-mutant cell lines tested, whereas its effects on AKT activation were dissimilar in the different cell lines. Our data suggest that EGFR is functional but usually inactive in EGFR high-expressing cell lines. Basal EGFR expression unlikely represents a biomarker for predicting the sensitivity to vemurafenib in melanoma, but EGFR activation might represent a mechanism of vemurafenib resistance in a subset of melanoma cells.

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.

Targeting the epidermal growth factor receptor (EGFR) pathway is an important approach for a variety of tumors. This study assessed the effect of cetuximab, an anti-EGFR monoclonal antibody, on three gastric cancer cell lines with different phenotypes in vitro and in a therapeutic orthotopic murine gastric cancer model.Three human gastric cancer cell lines (AGS, MKN-45, NCI-N87) were evaluated for cell surface EGFR expression, and K-ras and BRAF mutations. In vitro, the effects of cetuximab, carboplatin, irinotecan, and docetaxel were investigated. Orthotopic tumors derived from MKN-45 and NCI-N87 were established in nude mice. After 4 weeks, the animals received cetuximab (1 mg/kg, weekly i.p.) or carboplatin (20 mg/kg, weekly i.p.), or both agents. The volume of the primary tumor and local and systemic tumor spread were determined at autopsy at 14 weeks. Tumor sections were immunostained for EGFR, as well as stained for CD31 to analyze microvessel density.Cell surface expression of EGFR was found only in AGS and NCI-N87 cells. AGS cells displayed a codon 12 K-ras mutation, and all three cell lines were BRAF wild-type. In vitro, cetuximab significantly reduced cell viability and proliferation only in EGFR-positive/K-ras wild-type NCI-N87 cells (-48%). In vivo, cetuximab in combination with carboplatin synergistically reduced tumor volume (-75%), dissemination (-63%), and vascularization (-47%) in NCI-N87 xenografts. Tumors derived from EGFR-negative MKN-45 cells were unaffected by cetuximab.Cetuximab is effective in K-ras wild-type, EGFR-expressing gastric cancer cell lines and xenografts. In vivo, the combination of cetuximab with carboplatin displayed synergistic antitumor activity.

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.

In an earlier study, we described a patient who developed an anti-tyrosinase T-cell response leading to long-term tumor control. Here we analyzed this response with regard to T-cell receptor (TCR) Vbeta family usage and clonality in order to further elucidate the nature of the T cell response in this patient. For identification of expanded specific cytotoxic T-cell (CTL) clones, tetramer enrichment of tyrosinase reactive T-cells was followed by comparative quantitative reverse transcribed PCR (qRT PCR) quantification of all TCR Vbeta-families and sequencing of family Vbeta4 elevated in the enriched fraction. The predominant specific clone was quantified by clonotypic qRT PCR in multiple samples from blood, bone marrow, and tumor tissue. FACS analyses with staining of TYR.A2 and TCR Vbeta4 were performed. Epitope specific enrichment revealed an isolated increase of Vbeta-family 4. FACS analysis showed a shift of specific CTLs to Vbeta-family 4 during tumor regression with a maximum of 80% of all TYR.A2 specific cells belonging to this family. Sequencing revealed a single predominant clone against polyclonal background coding for identical CDR3 loops. The predominant clone was highly expressed in bone marrow and tumor tissue, and was detectable in blood over a period of ten years. Considering the results of previous studies showing a specific effector phenotype in blood and a specific memory compartment in bone marrow of this patient, this data implicate the predominant clone featured all attributes of a sufficient CTL response including homing capacity and memory formation resulting in long term clonal persistence and tumor control.

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.

Numerous cancers are driven by mutationally activated kinases, many of which can be employed to define subsets of cancers and therefore patients most likely to benefit from treatment with rationally designed targeted therapies. Defining the landscape of genetic alterations that participate in cancer transformation provides insight into the diversity of clinical responses observed to targeted treatments. Here, we identified concurrent mutational inactivation of the tumor suppressors TP53 and PTEN as a mechanism of resistance to BRAF inhibitors in melanomas harboring mutant V600EBRAF. Defects in TP53 function and PTEN loss occur in 17% and 12% of malignant melanomas respectively, rendering cells dependent on CHK1 to maintain normal cell cycle progression. We demonstrate that CHK1 inhibition increases sensitivity to a variety of alkylating agents and targeted therapies in V600EBRAF mutant, TP53/PTEN deficient cell lines and patient derived xenografts. Inhibition of CHK1 concurrently with the induction of either DNA damage or replication stress leads to ‘‘mitotic catastrophe’’ and cell death in our in vitro and in vivo models of TP53/PTEN deficient tumors. In the case of externally applied cellular stress from chemotherapeutics, our results suggest CHK1 inhibition may sensitise this genetically defined subset of melanomas to such treatments. Our results provide some insight into the heterogeneity of clinical outcomes observed when treating BRAF mutant melanomas with BRAF inhibitor and suggest a need for comprehensive screening of TP53 and PTEN inactivation in these patients. Citation Format: Elena Galvani, Kelly Brooks, Franziska Baenke, Maria Romina Girotti, Gabriela Gremel, Amit Kumar Mandal, Amaya Viros, Clare McManus, Matthew Smith, Kok Haw Jonathan Lim, Rebecca Lee, Alberto Fusi, Paul Lorigan, Richard Marais. Simultaneous inactivation of TP53 and loss of PTEN diminish response to targeted therapy in V600EBRAF mutant melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2683. doi:10.1158/1538-7445.AM2015-2683

Plasticity in T-cell populations during response and onset of resistance to targeted therapy has been well documented. More recently, macrophage-derived tumor necrosis factor has been described to drive resistance to MAPK pathway inhibition, yet little is known on the adaptation of tumor-associated macrophage populations to treatment and disease progression in human melanoma. To test for alterations in macrophage abundance following resistance to MAPK pathway inhibition, we applied a fluorescence-based co-staining strategy on melanoma specimens, representing tissue collected from patients before treatment and at relapse. In addition, we established in vitro co-culture systems to study the paracrine effects of melanoma cells on various aspects of monocyte differentiation and macrophage activation in the background of MAPK inhibition and resistance. Resistance to targeted therapy was found to be associated with an increase in the number of tumor-resident macrophages. In vitro co-culture of melanoma cell lines with monocytes isolated from the peripheral blood of healthy donors or with CSF1-differentiated macrophages in the presence or absence of MAPK inhibition led to profound changes in monocyte/macrophage marker expression, indicating a direct interplay between the two cell populations upon MAPK pathway inhibition. Our data show that macrophages play an integral role in the changing immune-microenvironment during the course of MAPK pathway inhibition. Elucidation of mechanisms driving these processes will result in strategies to optimize treatment combinations and consecutive treatment outcomes for melanoma patients. Note: This abstract was not presented at the meeting. Citation Format: Gabriela Gremel, Maria Romina Girotti, Amaya Viros, Garry Ashton, Helen Bradley, Elena Galvani, Rebecca Lee, Alberto Fusi, Paul Lorigan, Richard Marais. Implications of MAPK pathway inhibition on monocytes and tumor-associated macrophages in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5024. doi:10.1158/1538-7445.AM2015-5024

2583 Background: The clinical utility of agents that target EGFR and VEGFR signaling in Non-Small Cell Lung Cancer (NSCLC) is limited by resistance due to emergent alternative growth stimulatory pa...

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.

5572 Background: The PI3K/PTEN/AKT/mTOR pathway is one of the most targeted in human cancers and alterations in the axis are frequent events in squamous cell carcinoma of the head and neck (HNSCC). Specific genetic variations in this pathway have been associated with variation in recurrence, survival and response in lung cancer and esophageal cancer. The aim of this study was to determine whether single nucleotide nucleotide polymorphisms (SNPs) in AKT1, AKT2, FRAP1, PI3KCA and PTEN identified in other cancers were associated with treatment response and clinical outcome in patients with HNSCC. Methods: Genomic DNA was extracted from FFPE tissue specimens of 45 patients with recurrent or initially metastatic HNSCC who received a combined treatment with docetaxel and cetuximab in a phase II study. Eleven single SNPs in the genes AKT1, AKT2, FRAP1 (mTOR), PIK3CA and PTEN were genotyped by means of Real Time PCR system or by direct sequencing and analysed for association with response to therapy and survival. Results: None of the SNPs was related to treatment response. Two SNPs (AKT2:rs8100018 and PTEN:rs12569998) were associated with variation in progression risk. The AKT2:rs8100018 homozygous variant resulted in increased risk, with a HR of 4.83 (95% CI, 1.11-21.03) and the PTEN:rs12569998 homozygous variant in an increased risk, with a HR of 2.36 (95% CI, 1.24-4.50). AKT2:rs8100018 and PTEN:rs12569998 genetic variation had an additive effect on risk of tumor progression. The AKT2:rs8100018 homozygous variant was significantly associated with overall survival (OS) with HR of 3.57 (95% CI, 1.06-12.00) while presence of one of the two variant alleles of AKT1:rs3803304 with a lower risk of death (HR: 0.51; 95% CI, 0.27-0.97). Conclusions: We found significant associations between common genetic variants in the PI3K/PTEN/AKT/mTOR pathway and outcome in patients with HNSCC. Despite the small sample size, patient cohort and treatment were homogeneous. As a consequence, evaluation of SNPs as a host factor may be considered in addition to tumor-specific mutations for personalized treatment development.