Evolving topics in cancer immunotherapy
O3 Altering the gut microbiota to improve responses to immune checkpoint blockade
Connie P. M. Duong1,2, Marie Vetizou1,2, Laurence Zitvogel1,2,3,4
1Gustave Roussy Cancer Campus, Villejuif, France, 2INSERM Unit U1015, Villejuif, France, 3Université Paris Sud, Université Paris-Saclay, Faculté de Médecine, Le Kremlin Bicêtre, France, 4Center of Clinical Investigations in Biotherapies of Cancer 507, Villejuif, France
Correspondence: Connie P. M. Duong - CONNIE.DUONG@gustaveroussy.fr
Journal of Translational Medicine 2016, 15(Suppl 1):O3
Background: The targeting of the immune checkpoint CTLA4 in melanoma patients has led to increased objective response rates and subsequent approval by the EMA and FDA. However, studies have also reported that some patients have experienced immune-related adverse events, often occurring at sites exposed to commensal microbiota, leading to cessation of treatment. Our lab and others have demonstrated that the gut microbiota impacts the efficacy of checkpoint blockade therapy [1, 2]. We have shown that Bacteroides species play an important role on the efficacy of CTLA4 blockade in the treatment of melanoma. Analysis of feces from metastatic melanoma patients led to the identification of three clusters (A, B and C) based on genus composition. Here we present a follow up study, where we hypothesise that compensating Cluster B patient transplanted mice with live or immunogenic bacteria, termed oncomicrobiotics, can increase the efficacy of anti-CTLA4 treatment. Given recent data showing that the co-blockade of CTLA4 and PD1 has a significantly greater response rate compared to targeting CTLA4 alone , we also wanted to investigate the impact of microbiota on this combination.
Materials and methods: To test this, SPF mice were treated with or without a cocktail of antibiotics (ATBx) composed of ampicillin, streptomycin and colistin. Mice were subsequently gavaged with patient feces from different clusters and inoculated with the sarcoma cell line, MCA205. Mice were then treated with five doses of anti-CTLA4 Ab, gavaged with defined species of oncomicrobiotics and monitored for tumor growth and survival. In order to evaluate the effect of the gut microbiota on the combination, mice were treated with or without ATBx, inoculated with either MCA205 or the melanoma cell line RET, and treated with anti-CTLA4, anti-PD1, or a combination of both Abs.
Results: Consistent with our previous data, mice which received feces from Cluster C patients had a significantly greater response to CTLA4 blockade compared to mice which received Cluster B feces. Mice that has been colonised with Cluster B feces had a significantly greater response to anti CTLA4 therapy when compensated with oncomicrobiotics. We also found that the efficacy of anti CTLA4 and anti PD1 blockade is partially dependent on an intact microbiome, as dysbiotic mice had a significantly reduced response to the combination treatment.
Conclusions: It is anticipated that this work will increase the therapeutic coverage of immune checkpoint blockade treatment when appropriate oncomicrobiotics are co-administered, resulting in increased durable responses in patients with metastatic melanoma.
1. Vetizou et al. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science. 2015;350:1079–84.
2. Sivan et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. Science. 2015;350:1084–89.
3. Postow et al. Nivolumab and Ipilimumab versus Ipilimumab in Untreated Melanoma. NEJM. 2015;37(21):2006–17.
System biology session: Molecular
O4 Inhibition of Stearoyl-Coa desaturase 1 (SCD1) enzymatic activity reverts BRAFi and MEKi-induced selection of cancer stem cells in BRAF-mutated malignant melanoma
Maria Elena Pisanu1,2, Alessia Noto2,3, Luigi Fattore1,2, Debora Malpicci2,4, Gennaro Ciliberto1, Rita Mancini2,3
1Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione G. Pascale”, Naples, Italy, 2Dipartimento di Chirurgia “P. Valdoni”, Sapienza Università di Roma, Rome, Italy, 3Dipartimento di Medicina Clinica e Molecolare, Sapienza Università di Roma, Rome, Italy, 4Dipartimento di Medicina Sperimentale e Clinica, Università degli Studi di Catanzaro “Magna Graecia”, Catanzaro, Italy
Correspondence: Maria Elena Pisanu - email@example.com
Journal of Translational Medicine 2016, 15(Suppl 1):O4
Background: Therapy of melanoma has been improved by the advent of immunological checkpoint inhibitors and by targeted therapies with kinase inhibitors . However, the efficacy of targeted therapies against BRAFV600 mutations is hampered by the development of acquired resistance [2–3]. Treatment failures in melanoma patients have been attributed in part to the presence of cancer stem cells (CSCs). Observations that CSC have a distinct biology when compared to that of the bulk tumor cells and, more importantly, are resistant to chemotherapies, suggest their involvement in invasion, metastasis and relapse . We previously demonstrated that lung CSCs are enriched for the expression of SCD1, a key enzyme of lipid metabolism involved in the conversion of saturated into mono-unsaturated fatty acids, and that its inhibition suppresses the ability to form spheroids and selectively kills CSCs. In this study we investigated the involvement of SCD1 in melanoma stem cells survival and the interplay between BRAF/ERK inhibitors (BRAF/MEKi) and lipid metabolism.
Materials and methods: Bioinformatics analysis of published melanoma datasets was performed using online tool (http://www.cbioportal.org/). SCD1 gene expression data of 479 patients were downloaded from TCGA and correlated with survival. Combination of BRAF/MEKi and the SCD1 inhibitor MF438 were tested by spheroid forming assays on two BRAF-mutated melanoma cell lines (M14 and WM115) grown in selective 3D medium. SCD1, Nanog, Oct4 expression was studied by WB and RT-PCR.
Results: Bioinformatics analysis of public datasets was carried out to assess if SCD1 can act as prognostic factor in melanoma. By using online tool we found that overall survival of patients affected by melanoma was inversely correlated to SCD1 expression. This finding led us to measure SCD1 expression in CSCs obtained from WM115 and M14 cell lines. Preliminary results showed that SCD1 expression is upregulated in spheroids derived from both lines. Moreover SCD1 overexpression was associated with enrichment of stemness markers. Exposure to high doses 10–20 μM of Vemurafenib or MEK162 induced increased spheroid formation. Based on these evidences we analyzed the functional role of SCD1 by inhibiting its activity using MF438. We found that spheroids were resistant to BRAF/MEKi and expressed high levels of Oct4 and Nanog. Furthermore we observed that combining BRAF/MEKi with MF438 inhibited synergistically spheroid growth.
Conclusions: Our results suggest that treatment of BRAF-mutated melanoma cells with BRAF/MEKi selects for cells with stem cell features and that this phenomenon is counteracted by SCD1 inhibitors. These findings have potential implication of the development of new combination therapies.
1. Flaherty KT. BRAF inhibitors and melanoma. Cancer J. 2011;17(6):505–511.
2. Lo RS. Combinatorial therapies to overcome B-RAF inhibitor resistance in melanomas. Pharmacogenomics. 2012;13(2):125–8.
3. Corcoran RB, Settleman J, Engelman JA Potential therapeutic strategies to overcome acquired resistance to BRAF or MEK inhibitors in BRAF mutant cancers. Oncotarget 2011;2(4):336–46.
4. Grasso C, Anaka M, Hofmann O, Sompallae R, Broadley K, Hide W, Berridge MV, Cebon J, Behren A, McConnell MJ Iterative sorting reveals CD133+ and CD133-melanoma cells as phenotypically distinct populations. BMC Cancer 2016;16(1):726.
5. Noto A, Raffa S, De Vitis C, Roscilli G, Malpicci D, Coluccia P, Di Napoli A, Ricci A, Giovagnoli MR, Aurisicchio L, Torrisi MR, Ciliberto G, Mancini R Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cells. Cell Death Dis. 2013;4:e947.
O5 Screening of malignant melanoma (MM) by miRNA: preliminary data on incidence after initial clinical evaluation
Marcella Occelli1, Carolina Cauchi1, Grazia Sciancalepore2, Cristiana Lo Nigro1, Michela Rovera3, Chiara Varamo1, Daniela Vivenza1, Zelda Seia4, Stefania Palazzini4, Fabiana Errico4, Davide Basso4, Laura Quaranta4, Giuseppe Forte2, Fulvio Lavagna5, Silvia Violante1, Paolo Bosio6, Laura Lattanzio1, Marco Carlo Merlano1
1Oncologia Medica, Azienda Ospedaliera Santa Croce e Carle, Cuneo, Italy, 2Anatomia Patologica, Azienda Ospedaliera Santa Croce e Carle, Cuneo, Italy, 3CAS, Azienda Ospedaliera Santa Croce e Carle, Cuneo, Italy, 4LILT, Cuneo, Italy, 5SS Day Surgery, Azienda Ospedaliera Santa Croce e Carle, Cuneo, Italy, 6Chirurgia Generale, Azienda Ospedaliera Santa Croce e Carle, Cuneo, Italy
Correspondence: Marcella Occelli - firstname.lastname@example.org
Journal of Translational Medicine 2016, 15(Suppl 1):O5
Background: The incidence of MM progressively increases and today 16 cases/100,000 cases per year are expected in Italy. However, there are major differences between geographical areas. In northern Italy the risk is double of that in the southern regions. The screening campaigns led to the identification of many early forms of MM, resulting in increased incidence of the disease, but have not changed mortality. This is possibly related to the identification of melanomas that would have remained silent. It should be remembered that the visit and the dermoscopy are operator-dependent methods and that requires long experience for their optimal use. Even in the most experienced hands, the sensitivity of these methods does not exceed 85% and 90%, respectively. The identification of new effective screening methods, able to eliminate the existing limits, is needed. MM is the skin cancer with the worst prognosis, especially when discovered at advanced stages. Moreover, it would be clinically relevant to identify in advance the melanoma lesions at risk of recurrence.
Materials and methods: In 2014 we have activated an ongoing experimental study to investigate the possible role of miRNA in MM screening tool. All the subjects clinically screened (including dermatoscopy) by the “Lega Italiana per la Lotta contro i Tumori” (LILT) are invited to join the study. They undergo a blood sample collection and a panel of 15 miRNAs is analyzed. The primary objective is to compare the level of miRNAs in patients with MM and non-neoplastic skin pigmented lesions, to evaluate their diagnostic value. The study foresees to enroll 700 subjects, to find at least 100 MM.
Results: From September 2014 to April 15, 2016, we accrued 546 subjects, who had a suspicious skin lesion. 5 people have not performed excision (patient’s refusal or dermatological second opinion); 87 (16%) had a histological diagnosis of MM. Specifically we found 37 in situ MM, 50 infiltrating, including 15 pT1a, 19 pT1b, 4 pT2a, 2 pT2b, 1 pT3a, 4 pT3b, 2 pT4b. One patient was diagnosed with multifocal mucosal melanoma; in 2 MM patients pathological staging is under progress.
Conclusions: The study is ongoing and biomolecular data are not yet available. However, it gives in a prospectic way, preliminary information about the incidence of MM in a population clinically screened and carrying suspected skin lesions.
Acknowledgements: This project is supported by CRC Foundation and LILT.
System biology session: Immunology
O6 Regulation of T cell sensitivity by TCR-proximal signaling components during anti-melanoma responses
Duane Moogk1, Shi Zhong1,2, Zhiya Yu3, Ivan Liadi4, William Rittase5, Victoria Fang1,6, Janna Dougherty1, Arianne Perez-Garcia1,7, Iman Osman1,8, Cheng Zhu5, Navin Varadarajan4, Nicholas P. Restifo3, Alan Frey9, Michelle Krogsgaard1,10
1Laura and Isaac Perlmutter Cancer Center New York University School of Medicine, New York, NY, 10016, USA, 2Life Sciences Center, Xiangxue Pharmaceutical Co., Ltd. GuangZhou, China, 3Center for Cancer Research, National Cancer Institute, US National Institutes of Health, Bethesda, MD, 20892, USA, 4Department of Chemical and Biomolecular Engineering, University of Houston, TX, 77004, USA, 5George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia, 30332-0405, USA, 6NYU Medical Scientist Training Program, New York, NY, 10016, USA, 7Kite Pharma, Santa Monica, CA, 90404, USA, 8Ronald Perelman Department of Dermatology, NYU School of Medicine, New York, NY, 10016, USA, 9Department of Cell Biology, New York University School of Medicine, New York, NY, 10016, USA, 10Department of Pathology, New York University School of Medicine, New York, NY, 10016, USA
Correspondence: Michelle Krogsgaard - Michelle.Krogsgaard@nyumc.org
Journal of Translational Medicine 2016, 15(Suppl 1):O6
Background: Immunotherapies for cancers, including melanoma, have made great strides in recent years, yet new and improved approaches are required to achieve more durable responses in a greater number of patients. The in vitro expansion phase of adoptive T cell therapy prior to reinfusion into the patient provides the opportunity to genetically enhance T cell subsets to improve in vivo performance. While the most common genetic modification is the incorporation of engineered antigen-specific TCRs or chimeric antigen receptors [1, 2], modification to signaling pathways in T-cell memory subsets in order to enhance T cell sensitivity is an underexplored strategy. This is mainly because contributions of TCR signaling components that confer differences in activation sensitivity and functional outcomes between CD8+ T
Materials and methods: To understand how TCR-proximal signaling differs significantly between T-cell memory subsets, we derived TCM and TEM  from the humanized TCR-transgenic melanoma mouse model (JR209) . We quantified differences in TCR activation and feedback regulation by novel live-cell imaging technologies, phospho-specific protein assays and used modeling of early TCR signaling to reveal the physiological significance of these differences .
Results: One of the critical steps of T cell triggering is the coordinated phosphorylation and binding of CD3 and Zap-70 by Lck following TCR ligation by Pmhc [6, 7]. Here, we show that T
possess differential constitutive Lck activities . Immediately proximal to Lck signaling, we observed enhanced Zap-70 phosphorylation in TEM following TCR ligation compared with TCM. Further, we observed increased intracellular calcium influx and cytotoxic effector function in TEM compared with TCM, and provide evidence that this results from a lower probability of TCM reaching threshold activation signaling due to the decreased magnitude of TCR-proximal signaling. We show that the differences in Lck constitutive activity between CD8+ T
are driven in part by differential regulation by SH2 domain-containing phosphatase-1 (Shp-1) and C-terminal Src kinase (Csk). We demonstrate that inhibition of Shp-1 results in increased constitutive Lck and cytotoxic activity in TCM to levels similar to that of TEM.
Conclusions: Together, this work demonstrates that differential activities of TCR-proximal signaling components may contribute to establishing the divergent effector properties of TCM and TEM. Inhibition of negative regulatory molecules, for example Shp-1 or Csk, or generalized augmentation of T cell sensitivity with miRNA offer potential therapeutic approaches in T cell immunotherapy but must be considered in the context of specificity and optimal targeting.
1. Kershaw MH et al. Clinical application of genetically modified T cells in cancer therapy. Clin Transl Immunology. 2014;3(5):e16.
2. Hinrichs CS, Rosenberg SA. Exploiting the curative potential of adoptive T-cell therapy for cancer. Immunol Rev. 2014;257(1):56–71.
3. Klebanoff CA et al. IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8 + T cells. Proc Natl Acad Sci USA. 2004;101(7):1969–74.
4. Yu Z et al. Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance. J Clin Invest. 2004;114(4):551–60.
5. Moogk D et al. Constitutive Lck activity drives sensitivity differences between CD8+ memory T cell subsets. J Immunol. 2016;197(2):644–54.
6. Straus DB, Weiss A. Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor. Cell. 1992;70(4):585–93.
7. Iwashima M et al. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science. 1994;263(5150):1136–9.
8. Nika K et al. Constitutively active Lck kinase in T cells drives antigen receptor signal transduction. Immunity. 2010;32(6):766–77.
O7 Tumor-infiltrating immune cells as potential predictive markers of response to treatment and survival in metastatic melanoma patients receiving ipilimumab
Timea Balatoni1, Anita Moho2, Timea Sebestyén3, Anita Varga4, Judit Oláh4, Zsuzsanna Lengyel5, Gabriella Emri6, Gabriella Liszkay1, Andrea Ladányi7
1Department of Oncodermatology, National Institute of Oncology, Budapest, Hungary, 21st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary, 3Department of Pathology, St. John’s Hospital, Budapest, Hungary, 4Department of Dermatology and Allergology, Albert Szent-Györgyi Medical Center, University of Szeged, Szeged, Hungary, 5Department of Dermatology, Venerology and Oncodermatology, University of Pécs, Pécs, Hungary, 6Department of Dermatology, University of Debrecen, Debrecen, Hungary, 7Department of Surgical and Molecular Pathology, National Institute of Oncology, Budapest, Hungary
Correspondence: Andrea Ladányi - email@example.com
Journal of Translational Medicine 2016, 15(Suppl 1):O7
Background: Immunotherapeutic modalities of cancer treatment have been increasingly gaining ground in the past few years. Antibodies targeting immune regulatory pathways showed unprecedented clinical efficacy in patients with advanced cancers. Nevertheless, generally only a smaller proportion of patients benefit from these therapies, prompting a search for biomarkers that could help identify patients likely to benefit from the treatment. Although several candidates have been suggested, no validated predictive biomarkers are available yet.
Materials and methods: Our study was performed on archived paraffin blocks of surgical samples excised within one year before ipilimumab therapy from patients with metastatic melanoma (30 patients, 1–25 lesions per patient). Eighty-six samples were analyzed, 52 nodal and 34 subcutaneous/cutaneous metastases. Immunohistochemical analysis was performed for detection of 10 immune cell markers (CD8, CD45RO, CD20, CD134, CD137, FOXP3, PD-1, CD16, CD68 and NKp46). Intratumoral density of the labeled cells was determined and evaluated in relation to response to ipilimumab treatment and disease outcome.
Results: For most of the markers studied, median immune cell densities were at least 2 times higher in lymph node metastases compared to subcutaneous/cutaneous ones, therefore, the prognostic and predictive associations of immune cell infiltration were evaluated separately in the two groups of metastases as well as in all samples together. Thirteen patients were considered responders showing complete or partial response, or stable disease for at least 6 months. Higher prevalence of immune cells expressing the FOXP3, CD8, CD20, NKp46, or CD134 marker was seen in lymph node metastases of the responders compared to non-responders. Kaplan–Meier analysis of survival revealed that high mean density of immune cells in nodal metastases was associated with significantly longer progression-free and overall survival in the case of the majority of cell types studied. In subcutaneous/cutaneous metastases, on the other hand, correlation with treatment response or with survival was limited to the CD16, CD68 and NKp46 markers.
Conclusions: Our findings corroborate previous results indicating stronger response to ipilimumab treatment in cases of an immunologically active tumor microenvironment. Studies on larger patient cohorts are required to prospectively validate infiltrating immune cell densities as biomarkers of ipilimumab efficacy and, possibly, to investigate their predictive value in the case of anti-PD-1/PD-L1 agents as well as of other immunotherapy approaches.
The work was supported by the National Research, Development and Innovation Office NKFI Grant 105132.
O8 Identification of five circulating microRNAs with high diagnostic values in cutaneous melanoma
Beatrice Polini1, Stefano Fogli1, Sara Carpi1, Barbara Pardini2, Alessio Naccarati2, Nevio Dubbini3, Maria Cristina Breschi1, Antonella Romanini3, Paola Nieri1
1Department of Pharmacy, University of Pisa, Pisa, Italy, 2Human genetic foundation (HUGEF), Torino, Italy, 3Department of Medical Oncology, University Hospital of Pisa, Italy
Correspondence: Beatrice Polini - firstname.lastname@example.org
Journal of Translational Medicine 2016, 15(Suppl 1):O8
Background: Melanoma is the fourth and sixth most common malignancy in men and women, respectively, and the 5-year survival rate critically depends on disease stage (American Cancer Society, 2016). MicroRNAs (miRNAs) are small, non-coding, single-stranded RNAs endogenously produced by the cells, which regulate the expression of hundreds of target genes. Circulating miRNAs actively secreted by tumor cells or released as the consequence of tumor cell death, have been proposed as potential biomarkers in cancer patients because of their stability in body fluids, resistance to endogenous RNase and constant expression in healthy individuals (Mitchell et al. 2008; Chen et al. 2008).
Materials and methods: In the current study, the expression profiles of a selected panel of circulating miRNAs were analysed in plasma samples derived from patients and healthy donors to identify possible candidate biomarkers for diagnosis, prognosis and/or surveillance of human cutaneous melanoma. The study protocol was approved by local Ethics Committee and conducted in accordance to the principles of the Declaration of Helsinki. Blood samples were collected from melanoma patients (n = 30) at different disease stages, and from healthy age- and sex-matched volunteers (n = 32). Plasma miRNAs were isolated by miRNeasy Serum/Plasma Kit (Qiagen) and real-time PCR were carried out using miRCURY LNATM Universal RT microRNA PCR system (Exiqon, Denmark). Data analysis was carried out using two different strategies for normalization: Global Mean Normalization (GMN) and NormFinder model.
Results: The GMN approach and NormFinder algorithm provided 13 and 7 significantly dysregulated miRNAs (p < 0.05), respectively, and those that resulted significantly dysregulated after normalizations and the Bonferroni correction were selected. Circulating miR-15b-5p (p = 1.34e−05), miR-149-3p (p = 3.40e−12), and miR-150-5p (p = 2.85e−12) were up-regulated, while miR-193a-3p (p = 1.30e−06) and miR-524-5p (p = 2.41e−05) were down-regulated in patients (regardless of disease stage) compared to healthy controls. Linear regression and following receiving operator curves (ROC) analyses were performed to evaluate the diagnostic value of these five selected miRNAs (i.e., the ability to discriminate between cases and controls). The area under ROC curve (AUCs) for individual miRNAs ranged from 0.801 to 0.951. Although the predictive power of all selected miRNAs was clearly demonstrated, miR-150-5p and miR-149-3p gave the best performance (AUCs of 0.9489, 95% CI from 0.8852 to 1.017 and 0.9510, 95% CI from 0.8852 to 1.017, respectively). Noteworthy, predictive performance was further improved when considering the double combination of miR-150-5p and miR-149-3p. The double classifier has indeed an increased area under ROC curve (AUC) of 0.966 (95% CI 0.938–0.994) with 90% sensitivity, 68% specificity.
Conclusions: In conclusion, findings of the current pilot study identified five circulating miRNAs in melanoma patients, of which three detected for the first time as circulating in this type of cancer, as potential diagnostic biomarkers with high sensitivity and specificity. In particular, the combined miR-149-3p, miR-150-3p and miR-193a-3p signature showed a high capacity to discriminate between healthy subjects and affected individuals. This feature make the signature suitable to be used in controversial melanoma diagnosis. Indeed, these preliminary data that identify a new panel of three miRNAs, including two identified for the first time as circulating in melanoma, deserve to be validated in a larger number of patients, in order to confirm their diagnostic power not only as a possible support to controversial pathology reports histological results but also in early diagnosis of melanoma.
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3. Lynam-Lennon N, Maher SG, Reynolds JV. The roles of microRNA in cancer and apoptosis. Biol Rev Camb Philos Soc. 2009;84:55–71
4. Mitchell PS, Parkin RK, Kroh EM et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci. 2008;105:10513–18.
5. Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 2008;18: 997–1006.
6. Segura MF, Belitskaya-Levy I, Rose AE, et al. (2010) Clin Cancer Res 16: 1577–86
O9 Impact of phosphoinositide 3 kinase and vitamin D3 nuclear receptor single nucleotide polymorphisms on the outcome of malignant melanoma patients
Francesca Morgese1, Davide Soldato1, Silvia Pagliaretta1, Riccardo Giampieri1, Donatella Brancorsini2, Silvia Rinaldi1, Mariangela Torniai1, Anna Campanati3, Giulia Ganzetti3, Annamaria Offidani3, Alfredo Giacchetti4, Giuseppe Ricotti4, Agnese Savini1, Azzurra Onofri1, Francesca Bianchi1, Rossana Berardi1
1Medical Oncology, Università Politecnica delle Marche, Azienda Ospedaliero-Universitaria Ospedali Riuniti Umberto I-GM Lancisi-G Salesi, Ancona, Italy, 2Section of Pathological Anatomy and Histopathology, Deparment of Neuroscience, Università Politecnica delle Marche, Azienda Ospedaliero-Universitaria Ospedali Riuniti Umberto I-GM Lancisi-G Salesi, Ancona, Italy, 3Dermatology Clinic, Università Politecnica delle Marche, Azienda Ospedaliero-Universitaria Ospedali Riuniti Umberto I-GM Lancisi-G Salesi, Ancona, Italy, 4U.O. Dermatologia, INRCA/IRCCS, Ancona, Italy
Correspondence: Francesca Morgese - email@example.com
Journal of Translational Medicine 2016, 15(Suppl 1):O9
Background: Nowadays, the biomolecular mechanisms at the basis of malignant melanoma development and progression are still poorly understood. Although several studies were conducted in order to associate single nucleotide polymorphisms (SNPs) frequencies with the carcinogenesis and tumors outcome, malignant melanoma literature data are inconclusive [1-14].
In our study we evaluate the impact of different genotypes for phosphoinositide 3 kinase (PI3K) and vitamin D3 nuclear receptor (VDR) SNPs on melanoma patients’ outcome.
Materials and methods: Genomic DNA of 88 patients was extracted from blood and Formaldehyde Fixed-Paraffin Embedded samples. Gene polymorphisms were determined by Real-Time Polymerase chain reaction (PCR) using TaqMan assays . We selected polymorphisms of the regulatory and catalytic subunit of PI3K encoded by PIK3CA  and PIK3R1  genes, respectively. In particular, we analyzed rs2699887C > T of PIK3CA and rs3730089G > A of PIK3R1 SNPs. Furthermore we considered the following VDR SNPs: rs2228570A > G (Fok1), rs731236A > G (Taq1) and rs1544410C > T (Bsm1).
Progression free survival (PFS) and overall survival (OS) were estimated with the Kaplan–Meier method and with Mantel–Haenszel log-rank test .
Results: The statistical analysis for rs2699887C > T of PIK3CA, rs3730089G > A of PIK3R1, Taq1 and Bsm1 of VDR didn’t result in statistical significant differences in PFS and OS.
On the contrary, Fok1 of VDR showed a significant difference in PFS after the first line therapy in the analyzed population (median PFS = 21.2 months of homozygous recessive genotype vs. 3.3 months of homozygous dominant and heterozygous ones, p = 0.03). In particular, in homozygous recessive patients for Fok1 SNPs of VDR a high rate of histological regression and BRAF mutation were observed. Furthermore, more efficacy of BRAF +/- MEK inhibitors therapies vs. homozygous dominant and heterozygous ones was shown.
Conclusions: Few studies, from the published literature, associate malignant melanoma patients’ outcome with VDR SNPs [7, 19, 20]. Our study demonstrated a significant correlation between homozygous recessive genotype of Fok1 SNPs of VDR gene and an increased PFS in patients who underwent a first line therapy with BRAF inhibitors.
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3. Jurutka PW, Remus LS, Whitfield GK, Thompson PD, Hsieh JC, Zitzer H, Tavakkoli P, Galligan MA, Dang HT, Haussler CA, Haussler MR. The polymorphic N terminus in human vitamin D receptor isoforms influences transcriptional activity by modulating interaction with transcription factor IIB. Mol Endocrinol. 2000;14(3):401–20.
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5. Haussler MR, Jurutka PW, Haussler CA, Hsieh J-C, Thompson PD, Remus LS, Selznick SH, Encinas C, Whitfield GK. VDR-mediated transactivation: interplay between 1,25(OH)2D3, RXR heterodimerization, transcription (co)factors and polymorphic receptor variants. In: R. Norman, R. Bouillon, and M. Thomasset (eds.), Vitamin D. Chemistry, Biology and Clinical Applications of the Steroid Hormone. 1997:210–17.
6. Haussler MR, Whitfield GK, Haussler CA, Hsieh JC, Thompson PD, Selznick SH, Dominguez CE, Jurutka PW. The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J. Bone Miner. Res.1998;13:325–49.
7. Orlow I, Reiner AS, Thomas NE, Roy P, Kanetsky PA, Luo L, Paine S, Armstrong BK, Kricker A, Marrett LD, Rosso S, Zanetti R, Gruber SB, Anton-Culver H, Gallagher RP, Dwyer T, Busam K, Begg CB, Berwick M; GEM Study Group. Vitamin D receptor polymorphisms and survival in patients with cutaneous melanoma: a population-based study. Carcinogenesis. 2016;37(1):30–8.
8. Evans SR, Houghton AM, Schumaker L, Brenner RV, Buras RR, Davoodi F, Nauta RJ, Shabahang M. Vitamin D receptor and growth inhibition by 1,25-dihydroxyvitamin D3 in human malignant melanoma cell lines. J Surg Res. 1996;61(1):127–33.
9. Shabahang M. Vitamin D receptor and growth inhibition by 1,25-dihydroxyvitamin D3 in human malignant melanoma cell lines. J Surg Res. 1996;61(1):127–33.
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11. Osborne JE, Hutchinson PE. Vitamin D and systemic cancer: is this relevant to malignant melanoma? Br J Dermatol. 2002;147(2):197–213.
12. Gandini S, Raimondi S, Gnagnarella P, Doré JF, Maisonneuve P, Testori A. Vitamin D and skin cancer: a meta-analysis. Eur J Cancer. 2009;45(4):634–41.
13. Zhao XZ, Yang BH, Yu GH, Liu SZ, Yuan ZY. Polymorphisms in the vitamin D receptor (VDR) genes and skin cancer risk in European population: a meta-analysis. Arch Dermatol Res. 2014;306(6):545–53.
14. Hou W, Wan X, Fan J. Variants Fok1 and Bsm1 on VDR are associated with the melanoma risk: evidence from the published epidemiological studies. BMC Genet. 2015;16:14.
16. Bader AG, Kang S, Zhao L, Vogt PK. Oncogenic PI3 K deregulates transcription and translation Nature Reviews Cancer. 2005;5:921–929.
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