Abstract Even the most therapy responsive melanomas eventually evade immune checkpoint inhibition. Interestingly, initial responses seem to benefit from localized inflammation, but this can become prohibitive when treating advanced disease. Subsequently, there is an urgent need to elucidate how inflammation impacts the tumor microenvironment (TME). Here, inhibition of EGFR and Ras/Raf/MEK is shown to drive the release of a pro-inflammatory secretome that activates the TME to promote progression. This secretome was enriched with immune recruiting cytokines and members of the transforming growth factor beta (TGFβ) superfamily. Both healthy and cancer associated fibroblasts (CAFs) were hyperactivated in response to the inflamed secretome with induction of the inflammatory marker, alpha smooth muscle actin (αSMA). Pharmacologic inhibition of TGFβ receptor type I (TGFβRI) using SB431542 or clinically relevant TEW-7197, diminished CAF αSMA phenotype, disrupted TGFβ and phospo-SMAD2/3 signaling, and blunted CAF expression of tumor promoting extracellular matrix and adhesion molecules. Invasion of melanoma spheroids was enhanced ~2.5-fold in CAF coculture transwells. However, treatment with MEK inhibitor AZD6244 and TEW-7197 only limited invasion of mixed melanoma and CAF multi-cell spheroids in vitro. Prompted by this, the extracellular matrix of patient-derived and melanoma xenograft tumors was analyzed for inflammatory protein expression. Micrometastatic foci were flanked by peri-tumor αSMA+ CAFs and enriched for inflammatory Tenascin-C (TNC) at the invasive front. The analysis of human melanoma tissue microarrays corroborated the experimentally observed expression of αSMA CAFs and TNC which statistically increase with malignant tumor grade. Conversely, the anti-invasive Decorin (DCN), decreased in malignant melanoma while collagen-1 increased during late stage tumor fibrosis. These findings prompted the use of ex vivo human skin organ cultures (SOCs) to define how cell matrix impacted melanoma. Addition of DCN to the SOC eliminated melanoma vertical invasion whereas pathological TNC promoted progression. Thus, a BRAFV600E resistant patient-derived metastatic cell line was used to establish intrasplenic xenografts that were treated after one week using AZD6244 and TEW-7197. Both monotherapy and combination therapy were well tolerated and resulted in reduced splenic tumor burden, αSMA staining, and halted metastasis to the liver. Citation Format: Andrew M. Bradshaw, Erica Kuo, Jelena Grahovac, Kyle Sylakowski, Cindy Sander, Howard Edington, John M. Kirkwood, Alan Wells. The therapeutically inflamed tumor microenvironment drives melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3169.

Purpose Immunotherapy has demonstrated efficacy in treatment of intracranial metastasis from melanoma, calling into question the role of intracranial radiotherapy (RT). Herein, we assessed the utilization patterns of intracranial RT in patients with melanoma brain metastasis and compared outcomes in patients treated with immunotherapy alone versus immunotherapy in addition to intracranial RT. Methods We queried the National Cancer Database (NCDB) for patients with melanoma brain metastases treated with immunotherapy and intracranial RT or immunotherapy alone. Multivariable logistic regression identified variables associated with increased likelihood of receiving immunotherapy alone. Multivariable Cox regression was used to identify co-variates predictive of overall survival (OS). Propensity matching was used to account for indication bias. Results We identified 528 and 142 patients that were treated with combination therapy and immunotherapy alone, respectively. Patients with lower comorbidity score were more likely to receive immunotherapy alone. Extracranial disease and treatment at a non-academic center were associated with worse OS. Median OS for all patients was 11.0 months. Treatment with stereotactic radiosurgery (SRS) in addition to immunotherapy was superior to immunotherapy alone, median OS of 19.0 versus 11.5 months (p = 0.006). Whole brain radiation therapy (WBRT) in combination with immunotherapy performed worse than immunotherapy alone, median OS of 7.7 versus 11.5 months (p = 0.0255). Conclusions For melanoma patients requiring WBRT, immunotherapy alone may be reasonable in asymptomatic patients. For those eligible for SRS, combination therapy may provide better outcomes. Results of ongoing prospective studies will help provide guidance regarding the use of radioimmunotherapy in this population.

11. Nicole J. Martucci, Mary-Claire Cotner, Bharat Bhushan, Wendy M. Mars and George K. Michalopoulos. Nuclear Phosphoinositide 3-kinase delta (PIK3CD) Increases with Conditional Deletion of Hepatocellular Integrin Linked Kinase 12. Farzaneh Moghadam, Jeremy J Velazquez, Ryan LeGraw, Nan Cher Yeo, Chenxi Xu, Jin Park, Alejandro Chavez, Mo R Ebrahimkhani and Samira Kiani. CRISPR-based transcriptional repression to perform immunomodulation in vivo

Surgical meshes have become the standard procedure for a variety of surgical applications with 20 million meshes being implanted each year. The popularity of mesh usage among surgeons is backed by the multiple studies that support its functionality as a tool for improving surgical outcomes. However, their use has also been associated with infectious surgical complications and many surgeons have turned to biologic meshes. While there have been several studies investigating synthetic meshes, there is limited data comparing synthetic and biologic meshes in vitro in an infection model. This study evaluates the in vitro susceptibility of both synthetic and biologic meshes to single-species methicillin-resistant Staphylococcus aureus (MRSA) biofilms. This research compares biofilm biomass, average thickness, and coverage between the three meshes through florescent in situ hybridization (FISH), confocal scanning microscopy (CSLM), and image analysis. We also report the varying levels of planktonic and attached bacteria through sonication and cfu counts. While the data illustrates increased biofilm formation on biologic mesh in vitro, the study must further be investigated in vivo to confirm the study observations.

Cancer mortality ensues from metastatic growths. Cancers use two strategies to allow for this unrelenting expansion. The first way is that early metastases are often cryptic or dormant, being invisible to both innate suppressive actions and undetected clinically. Second, both the micrometastases and later clinically lethal growths are resistant to therapies, whether standard chemotherapies, targeted biologics, or even immunotherapies. These two modes of resistance necessitate new approaches to treatments if we are to eliminate mortality from solid tumors. However, to develop such therapeutic strategies, we first need to better understand the cellular behaviors and molecular events that enable the resistances. Herein, we present a comprehensive model of changing methods of avoidance and resistance that occur during tumor progression, and doubly confound treatment by mixing survival strategies throughout the continuum creating moving targets. Melanoma is presented as the model cancer, as it is being targeted by all three types of agents for disseminated disease, with breast and prostate cancer as two other key carcinomas.

Herein, we present a case of a 76-year-old Caucasian man with a very large fungating, ulcerating mass, involving the right neck and parotid area, which developed while he was being treated for chronic lymphocytic leukemia/small lymphocytic lymphoma.Resection of the fungating right neck tumor, right modified radical neck dissection, and right superficial parotidectomy with flap reconstruction were performed.The final pathological diagnosis was high-grade leiomyosarcoma of the skin and the subcutaneous tissue, with invasion into the skeletal muscle, skin, and soft tissue.Additionally, the sarcoma had metastasized to the lymph nodes that were involved diffusely by lymphoma.The most interesting fact for this case is coincidence of three rare occurrences which were soft tissue sarcomas of subcutaneous leiomyosarcoma form and its metastasis to same lymph nodes that were involved with lymphoma.

The modification of proteins with reactive handles has facilitated the use of these biomolecules in diverse fields including drug delivery, diagnostics, environmental remediation, and cell culture matrices. Recently, a new "click" type reaction, sulfur(VI) fluoride exchange (SuFEx), was reported between sulfuryl fluoride and various organic moieties (e.g. hydroxyl, amine) to yield a sulfamoyl fluoride (-NSO2F) or fluorosulfate (-OSO2F) moiety under biphasic conditions. The model protein bovine serum albumin (BSA) was reacted with SO2F2 gas under biphasic conditions to yield -SO2F functionalized protein. The resultant BSA-SO2F was characterized using gel electrophoresis, mass spectroscopy and Fourier transmission infrared spectroscopy to confirm the addition of -SO2F functional group. SuFEx modification of BSA caused a marked change in the pH dependent size and zeta potential of the protein as well as increased the protein's denaturation temperature. Crucially, BSA-SO2F was demonstrated to be biocompatible after 72 h incubation with A549 lung endothelial cells. Due to the unique and elective reactivity of the -SO2F group with amines, BSA-SO2F could be self-condensed to form a biocompatible hydrogel that was used in co-culture with HEK 293 cells. In addition, polyethylene glycol (PEG) with reactive -OSO2F groups at chain end was conjugated with BSA under various pH conditions through SuFEx chemistry. This communication, to our knowledge, is the first report of the application of the SuFEx in the field of bioconjugates. (C) 2016 Elsevier Ltd. All rights reserved.

Post-polymerization functionalization is a critical tool in preparing functional materials. The critical aspects of post-polymerization reactions are high yields, short reaction times, simple purification, near stoichiometric amounts of reactants, and facile reaction conditions. Polymeric epoxides (i.e. poly(glycidyl methacrylate)) represent an underutilized class of functionalized polymers due to long reaction times, high temperatures, and large excess of reactants to drive the reaction to high conversion in a reasonable time frame. In this manuscript we describe the use of a novel solvent trifluoroethanol (TFE) for the ring opening of amines with poly(glycidyl methacrylate). We demonstrate that TFE gives faster reaction times and higher yields than traditional solvents used for the ring opening reaction. We utilized TFE to prepare dual functionalized polymers that could be “clicked” using strain promoted azide alkyne cycloaddition and an amine-bearing drug ring opening of the polymeric backbone.

The currently-used modes of administration of immunotherapeutic agents result in their limited delivery to the lymph nodes and/or require repetitive ultrasound-guided nodal injections or microsurgical lymphatic injections, limiting their feasibility. Here, we report on the feasibility and safety of a new method of long-term repetitive intralymphatic (IL) infusion of immune cells, using implantable delivery ports.

Treating burn-related injuries remains challenging, because of donor site morbidity, bacterial infection, and major scarring. Recent advances have led to the development of promising natural and synthetic polymer scaffolds, the objective of which is to regenerate skin using a "top down" approach, in which the top layers of the skin populated by fibroblasts and keratinocytes are regenerated first. An often-overlooked component of the integumentary system is the subcutaneous adipose tissue, which has been implicated in a variety of activities, including wound healing. We have created a novel coating composed of collagen VI (col VI) and heparin sulfate (HS) that promotes adipocyte attachment and adipogenesis at a rate significantly faster at both early and late time points (5 and 23 days), compared to a noncoated control. In addition, Vicryl, which is a biodegradable surgical implant mesh, was coated with the optimized col VI-HS ratio. Coated Vicryl had an increased number of attached adipocytes with more-pronounced growth, when compared to uncoated Vicryl. These findings suggest that the combination of optimized col VI-HS coating and the fibrous woven Vicryl may have excellent potential in developing skin graft applications using a paradigm-shifting "bottom-up" approach.

We evaluated neoadjuvant ipilimumab in patients with surgically operable regionally advanced melanoma in order to define markers of activity in the blood and tumor as assessed at baseline (before ipilimumab) and early on-treatment. Patients were treated with ipilimumab (10 mg/kg intravenously every 3 weeks ×2 doses) bracketing surgery. Tumor and blood biospecimens were obtained at baseline and at surgery. Flow cytometry and immunohistochemistry for select biomarkers were performed. Thirty five patients were enrolled; IIIB (3; N2b), IIIC (32; N2c, N3), IV (2). Worst toxicities included Grade 3 diarrhea/colitis (5; 14%), hepatitis (2; 6%), rash (1; 3%), elevated lipase (3; 9%). Median follow up was 18 months: among 33 evaluable patients, median progression free survival (PFS) was 11 months, 95% CI (6.2–19.2). There was a significant decrease in circulating myeloid derived suppressor cells (MDSC). Greater decrease in circulating monocyte gate MDSC Lin1−/HLA-DR−/CD33+/CD11b+ was associated with improved PFS (p = 0.03). There was a significant increase in circulating regulatory T cells (Treg; CD4+CD25hi+Foxp3+) that, unexpectedly, was associated with improved PFS (HR = 0.57; p = 0.034). Baseline evidence of fully activated type I CD4+ and CD8+ antigen-specific T cell immunity against cancer-testis (NY-ESO-1) and melanocytic lineage (MART-1, gp100) antigens was detected and was significantly potentiated after ipilimumab. In tumor, there was a significant increase in CD8+ T cells after ipilimumab (p = 0.02). Ipilimumab induced increased tumor infiltration by fully activated (CD69+) CD3+/CD4+ and CD3+/CD8+ T cells with evidence of induction/potentiation of memory T cells (CD45RO+). The change in Treg observed within the tumor showed an inverse relationship with clinical benefit and greater decrease in tumor MDSC subset Lin1−/HLA-DR−/CD33+/CD11b+ was associated with improved PFS at one year. Neoadjuvant evaluation revealed a significant immunomodulating role for ipilimumab on Treg, MDSC and effector T cells in the circulation and tumor microenvironment that warrants further pursuit in the quest for optimizing melanoma immunotherapy.

e20029 Background: The risk of lymph node involvement for cutaneous melanoma increases with tumor thickness - 5% for Breslow thickness ≤1mm and 34% for T4 or ulcerated lesions. SLNB is standard of care for cutaneous melanoma of Breslow thickness >1mm. Positive SLNB is followed by completion lymph node dissection (CLND) of the affected nodal basin. SLNB has a high negative predictive value (NPV) but false negative (FN) rates vary widely (3-12.5%). Methods: Data were collected on 3,600 patients (pts) who underwent SLNB for cutaneous melanoma from 1996 to 2012 using the UPCI Cancer Registry. Cancer registry records were reviewed for primary tumor characteristics, demographic details, and outcomes. The influence of baseline characteristics on SLNB positivity was assessed using Cox proportional hazards analysis. Results: 3,600 SLNB performed from 1996 to 2012 of which 969 SLNB (969 pts) reviewed as a pilot. Primary tumor characteristics and demographic details obtained. SLN metastases were detected in 169 pts (17.4%). 134 CLND were performed with 31 pts not having CLND secondary to refusal or early distant metastatic disease. Following CLND, 48 recurrences were observed: recurrence pattern was predominantly cutaneous (19/48 pts, 39.6%), although ipsilateral recurrences in the prior CLND bed were noted (14/48 pts, 29.2%). Visceral recurrences comprised the remainder [non-pulmonary 7/48 pts (14.6%), pulmonary 5/48 pts (10.4%) and CNS 2/48 pts (4.2%)]. Most CLNDs followed by ipsilateral regional nodal recurrences were performed outside tertiary care centers. PFS and OS were significantly worse with increasing stage and SLNB status. Conclusions: AJCC, ASCO and SSO guidelines recommend SLN biopsy for intermediate-thickness melanomas (1-4mm Breslow) to optimize staging accuracy. Incidence of regional recurrence after CLND has been estimated at 30-35%. In this large and uniformly treated series of patients at a major melanoma referral center, SLN were involved in 17.2% and median PFS after CLND was 22mos. Biomarkers associated with SLNB and CLND positivity are currently being evaluated in prospectively banked specimens from patients (07-133) and under the aegis of the SPORE in Skin Cancer (P50CA121973).

9043 Background: For clinical stage I and II node negative melanoma the histologic status of the sentinel lymph node (SLN) is the most significant predictor of survival. Molecular characterization of node positive and node negative SLNs through gene expression profiling may improve the understanding of the molecular mechanisms of metastasis and identify specific gene signatures for SLN+/SLN- that correlate with clinical outcome. Methods: We characterized 15 SLN+ and 15 SLN- melanoma patients (T3a/b, T4a/b) who underwent SLN dissection for routine staging using transcriptome profiling analysis on 5μ sections of fresh LN samples. The primary endpoint was mRNA expression profiling using the U133A 2.0 Affymetrix gene chips. Significance Analysis of Microarrays v.4 was used to perform non-parametric analysis and statistical comparison for each transcript corrected for false discovery rate (q value) to control for type 1 errors arising from multiple tests. Pathway analysis was performed using Ingenuity Pathway Analysis software. Results: Tumor size ranged from 1-2mm in size. Twenty-one genes were expressed at significantly (q value <0.056) higher levels in SLN+ vs SLN-. These included CCNA2, CEP55, DCT, FEN1, HMMR, MLANA, PAICS, PBK, POSTN, RAB27A, RRM2, SHCBP1, SILV, TYR, CENPE, CCL20, CHEK1, LINS, TPX2, TTK, TYRP1. Pathway analysis (39 genes >1.4 fold; q < 0.12) identified the top disease categories as “Cancer” (26 genes, p = 5.7x10-9) and “Dermatological” (14 genes, p = 3.9x10-6). The top functional categories included “Cell Cycle” (21 genes p= 2.3x10-12) and “Cell Growth and Proliferation” (26 genes p=9x10-9). The relevant canonical pathways were “Eumelanin Biosynthesis” (p=3x10-5) and “Cell Cycle: G2/M DNA Damage Checkpoint Regulation” (p=1.2x10-4). Conclusions: We identified a 21 gene signature that is consistent with metastatic melanoma and its microenvironment and is differentially expressed in SLNs that are tumor involved. These gene families provide a signature of nodal involvement that may assist in diagnosis, and may be further explored towards prediction of outcome and development of therapy. A validation study is ongoing with clinical outcome association analyses as follow up is mature.

Background: Melanoma regional lymph node and in-transit metastases (stage IIIB-C) carry a high relapse and mortality risk. Neoadjuvant ipilimumab may modulate the host suppressor immune response, enhance antitumor immunity and improve the clinical outcome. Methods: Patients with clinically palpable stage IIIB-C melanoma were treated with induction ipilimumab (10 mg/kg IV q3weeks x2doses) preoperatively and had definitive lymphadenectomy (week ≥ 6), followed by 2 maintenance doses of ipilimumab (q3 weeks). Tissue samples were obtained at baseline and at definitive surgery (after 2 doses of ipilimumab) and blood (serum/PBMC) collected at baseline, 6 weeks, then at 3, 6, 9, 12 months and/or progression. Circulating T-regulatory cells (T-regs) and myeloid derived suppressor cells (MDSC) were monitored utilizing multicolor flow cytometry comparing preoperative (6 weeks) and baseline samples. Results: Twenty nine patients (20 male, 9 female), age 40-87 (median 54) have been enrolled since February/2010 (24 cutaneous primary, 1 unknown and 4 mucosal). Five had AJCC stage IIIB (N2b, N2c) and 24 had IIIC (N3) melanoma. Ninety one cycles have been delivered (median 4). Grade 3 (worst) toxicities include diarrhea/colitis (5 patients; 17%), hepatic enzyme elevations (2; 7%), rash (2; 7%), lipase (1; 3%), all manageable. Median follow-up is 9.7 months and 18 patients (62%) continue disease free. Median PFS is 15.5 months, 95% CI = (8.1, -). The probability of 6 and 12 months PFS is 84.2% (95% CI=0.63, 0.94) and 54.1% (95% CI=0.31, 0.73) respectively. There is a significant increase in the frequency of circulating T-regs (CD4+CD25hi+ Foxp3+; p=0.023 CD4+CD25hi+CD39+; p=0.001) from baseline to 6 weeks. In parallel there is a significant decrease in circulating MDSCs: (1) monocytic: HLA-DR+/low/CD14+; p= 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 5375. doi:1538-7445.AM2012-5375

8533 Background: Neoadjuvant ipilimumab (ipi) for stage IIIB-C melanoma may improve the clinical outcomes and provide access to pre/post ipi blood and tumor to gain insight into host effector and suppressor immune response mechanisms. Methods: Patients were treated with ipi (10 mg/kg IV q3weeks x 4doses total) bracketing definitive surgery. Tissue samples were obtained at baseline and at definitive surgery (week ≥ 6) and serum/PBMC collected at baseline, 6 weeks, then at 3, 6, 9, 12 months and/or progression. Flow cytometry was used to monitor the host effector and suppressor immune response in blood and evaluable tumor. Results: Thirty pts (21 male, 9 female), age 40-87 were enrolled (25 cutaneous primary, 1 unknown, 4 mucosal). Six had AJCC stage IIIB (N2b, N2c) and 24 IIIC (N3) melanoma. Ninety-three cycles have been delivered (median 4). Worst toxicities included grade 3 diarrhea/colitis (5 patients; 17%), hepatic enzyme elevations (2; 7%), rash (2; 7%), lipase (1; 3%), all manageable. Median follow up is 14 months: among 29 evaluable pts 15 (52%) continue disease free. Median PFS is 15.5 months, 95% CI = (8.1,-). The probability of 6 and 12 month PFS is 82.4% (95% CI=0.63, 0.92) and 53% (95% CI=0.31, 0.70) respectively. Peripherally, a significant increase in frequency of circulating T-regs (CD4+CD25hi+ Foxp3+; p=0.02 CD4+CD25hi+CD39+; p=0.001) from baseline to 6 weeks was observed. Greater increases in T-regs were associated with improved PFS (p=0.045; HR=0.54). Significant decreases in circulating MDSCs, were observed in monocytic HLA-DR+/low/CD14+ MDSC subtype (p<0.0001). Spontaneous in vivo cross presentation was observed resulting in Th1 CD4 and CD8 antigen specific T-cell immunity (gp-100, MART-1, NY-ESO-1 peptides) with increase in frequency after ipi. Activated TIL in tumor increased after ipi (CD3+/CD4+/CD69+;p=0.06 and CD3+/CD8+/CD69+) with significant induction/potentiation of T-cell memory (CD8+/CD45RO+/TNF-α+;p=0.03). Conclusions: Neoadjuvant ipi exhibits promising clinical activity and significantly modulates the host effector and suppressor immune response. Full analysis of this completed trial and its correlates will be presented. Support: BMS, P30CA047904 and P50CA121973.

TPS8606 Background: Increasing incidence and the poor prognosis of advanced melanoma argue for prevention efforts. Primary prevention and ultraviolet radiation (UVR) reduction have failed to gain traction in the population, making chemoprevention increasingly attractive. Sulforaphanes (SFN) are bioactive cruciferous compounds rich in the Brassica family, especially broccoli sprouts. Topical broccoli sprout extracts (BSE) decrease UVR erythema response in human skin, and may protect against UVR DNA damage. We have shown SFN inhibition of STAT3, which is constitutively activated in melanoma. We have therefore initiated a pilot study to evaluate BSE SFN in relation to melanoma progression biomarkers, and expression of STAT proteins of atypical melanocytic nevi. Methods: Melanoma patients with >2 atypical nevi are eligible for this trial, which aims to define the safety and clinico-pathological response to oral BSE-SFN. Secondary objectives are documentation of pharmacokinetics and pharmacodynamics of BSE-SFN, and the modulation of STAT 1/3 expression in atypical nevi and normal skin. Baseline photo-documentation and atypical nevus biopsy is performed to assess histopathological atypia and STAT 1/3 expression. Adjacent normal skin biopsies will establish baseline skin SFN levels. Six subjects per group will be accrued at daily dosages of 50, 100, and 200 μM. Subjects must abstain from dietary glucosinolates and isothiocyanates for the study duration and maintain food diaries. Following 27 days of BSE-SFN, blood samples, photography of index atypical nevi, and biopsies of selected atypical nevi and normal skin will be obtained. The three dosage groups will be analyzed for changes in atypia, SFN localization histopathology, and STAT 1/3 expression in the nevi and skin harvested at baseline and at study completion. This trial, UPCI 10-114, has received an IND and is IRB-approved. Accrual is planned to be completed during 2012-13.

76 Background: Neoadjuvant ipilimumab (Ipi) for stage IIIB-C melanoma may improve the clinical outcome and provide access to pre/post Ipi blood. Methods: Pts were treated with Ipi (10mg/kg IV q3wks x 2 doses) bracketing definitive surgery. Tissue samples were obtained at baseline and at surgery (wk ≥ 6) and serum/PBMC collected at baseline, 6 wks, 3, 6, 9, 12 mos and/or progression. Flow cytometry was used to monitor the host immune response in blood and evaluable tumor. IHC for select markers was also performed. Baseline and wk-6 serum cytokines were tested by xMAP multiplex technology (Luminex Corp). Results: 31 pts were enrolled, 6 had stage IIIB (N2b, N2c), and 25 IIIC (N3) melanoma. Worst toxicities (N=31 pts) included grade 3 diarrhea/colitis (5 pts; 16%), hepatitis (2; 6%), rash (1; 3%), lipase (2; 6%), all manageable. Median f/u was 19 mos: among 29 evaluable pts, median PFS was 12.9 mos, 95% CI = (7.4,-). Only 2 pts died. Peripherally, a significant increase in circulating T-regs (CD4+CD25hi+ Foxp3+; p=0.02 CD4+CD25hi+CD39+; p=0.001) from baseline to 6 wks was observed. Significant decreases in circulating MDSCs, were observed in monocytic HLA-DR+/low/CD14+ MDSC (p<0.0001). Greater increases in T-regs were associated with improved PFS (p=0.034; HR=0.57). Spontaneous in vivo cross-presentation was observed resulting in Th1CD4+ and CD8+ antigen specific T-cell immunity (gp-100, MART-1, NY-ESO-1 peptides). Significant fold increase (3-10-fold) in CD3+/CD4+/INF-γ+ antigen specific T cells was seen only in pts who were progression free at 6 mos. Baseline serum IL-17 correlates with grade 3 diarrhea (p=0.02). In tumor, Tregs appeared higher at wk 6 in PD group while the opposite in clinical benefit group (p=0.09). In tumor, Ipi induced TIL T-cell activation as evidenced by CD69 in the absence of other in vitro stimulation and induced T cell memory (CD45RO+) and not naïve (CD45RO-). By IHC, there was significant increase in CD8+ TIL after ipilimumab (p=0.02). Conclusions: Neoadjuvant ipi exhibited promising clinical activity and significantly modulated the host effector and suppressor immune response. Functional studies and prediction modeling analyses of biomarker findings are ongoing.

8536 Background: Regional lymph node metastasis from melanoma has a high relapse and mortality risk. Neoadjuvant ipilimumab (ipi) may improve the clinical outcome and through access to tumor before and after ipi may allow new insight into the biologic and immunologic response. METHODS Pts (stage IIIB-C melanoma) were treated with ipi (10 mg/kg IV q3weeks x2doses) as preoperative induction followed by lymphadenectomy (at week ≥ 6) and 2 additional doses of maintenance ipi (3 weeks apart). Tissue samples were obtained at baseline and at definitive surgery and blood (serum/PBMC) collected at baseline, 6 weeks, 3, 6, 9, 12 months and/or progression for immunologic/mechanistic studies comparing pre- and post-treatment tumor and blood features. RESULTS Seventeen pts (14 M, 3 F), age 44-87 have been enrolled since Feb/2010 (14 cutaneous primary and 3 mucosal). Two had stage IIIB (1N2b, 1N2c) and 15 IIIC (N3) melanoma. Seven pts had recurred after receiving adjuvant IFN. Fifty three cycles have been delivered (median 4). Grade 3 (worst) toxicities include diarrhea/colitis (6 pts; 35%), hepatic enzyme elevations (1; 5%), rash (1; 5%), lipase (1; 5%), all manageable. Among 16 evaluable pts (surgery is pending for1), median follow-up is 7.9 months and 13 pts (81%) continue disease free. The probability of 6 and 12 months PFS is 84.4% (95% CI=0.50, 0.95) and 63.3% (95% CI=0.17, 0.88) respectively. There is significant increase in the frequency of circulating T-regulatory cells (CD4+CD25hi+ Foxp3+; p=0.009. CD4+CD25hi+CD39+; p=0.011) from baseline to 6 wks. In parallel there is significant decrease in circulating MDSCs: (1) monocytic: lin1neg/HLA-DRneg/ CD33+/CD11b+; p=0.038, (2) other monocytic: HLA-DR+/low/CD14+; p<0.0001 and less significant for (3) lymphoid: Lin1neg/HLA-DR-/CD33+/CD11b+; p=0.067. Immune monitoring comparing baseline and 6 wk tumor samples is ongoing. CONCLUSIONS Our preliminary results show that neoadjuvant ipilimumab is clinically promising and immunologically appears to significantly modulate host immune responses. Ongoing functional and tissue biomarker studies will seek correlations between systemic findings and the tissue impact of ipilimumab that are uniquely assailable using the neoadjuvant model.