Melanoma Relapse Research Articles

TREATMENT OF DOGS WITH ORAL MELANOMA RECURRENCE BY DIODE LASER EXCISION

Introduction: Treatment of oral melanomas utilizes the surgical excision-resection (Culp et al., 2013) and/or radiation therapy (Proulx et al., 2003), chemotherapy with carboplatin (Brockley et al., 2013), immunotherapy (Ottnod et al., 2013). Treatment based on surgical excision is usually palliative (Freeman et al., 2003). Aims: In the literature even though there are data concerning the prognosis of oral melanomas in dogs after surgery, are missing data after laser excision. Taking into account these findings we wished to present our experience regarding three cases of oral melanoma recurrence and immediate and long term laser surgery results. Materials and Methods: The casuistry consisted of three dogs with recurrent oral malignant melanomas, subjected to surgical reintervention. The initial diagnosis was melanotic melanoma in stage I or II. The animals were brought back at different time intervals from originally excision with electric scalpel. Before reintervention, dogs were subjected to clinical, paraclinical exam and biopsy. Excision of the tumor mass was made with an optical fiber hawing a diameter of 400µm, at a power of 10W and a wavelength of 940 nm with a diode laser. At 1, 2, 3, 6 and 12 months after laser reintervention the dogs were reexamined. Results: Average time in which appeared canine oral melanoma relapse was 58.6 days. After reexamination all cases where reinstatement in stage I. Operators times were held in conditions of comfort with wide access, minimum bleeding, effective hemostasis. After surgery at 24 hours on the intervention place a slight local redness, without swelling and bleeding was observed. Palpation revealed initially also a slight local sensitivity which completely disappeared in 48 hours. There were no grasping and chewing disturbances. Macroscopic healing occurred in 7-9 days. At last recheck performed at 12 months there were no evidences of tumour recurrence or metastasis. Conclusion: Diode laser excision of oral malignant melanoma in dogs can be an alternative palliative procedure to invasive surgical resection procedures. The average of free recurrence and metastasis time after laser surgery has exceeded 360 days in these three cases.

Expression of PIWIL3 in primary and metastatic melanoma.

The PIWI-interacting RNA machinery in malignant melanoma (MM) has not been sufficiently studied. We aimed to investigate the PIWIL3 expression profiles in primary melanomas and metastases of MM including a correlation with clinical data. We studied 161 primary melanomas, 45 lymph node metastases, and 16 distant metastases of 183 patients with MM. We used immunohistochemistry to assess PIWIL3 protein expression in situ. The relationship between the immunoreactivity of PIWIL3 and clinical data was statistically evaluated. We observed a significantly (P=0.000059) higher median immunoreactivity score in primary melanomas (4.9; range, 0.1-6), lymph node metastases (5.1; range, 3.3-6), and distant metastases (5.6; range, 4.5-6). PIWIL3 was expressed significantly higher (P=0.0002) in primary nodular melanomas and acral melanomas (5.2; range, 3.4-6) when compared to other melanoma subtypes (4.7; range, 0.1-6). On univariate analysis, a significant positive correlation was observed between primary melanoma PIWIL3 expression and tumor thickness (r=0.2; P=0.014). On univariate and multivariate analysis, PIWIL3 did not prove to be an independent predictor for melanoma relapse or death. Our data indicate that PIWIL3 protein expression is elevated in more aggressive primary MM and metastatic disease. As also observed in other malignancies, PIWIL3 seems to play a role in MM progression.

289 - System Analysis of the Molecular Characteristics of the Adapted and Quiescent/Dormancy(QD) State in an in Vitro Model for Late Relapse of Metastatic Melanoma

It has long been known clinically that revitalized dormant cells lead to late tumor relapse and eath. Our unique in vitro cell model that evolved in response to three common clinical dosage approaches demonstrates that there are multiple cellular strategies leading to drug resistance that correspond to low but increasing doses, medium dose and high dose treatment and include respectively the induction of the phenomena of adaptive response, genetic selection, and dormancy. Publications of acquired drug resistance have shown that a few studies of genetic mutations and many of non-genetic kinase network rewiring underlie the basis for the first two phenomena. To our knowledge, the biological basis of dormancy and its role in drug resistance has not been characterized. Through RNA-Seq profiling and system biology analysis, we compared the gene expression in parental SK-Mel28 cells and adapted SK-Mel28A2-1b, and discovered a number of altered signal cascades: the TGF-β-SMAD9-FGF1, PDGF-MAPK-DUSP-HIF2α and EDNRB-GNAI2-PLCB4-PKA-MITF pathways. Induction of HIF2α was confirmed by qRT-PCR in adapted Mel28A2-1b and also in A375C2 cells. Up-regulation of HIF2α targets and the negative regulator EGLN1 and EGLN3 occurred. Induction of MiTF was not shown at the transcript level in the adapted cells. However in the dormant cells we observed melanin accumulation and hypothesize that both HIF2α and MITF are up-regulated in dormancy; both genes are subject to extensive redox regulation. The details of these novel findings suggest that Plx4032-induced dormant cells may be dependent on HIF2α/MiTF-mediated p21WAF1 up-regulation for maintaining the dormancy status.The study of dormancy using an appropriate in vitro model has the potential to understand clinical dormancy and late relapse mechanistically and may offer a novel translational runway to developing new therapies for melanoma and possibly other cancers.

Melanoma dormancy in a mouse model is linked to GILZ/FOXO3A-dependent quiescence of disseminated stem-like cells.

Metastatic cancer relapses following the reactivation of dormant, disseminated tumour cells; however, the cells and factors involved in this reactivation are just beginning to be identified. Using an immunotherapy-based syngeneic model of melanoma dormancy and GFP-labelled dormant cell-derived cell lines, we determined that vaccination against melanoma prevented tumour growth but did not prevent tumour cell dissemination or eliminate all tumour cells. The persistent disseminated melanoma tumour cells were quiescent and asymptomatic for one year. The quiescence/activation of these cells in vitro and the dormancy of melanoma in vivo appeared to be regulated by glucocorticoid-induced leucine zipper (GILZ)-mediated immunosuppression. GILZ expression was low in dormant cell-derived cultures, and re-expression of GILZ inactivated FOXO3A and its downstream target, p21CIP1. The ability of dormancy-competent cells to re-enter the cell cycle increased after a second round of cellular dormancy in vivo in association with shortened tumour dormancy period and faster and more aggressive melanoma relapse. Our data indicate that future cancer treatments should be adjusted according to the stage of disease progression.

Cancer stem cell as therapeutic target for melanoma treatment.

Human malignant melanoma is a highly aggressive skin tumor that is characterized by its extraordinary heterogeneity, propensity for dissemination to distant organs and resistance to cytotoxic agents. Although chemo- and immune-based therapies have been evaluated in clinical trials, most of these therapeutics do not show significant benefit for patients with advanced disease. Treatment failure in melanoma patients is attributed mainly to the development of tumor heterogeneity resulting from the formation of genetically divergent subpopulations. These subpopulations are composed of cancer stem-like cells (CSCs) as a small fraction and non-cancer stem cells that form the majority of the tumor mass. In recent years, CSCs gained more attention and suggested as valuable experimental model system for tumor study. In melanoma, intratumoral heterogeneity, progression and drug resistance result from the unique characteristics of melanoma stem cells (MSCs). These MSCs are characterized by their distinct protein signature and tumor growth-driving pathways, whose activation is mediated by driver mutation-dependent signal. The molecular features of MSCs are either in a causal or consequential relationship to melanoma progression, drug resistance and relapse. Here, we review the current scientific evidence that supports CSC hypothesis and the validity of MSCs-dependent pathways and their key molecules as potential therapeutic target for melanoma treatment.

Malignancies of the skin and immunomodulatory antirheumatic therapy

For the clinical practice there is uncertainty as to what degree the therapeutic immunomodulation of rheumatoid arthritis (RA) is associated with a weakening of protective tumor immunity. Neoplasms of the skin in particular are known to exhibit increased incidence rates in association with therapeutic immunosuppression in transplantation medicine; however, the immunosuppression required for the prevention of allogenic graft rejection is much more intensive and thus not directly transferable to the potential risks for an onset or relapse of melanoma or non-melanoma skin cancers (NMSC), e. g. spinocellular and basal cell carcinomas in association with the antirheumatic treatment of RA. This review covers the association of RA and its pharmacotherapy with conventional synthetic (cs) and biologic disease-modifying antirheumatic drugs (DMARD) on the basis of a systematic literature search. The incidence rates of NMSC are twice as high in biologic-naive RA patients compared to the general population, whereas the respective incidence rates for melanoma do not differ. A biologic treatment with tumor necrosis factor (TNF) blockers compared with administration of csDMARD only has a minor, if any effect on the increase of NMSC risk but is associated with a trend towards an elevated incidence rate of new onset melanoma although significance level was not reached in all of the reviewed studies. The data on non-TNF blocking biologics is sparse. Accordingly, it is inappropriate to draw any strong conclusions on potentially associated skin cancer risks from the present lack of safety signals. The consideration of individual risk factors, recommendations on sufficient UV protection and regular skin monitoring may serve to improve the safety of DMARD therapy in RA.

Abstract A003: Polyfunctional antitumor CD8 T cells obtained from a broad repertoire elicited by chemo-immunotherapy and preventing melanoma relapse depends on the activation of an AKT pathway sustained by ICOS

Abstract The identification of activation pathways linked to anti-tumor T-cell polyfunctionality, associated with clinical benefit, is of great relevance in the new era of immunotherapy and combined chemo-immunotherapy. Recently we have reported that Melan-A-specific CD8 T cells isolated from long-term surviving patients treated with DTIC injected in a tight window before peptide-vaccination plus IFN-α; possess higher anti-tumor reactivity and an enlarged T-cell repertoire, compared to cells isolated after vaccination alone, suggesting that DTIC before peptide-vaccination may favor a protective anti-tumor specific immune response (1). To identify the mechanisms enhancing the immune response induced by the combined therapy, we analyzed the endogenous and treatment-induced antigen specific CD8 T-cell response in a panel of Melan-A- and gp100-specific clones from five patients. To this purpose, we analyzed the maturation/differentiation phenotype of these clones (CCR7 and CD45RA), the co-stimulatory (CD27, CD28 and ICOS) and inhibitory (TIM-3, LAG-3 and PD-1) profile, in parallel with the polyfunctionality (IFN-γ; TNF-α; and Granzyme B) and the activation of AKT (pSer473-AKT). AKT activation was correlated with the differentiation profile (in term of CD28 and/or CD27 expression) and was associated with poor polyfunctionality and low ICOS expression in Melan-A-specific T cells isolated from the endogenous response of both treatments and from the peptide-vaccination-driven response. Conversely, in three patients the combined treatment with DTIC before peptide vaccination elicited tumor-specific CD8+ T cells displaying the hallmarks of differentiated and highly activated effector T cells. These clones are highly efficient in tumor killing, possess polyfunctional activity, up-regulate inhibitory receptors (with PD-1 expressed at the highest level), retain proliferative capability and activate an AKT pathway not-related to the expression of CD27/CD28 molecules and partially dependent on ICOS engagement. Strikingly, T-cell polyfunctionality elicited by the combined therapy was strictly dependent on this AKT activation, as demonstrated by the blockade with selective inhibitors, which occurred only in Melan-A-specific CD8+ T cells and not in cells specific for gp100, suggesting that the nature of the peptide is crucial for the activation of this pathway. We suggest that this phenotypic and functional T-cell signature related to a highly effective tumor-specific response is fine-tuned between TCR activation, co-stimulatory and inhibitory signals critical to preserve the self-tolerance during immunotherapy treatment. Of clinical relevance, these three patients treated with chemo-immunotherapy are clinically disease-free after 9 years of follow-up. The study represents a critical contribution for the comprehension of the mechanisms underlying the advantages of combined chemo-immunotherapy and paves the way for the identification of new biomarkers of T-cell activation that may be employed as markers of immune responsiveness. Reference: 1. Palermo B, Del Bello D, Sottini A, Serana F, Ghidini C, Gualtieri N, Ferraresi V, Catricalà C, Belardelli F, Proietti E, Natali PG, Imberti L, Nisticò P. Dacarbazine treatment before peptide vaccination enlarges T-cell repertoire diversity of melan-a-specific, tumor-reactive CTL in melanoma patients. Cancer Res. 2010 Sep 15;70(18):7084-92. Citation Format: Ornella Franzese, Belinda Palermo, Cosmo Di Donna, Isabella Sperduti, Mariangela Panetta, Maria Laura Foddai, Angela Santoni, Paola Nisticò. Polyfunctional antitumor CD8 T cells obtained from a broad repertoire elicited by chemo-immunotherapy and preventing melanoma relapse depends on the activation of an AKT pathway sustained by ICOS. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A003.

Bayesian Analysis of a Patient with either Recurrent Metastatic Melanoma or Ipilimumab-Induced Sarcoidosis

A patient with metastatic melanoma received treatment with adjuvant Ipilimumab and subsequently developed signs of sarcoidosis 6 weeks post completion of treatment. 18 F FDG PET CT was completed which described FDG avid hilar and mediastinal adenopathy and bilateral FDG avid lung nodules with low-grade bilateral uptake in the lower limbs. Differential diagnosis was either recurrent melanoma or sarcoidosis arising as an immune related adverse event attributable to Ipilimumab. Prior to initiation of anti- PD-1 treatment we assessed the merit of undertaking a biopsy to confirm a second relapse of metastatic melanoma. We approached a quantitative appraisal of the clinical dilemma arising in this case using Bayes' theorem and a meta-analysis of published literature of the accuracy of PET detection of recurrent cutaneous melanoma. Partly on this rationale a bronchoscopic biopsy of two lymph nodes was undertaken. Results found abundant histiocytic aggregates focally forming granuloma giving a diagnosis of Ipilimumab induced sarcoidosis. Therefore, positive findings on PET imaging in patients with melanoma treated with Ipilimumab can represent other entities including iatrogenic sequelae from immunotherapy. With the emergence immunotherapy. With the emergence of novel immunotherapy treatment for metastatic melanoma and an increase in the popularity of PET imaging as a modality for diagnosing metastatic disease this article highlights two important issues; the traditional role of biopsies in confirming metastatic disease and immune related adverse events associated with novel therapies.

1024 Non-tumour risk factors for cutaneous melanoma relapse

1024 Non-tumour risk factors for cutaneous melanoma relapse

3353 Seropositivity to tumour antigens as a potential new biomarker for melanoma relapse

3353 Seropositivity to tumour antigens as a potential new biomarker for melanoma relapse

Patients’ experiences of coping with nodal relapse of melanoma

Sheena Stothers and Eilis McCaughan describe a study to investigate the support needs of this unique patient group

Seropositivity to tumour antigens as a potential new biomarker for melanoma relapse.

e20049 Background: Current prognostic markers fail to identify many patients with stage II / III melanoma who go on to relapse. Immunity has been assumed to play a part in influencing risk of recur...

The role of host B7-H1 in the recurrence of B16 melanoma. (TUM9P.1013)

Abstract Our B16 melanoma mouse model, in which ~50% of mice receiving adoptive cellular therapy with cytolytic CD4+ T cells recognizing the tumor/self antigen tyrosinase protein 1 (TRP-1), exhibits tumor relapse following initial tumor regression. CD4+ T cells from tumor-bearing TRP-1 antigen knockout (tyrp1BwRAG-/-) transgenic mice, or tumor sensitized (t.s.) T cells, expressed low levels of the exhaustion marker PD-1 and were 15-25% Foxp3+. Due to this presensitization, B16 tumors recurred faster and more predictably in RAG-/- mice with t.s. CD4+ T cells than naïve TRP-1-specific CD4+ T cells. However, in RAG-/- mice lacking B7-H1 (i.e. PD-L1, the ligand for PD-1), relapses occurred more rarely and developed at slower rates following adoptive transfer of t.s. CD4+ T cells. When CD4+ T cells from mice bearing relapsing tumors, expressing high levels of PD-1 and Foxp3+, were transferred into either RAG-/- or B7-H1-/-RAG-/- mice, the treatment effect was completely abrogated. We previously reported that primary tumors required B7-H1 blockade or Treg depletion to be treated, and relapsing tumors required both treatments. Here we show that Treg cell depletion in B7-H1-/-RAG-/- mice partially restored treatment with fully exhausted CD4+ T cells and relapses took longer to occur. These data indicate that although multiple mechanisms are involved in tumor recurrence (Treg cells and PD-1), the expression of B7-H1 in the host rather than the tumor plays a major role in B16 melanoma relapse.

Sequential molecular analysis of circulating MCAM/MUC18 expression: a promising disease biomarker related to clinical outcome in melanoma.

MCAM/MUC18 is a cell adhesion molecule associated with higher incidence of relapse in melanoma. The purpose of our study was to evaluate its role as a promising disease biomarker of progression through sequential molecular MCAM/MUC18 RT-PCR assay on serial blood samples collected during the clinical follow-up of 175 melanoma patients in different American Joint Committee on Cancer (AJCC) stages. MCAM/MUC18 molecular detection, found at least once in 22 out of the 175 patients, was significantly associated with poor prognosis and death (p < 0.001), regardless of the AJCC stages. Positive expression, either if primarily present or later acquired, was associated with melanoma progression, whereas patients primarily negative or with subsequent loss gained clinical remission or stable disease, even if in advanced stages (p < 0.005). Six AJCC advanced stages always MCAM/MUC18 negative are in complete remission or with a stable disease (p < 0.007). Semiquantitative immunohistochemical MCAM/MUC18 staining on corresponding primary melanomas was related to peripheral molecular expression. Correlations between circulating molecular and tissutal immunohistochemical detection, primary tumour thickness, AJCC stages and clinical outcome were statistically evaluated using Student’s t test, ANOVA, Spearman’s rank correlation test, Pearson χ 2-test and McNemar’s test. In our investigation, MCAM/MUC18 expression behaves as a “molecular warning of progression” even in early AJCC patients otherwise in disease-free conditions. Achievement of this molecule predicted the emergence of a clinically apparent status, whereas absence or persistent loss was related to a stable disease or to a disease-free status. If confirmed in larger case series, MCAM/MUC18 molecular expression could predict good or poor clinical outcome, possibly becoming a promising prognostic factor.

Ipilimumab-induced Colitis with Concomitant Clostridium difficile Infection

Introduction: Ipilimumab (Anti-Cytotoxic T lymphocyte-associated antigen (CTLA)-4 antibody)-induced colitis is a rare immune-mediated adverse event. Prompt diagnosis and treatment can prevent life-threatening complications. We report a case of colitis caused by Ipilimumab with concomitant Clostridium difficile (C. diff) infection and successful treatment with systemic corticosteroids. Case Report: A 32-year-old female who was undergoing Ipilimumab therapy post cycle number four for relapse of malignant melanoma presented with bloody diarrhea for two days. The patient denied fever, nausea, and vomiting. Examination revealed a soft but tender abdomen in the left lower quadrant. Stool studies were positive for C. diff toxin. She was treated with PO Vancomycin. Diarrhea slowly improved with recurrent episodes of bright red blood. Repeat C. diff toxin on day 10 was negative. IV Solumedrol 40 mg q8hrs was started with immediate response and complete resolution of hematochezia and diarrhea. Colonoscopy on day 13 showed severe colitis with erythema, edema, and friability with a plethora of large crated ulcers in the left colon, predominantly the splenic flexure. Biopsy showed mucosal ulceration and granulation tissue with rare cryptitis. She remained asymptomatic for the next few days on IV steroids, and was transitioned to 60-mg prednisone with a slow taper of Vancomycin over three weeks. As an outpatient, she was switched to Budesonide with plan for repeat colonoscopy. Discussion: Colonic toxicity is an immune-mediated adverse event caused by a biological agent. With increased use of Ipilimumab to treat malignant melanoma, the incidence of colitis is rising (recorded in about 20% of the patients receiving this treatment). Typical symptoms include abdominal pain and diarrhea, sometimes bloody. Endoscopy can show segmental or diffuse colitis. Biopsies show nonspecific inflammatory infiltrate with predominant T-cells, cryptitis, and abscess formation. Fatal complications (5%) like perforation (<1%) can occur without prompt appropriate treatment. Systemic steroids are the treatment of choice. Limited or no response to steroids in a week should lead to the use of infliximab. Failure of medical treatment can necessitate partial or complete colectomy with diverting ileostomy. Few cases have been reported of concomitant C. diff colitis, which complicates the course. Our patient was initially treated only for C. diff. Lack of sustained response to antibiotics prompted initiation of corticosteroids. Colonoscopy showed significantly deep ulcerations, suggesting that perforation could have occurred without timely steroids. C. diff infection should not preclude use of systemic steroids if immune mediated colitis is suspected.

Combined ipsilateral neck and axillary lymphadenectomy for metastatic skin cancers: A case series and surgical tips

In the absence of distant disease simultaneous skin cancer metastasis to neck and axillary lymph nodes necessitates both an axillary and neck en block lymphadenectomy. Acombined ipsilateral neck and axillary lymph node dissection should involve an in-continuity dissection through the cervicoaxillary canal for optimal lymphatic and oncological clearance. Review of the literature reveals little published instruction on the procedure since the radical surgery performed by Bowden over 50 years ago. We present 4 cases where ipsilateral axillary and neck lymph node dissections were performed for metastatic melanoma and a case of apical axillary node dissection via a neck incision approach. Our surgical tips include performing apical axillary node dissection via the neck incision and consideration of clavicular osteotomy or clavicular excision. A transclavicular approach was taken in one patient who had an excellent functional outcome after a plate and screw fixation. One elderly patient required a middle third claviculectomy which reduced shoulder elevation but was not associated with functional impairment. We conclude the surgery is safe and associated with the usual morbidity ascribed with either an axillary or neck dissection undertaken in isolation. However, patients have a significant risk of disease relapse as would be expected due to the duel metastatic sites, multiple lymph node and neck involvement which are known to be independent poor prognostic factors on melanoma survival and relapse.

Relapse of melanoma after successful adoptive T‐cell therapy: escape through inflammation‐induced phenotypic melanoma cell plasticity

Advanced stage melanomas are refractory to most available therapies including recently approved targeted therapies. This is also true for most biological (IFN-a or IL-2) treatments and therapy involving adoptive transfer of melanoma-reactive T cells (de Souza et al., 2012). A number of mechanisms have been proposed to explain the lack of T-cell functional activity in vivo. Some of these mechanisms include emergence of antigen-loss tumor variants, induction of immune inhibitory molecules such as CTLA4 or PD1 and induction of T-reg (regulatory) cells or myeloid-derived suppressor cells (MDSC) or presence of immune-suppressive cytokines such as TGF-b or IL-10 (Grivennikov et al., 2010). A number of environmental factors can further contribute to genetic instability and induce epigenetic changes of progressing heterogeneous tumors. Presence of inflammatory factors at the tumor site can cause selection of preexisting tumor cell variants resulting in preferential growth of antigen-negative tumor variants or tumor cells that have lost MHC-class I molecules (Khong and Restifo, 2002). Immune editing or alterations of tumor antigens due to immune pressure can also be one of the reasons for lack of T-cell-mediated tumor cell death in vivo (Singh and Paterson, 2007). In clinical trials, adoptive cell therapies (ACT) with melanoma-reactive tumor-infiltrating lymphocytes (TILs) or polyclonal T cells engineered to express chimeric antigen receptor (CAR) with re-directed specificity to melanoma have shown great promise with dramatic regression of bulky tumors (Robbins et al., 2011). However, in most patients, initial regression of tumor cells was followed by appearance of new lesions with therapy resistant melanoma cells (Robbins et al., 2011). Until now, the exact nature of ACT-related tumor resistance and the cause of emergence of antigen-negative melanoma cells were largely unknown. In the current edition of Nature, Landsberg et al. describe a novel mechanism of T-cell-induced loss of melanocytic antigens as a possible cause of tumor cell resistance to ACT. To demonstrate therapy resistance, the authors have used a previously published genetically engineered hepatocyte growth factor (Hgf)-Cdk4 mouse melanoma model and anti-gp100-specific CTLs obtained from a TcR transgenic pmel-1 (gp100) mouse model. TNF-a, a proinflammatory cytokine predominantly secreted by activated T cells caused down-modulation of gp100 antigen expression on the mouse melanoma cells resulting in loss of recognition by anti-gp100-specific CTLs used in ACT. TNF-a also down-modulated pigmentrelated gene expression and tyrosinaserelated protein (TRP)-2 on melanoma cells. Interestingly, the pro-inflammatory environment of TNF-a did not downmodulate the expression of non-melanocytic antigens. On the contrary, there was an increased expression of nerve growth factor receptor (NGFR) on melanoma cells. Down-modulation of gp100 expression was reversible when the activity of TNF-a was neutralized or the resistant tumor cells were re-transplanted into a normal mouse lacking an inflammatory environment. The mechanism of non-genetic reversible antigen down-modulation is novel and has not been described before. This is in contrast to other genetic heterogeneityand immunoselection-related mechanisms described earlier. Understanding inflammation-induced plasticity and the non-genetic heterogeneity of melanoma cells are critical for the development of new therapeutic strategies for melanoma. The authors then corroborated their mouse studies using human melanoma cell lines where they report similar down-modulation of gp100 and MART1/Melan-A expression in the presence of TNF-a. Similar to mouse studies, human melanoma cell lines also showed up-regulation of NGFR expression in the presence of TNF-a. This was confirmed in melanoma patients’ tumor tissue sections with abundant infiltrating immune cells further supporting the clinical relevance of the above findings. The results of Landsberg et al. have important clinical implications if the phenotypic plasticity of melanoma cells in an inflammatory microenvironment is validated in a larger cohort of patients with melanoma after ACT. Down-modulation of gp100 expression and concurrent up-regulation of NGFR expression of melanoma cells in an inflammatory environment raises the possibility of using ACT with a cocktail of CAR-engineered T cells with dual or multiple specificities to target more than one melanoma antigen to ensure complete regression of tumor lesions. For this, a personalized approach of analyzing melanoma antigen expression on tumor cells before and after ACT therapy will be essential for the selection of CARengineered T cells with defined tumor antigen specificity. The proposed approach may enhance the long term disease-free survival of patients with metastatic melanoma.

Metabolic state of perifocal zone and resection line of skin melanoma.

e19049 Background: The state of metabolism in perifocal zone and morphologically intact skin along resection line is of importance for the processes of neoplasm metastasizing and relapse, including skin melanoma. Normalization of the state of free-radical processes, first of all along resection line, and then in neoplasm perifocal zone, should be a characteristic of pathogenically substantiated neoadjuvant influence. Methods: to study activity of free-radical processes in tissue of skin melanoma relapse, its perifocal zone (PZ) (1 cm from tumour edge) and in skin along resection line (3-5 cm from tumour edge) we investigated concentration of vitamins E and A, activity of superoxide dismutase (SOD), catalase, summary peroxidase activity (SPA), and malonic dialdehyde level (MDA) in patients with skin melanoma (pT2-3N1-2M0). The study group included 54 samples after treatment (chemotherapy and radiation). Control groups: 45 samples before treatment (CG1) and 27 samples of intact skin after plastic operations (CG2). Results: After treatment MDA concentration in tumour tissue increased by 33.4% in comparison with the CG1, but remained 17.7% lower than in CG2. Average increase in concentration of vitamins E and A was equal to 1.8 times, catalase activity decreased by 30.3% in comparison with parameters before treatment (p&lt;0.05). SOD/catalase coefficient was 1.3 times higher than in the CG1, but 3.4 times lower than CG2.More manifested changes in activity of free-radical processes were observed in PZ: MDA concentration level after treatment increased by 30% in comparison with the CG1, and differed from the one in CG2 only by 10% (p&lt;0.05). Catalase activity decreased by 1.5 times and didn’t significantly differ from the corresponding parameter in tumour tissue after treatment. Normalization of concentration level of vitamins A and E without change of their ratio was observed. There was 1.6-fold increase of SOD/SPA coefficient in comparison with the CG1, however it was 2.4 times lower than CG2 (p&lt;0.05). All parameters under study along resection line after treatment didn’t significantly differ from parameters in CG2. Conclusions: The performed treatment completely restores broken metabolism in skin along resection line, partially–in tumour perifocal zone.

Abstract 4406: Clinical efficacious combined chemo/immunotherapy differently activates AKT pathway and functionality of gp100 and Melan-A specific T cell clones

Abstract A phase II randomized clinical study is ongoing in our Institution to prove the clinical efficacy of dacarbazine (DTIC) one day before peptide-vaccination (Melan-A and NY-ESO-1) in the prevention of melanoma relapse in clinically disease-free HLA-A2 patients. In a previous pilot study, this combination therapy (using Melan-A and gp100 peptides) increased the number of tumor-reactive long-lasting effector-memory CD8+ lymphocytes. To identify the mechanisms enhancing the immune response, induced by DTIC combined with peptide-vaccination, we analyzed the endogenous and treatment-induced antigen specific CD8 T-cell response at the clonal level. We analyzed the sequence of the TCR α-chain of these clones and the molecular results were correlated with the expression of CD27/CD28 co-stimulatory molecule, AKT activation and anti-tumor lytic activity. The combination of chemo/immunotherapy elicited in Melan-A-specific, but not in gp100 clones, a renewal of high-avidity/tumor-reactive T-cell clones, with a broadening TCR diversity in long-surviving patients, suggesting that the selection of immune-resistant tumor variants may be circumvented by this combination, and thus prevent tumor recurrence in melanoma. In gp100 clones, AKT activation (pSer473-AKT) canonically correlates with CD28 and/or CD27 expression, independent of the treatment while in Melan-A clones, lacking CD27 and CD28 expression, non-canonical AKT activation was only observed after the combination therapy. The identification of the extracellular stimuli and signaling pathway responsible for DTIC-mediated activation of the AKT signaling are currently under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4406. doi:1538-7445.AM2012-4406

Local and intralesional therapy of in‐transit melanoma metastases

Regional relapse of melanoma may occur as satellite or in-transit metastases proximal to the primary tumor in the direction of the lymph flow. The management of in-transit metastases is challenging because the efficacy of treatment is largely dictated by the biological behavior of the patient's melanoma. This review examines local treatment modalities.