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Background Neoadjuvant immunotherapy utilizing novel combinations has the potential to transform the standard of care for locally/regionally advanced melanoma. We hypothesized that neoadjuvant ipilimumab in combination with high dose IFNα2b (HDI) is safe and associated with durable pathologic complete responses (pCR). Methods Patients with locally/regionally advanced melanoma were randomized to ipilimumab 3 or 10 mg/kg × 4 doses bracketing definitive surgery, then every 12 weeks × 4. HDI was given concurrently. We evaluated the safety and efficacy of the combination with ipilimumab 3 or 10 mg/kg. The impact on T-cell fraction and clonality were investigated in tumor and blood. Results Thirty patients (age 37–76), 15 each at 3 and 10 mg/kg, 18 male and 12 female were treated. Considering immune related adverse events (irAEs) of interest, more grade 3/4 irAEs were seen with ipilimumab 10 mg/kg versus 3 mg/kg (p = 0.042). Among 28 evaluable patients, 11 relapsed, of whom 5 died. Median follow-up for 17 patients who have not relapsed was 32 months. The radiologic preoperative response rate was 36% (95% CI, 21–54); 4 patients at ipilimumab 3 mg/kg and 6 at 10 mg/kg and 2 (at 10 mg/kg) later relapsed. The pCR was 32% (95% CI, 18–51); 5 patients at ipilimumab 3 mg/kg and 4 at 10 mg/kg and one (at 3 mg/kg) had a late relapse. In patients with pCR, T-cell fraction was significantly higher when measured in primary melanoma tumors (p = 0.033). Higher tumor T-cell clonality in primary tumor and more so following neoadjuvant therapy was significantly associated with improved relapse free survival. Conclusions Neoadjuvant ipilimumab-HDI was relatively safe and exhibited promising tumor response rates with an associated measurable impact on T-cell fraction and clonality. Most pCRs were durable supporting the value of pCR as a primary endpoint in neoadjuvant immunotherapy trials. Trial registration ClinicalTrials.gov, NCT01608594. Registered 31 May 2012. Electronic supplementary material The online version of this article (10.1186/s40425-018-0428-5) contains supplementary material, which is available to authorized users.

The purpose of this study was to learn whether molecular characterization through gene expression profiling of node-positive and node-negative sentinel lymph nodes (SLNs) in patients with clinical stage I and II melanoma may improve the understanding of mechanisms of metastasis and identify gene signatures for SLNs/SLNs that correlate with diagnosis or clinical outcome. Gene expression profiling was performed on SLN biopsies of 48 (24 SLN and 24 SLN) patients (T3a/b-T4a/b) who underwent staging of SLNs using transcriptome profiling analysis on 5 μm sections of fresh SLNs. U133A 2.0 Affymetrix gene chips were used. Significance analysis of microarrays was used to test the association between gene expression level and SLN status. Genes with fold change more than 1.5 and q value less than 0.05 were considered differentially expressed. Pathway analysis was performed using Ingenuity Pathway Analysis. The Benjamini and Hochberg method was used to adjust for multiple testing in pathway analysis. We identified 89 probe sets that were significantly differentially expressed (1.5-27-fold; q0.05). Upon performing the pathway analysis, it was found that 25 genes were common among the most significant and biologically relevant canonical pathways. The molecules and pathways that achieved differential expression of highest statistical significance were notably related to melanoma and its microenvironment and to signaling pathways implicated in immunosuppression and development of cancer. A 25-gene signature is significantly differentially expressed between SLN and SLN and is related to melanoma oncogenesis and immunosuppression. The identified expression profile provides a signature of melanoma nodal involvement. These findings warrant further investigation into the mechanisms of metastasis, melanoma metastasis diagnosis, and prediction of outcome.

Purpose: Patients with regionally advanced melanoma were treated with neoadjuvant ipilimumab in a previously reported study (PLOS One 2014). Gene expression profiles of tumors of treated patients were investigated for their association with immunotherapeutic benefit. Methods: Patients were treated with ipilimumab (10 mg/kg intravenously every 3 weeks × 2 doses) before and after surgery. Tumor specimens were obtained at baseline and at definitive surgery (weeks 6–8). Gene expression profiling was performed on the tumor biopsies of 27 patients. The primary endpoint was mRNA expression profiling using U133A 2.0 Affymetrix gene chips. Significance analysis of microarrays was performed to test the association of each gene with outcome. Pathway analysis was performed using Ingenuity Pathway Analysis software. The Benjamini and Hochberg method was used to adjust for multiple testing in the pathway analysis. Results: Pathway analysis identified biologically relevant pathways enriched with genes that are significantly associated with clinical outcome at baseline in relation to relapse-free survival (RFS) and disease non-progression (as assessed preoperatively at week 6) as well as early on-treatment (RFS and overall survival). The molecules and pathways that achieved differential expression of highest statistical significance were notably immune related. Association of the gene signature with clinical outcome overlapped between baseline and on-treatment specimens and across clinical endpoints tested. Conclusion: Gene expression profiling identified a signature reflecting an immune active and proinflammatory tumor microenvironment that derived clinical benefit from neoadjuvant ipilimumab at baseline and early on-treatment. These findings warrant further investigation in relation to ipilimumab and other immunotherapeutics.

181 Background: Neoadjuvant pembrolizumab at 200 mg in combination with high dose IFNα2b (HDI) for locally/regionally advanced or recurrent melanoma may improve the clinical outcomes of these high risk patients (pts), and provide access to blood and tumor pre/post pembro-HDI to illuminate the host effector and suppressor immune mechanisms. Methods: Pts were treated with pembro 200 mg IV every 3 weeks (wk) x 2 doses followed by definitive surgery, then every 3 wks for up to one year. HDI (20 MU/m²/d IV x 5 days (d)/wk for 4 wks then 10 MU/m²/d SC every other d TIW for 48 wks) was given concurrently. Tumor samples were obtained at baseline and at definitive surgery (wk 6-8) and serum/PBMC at baseline, 6 wks, 3, 6, 12 months (mo). Results: Twenty evaluable pts (14 male, 6 female, 14 cutaneous primary, 4 unknown, 3 mucosal), age 29-82 were treated. 5 had Stage IIIB (N1b, N2b, M2c), 11 IIIC (N3) and 4 IV (M1a, M1b) melanoma. Over 230 cycles have been delivered to date (median 14). Worst toxicities included grade (Gr) 3: fatigue (8; 40%), ↑CPK (5; 25%), hypophosphatemia (5; 25%), ↑lipase (3; 15%), lymphopenia (3; 15%), hypertension (2; 10%), diarrhea/colitis (1; 5%), arthralgia (1, 5%), syncope (1, 5%), hyponatremia (1, 5%), neutropenia (1; 5%), anemia (1, 5.00%) nausea (2, 10%), flu like symptoms (1, 5%). There were 3 Gr 4 events (CPK, hyperglycemia, lymphopenia). One suspected grade 5 event occurred 6 months after completion of therapy with autopsy evidence of pneumonia and myocarditis. Among 20 evaluable pts, 4 relapsed and 1 died. Median follow-up for pts who have not relapsed is 11 months. The radiologic preoperative response rate (WHO; unconfirmed) was 65%. The pathologic complete response rate (no viable tumor on histologic assessment) was 35%. Conclusions: Neoadjuvant pembro-HDI exhibited promising clinical activity. Longer follow up is underway in order to define the long term benefits and risks. Clinical trial information: NCT02339324.

9 Background: Patients with metastatic melanoma were treated with tremelimumab and IFN in a previously reported study (Tarhini. J Clin Oncol. 2012). Clonality of T-cell repertoire was analyzed in terms of clinical response both in TME and in peripheral blood. Methods: Patients received tremelimumab 15 mg/kg I.V. every 12 weeks. High dose IFN (HDI) was administered concurrently. Responses were assessed by RECIST as complete (CR) or partial (PR), stable disease (SD) or progression (PD). T-cell receptor beta chain (TCRB) repertoire was immunosequenced in peripheral blood mononuclear cells (PBMC) (N=33 patients) and tumor (N=18) utilizing Adaptive Biotechnologies immunoSEQ platform to determine repertoire clonality and T-cell fractions at pre-treatment (tumor, PBMC), one month (PBMC), and 3 months (PBMC). The clonality metric quantitates, the extent of mono- or oligo-clonal expansion by measuring the shape of the clone frequency distribution. Values range from 0 to 1; values approaching 1 indicate a nearly monoclonal population. Results: In pretreatment TME, T-cell clonality was significantly (p = 0.0008) different and greater in patients who achieved disease control (CR, PR, SD) versus those with PD. Further, there was a significant (p = 0.044) difference between the increased TCR fraction in TME in responders (CR, PR) and non-responders (SD, PD). There was a trend towards association between pretreatment TME T-cell clonality and overall survival (OS) (p = 0.24) and progression free survival (PFS) (p = 0.18) not reaching significance. Within the circulation (PBMC), no significant associations were found by examining the pretreatment samples. However, early on-treatment (day 29) there was significant association and decrease in T-cell clonality and OS (p = 0.005) and PFS (p = 0.003). Conclusions: T-cell clonality in the TME pretreatment is a promising biomarker of immunotherapeutic benefit in our study. While baseline PBMC clonality was not associated with clinical benefit, early on-treatment (day 29) was significantly associated. These findings require validation in an independent cohort and exploration in relation to other immunotherapeutics. Clinical trial information: NCT00610857.

Drug induced liver injury (DILI) can result either from dose-dependent direct hepatotoxicity or from an unpredictable dose-independent idiosyncratic reaction. Incidence of idiosyncratic DILI is estimated to be approximately 10–15 per 100,000 patient years. Here we report an extremely rare case of metronidazole induced delayed immune-allergic hepatocellular liver injury masquerading as autoimmune hepatitis. A previously healthy 54-year-old Caucasian male, who was treated with metronidazole forClostridium difficileassociated diarrhea, presented 3 months later with right upper quadrant abdominal pain. Laboratory tests revealed total bilirubin level of 12.7 mg/dL, direct bilirubin of 7.2 mg/dL, alanine aminotransferase (ALT) of 973 IU/L, aspartate transaminase (AST) of 867 IU/L, alkaline phosphatase (AP) of 96 IU/L, and an INR of 1.9, suggestive of hepatocellular pattern of injury. A detailed workup for hepatitis revealed no other etiology. A clinical diagnosis of metronidazole induced liver injury was made. With a persistent rise in his bilirubin and transaminase levels, the patient was started on oral prednisone. At the 2-week posthospitalization follow-up visit, the patient reported a significant improvement in his overall sense of being well and liver functions tests trended down substantially (total bilirubin 7.2 mg/dL, ALT 420 IU/L, AST 276 IU/L, AP 183 IU/L, and INR 1.5).

Background Patients with regionally advanced melanoma were treated with neoadjuvant ipilimumab in a previously reported study (Tarhini et al, PLOS One 2014). MicroRNA (miRNA) expression profiles of tumors of treated patients were investigated for their therapeutic predictive value. Methods Patients were treated with ipilimumab (10 mg/kg IV every 3 weeks x2 doses) bracketing surgery. Tumor specimens were obtained at baseline and following ipilimumab at definitive surgery (week 6-8). MiRNA expression profiling was performed on the tumor biopsies of 30 patients using Affymetrix miRNA array (v.4). Significance Analysis of Microarrays (SAM) was performed to test the association of each differentially expressed miRNA molecule with outcome. Targets of the selected miRNAs were obtained from miRTarBase (http://mirtarbase.mbc.nctu.edu.tw). Functional annotation analysis of the list of miRNA target genes were performed using DAVID (https://david.ncifcrf.gov). The FDR method was used to adjust for multiple testing in SAM and the functional analysis. Results An expression profile consisting of a 4-miRNA signature was associated with improved progression free survival. The signature consisted of miR-34c (previously reported to suppress cancer growth and invasion), miR-711 (reported as a prognostic marker in cutaneous T-cell lymphomas and to target and suppress Heat Shock Protein 70 highly expressed in melanoma), miR-641 (activates MAPK by targeting NF1 and cooperates with its host gene AKT2 in human cancer) and miR-22 (reported to function as a tumour suppressor). Functional annotation analysis of target genes for the 4-miRNA signature was statistically significantly enriched for various cancer-related pathways including regulation of cell proliferation (GO:0042147), regulation of apoptosis (GO:0042981), MAPK signaling pathway (hsa04010) and positive regulation of T cell activation (GO:0050870). Conclusions MiRNA expression profiling identified a 4-miRNA signature that is significantly associated with PFS in advanced melanoma patients treated with neoadjuvant ipilimumab. Preliminary results show that targets of these 4 miRNAs are biologically relevant and important. These findings warrant further investigation in relation to ipilimumab and other immunotherapeutics. Citation Format: Ahmad Tarhini, Priyanka Vallabhaneni, Theofanis Floros, William A. LaFramboise, Panayiotis V. Benos, Lucas Santana dos Santos. A tumor and immune related miRNA signature predicts progression-free survival of melanoma patients treated with ipilimumab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 473.

9585Background: Neoadjuvant ipilimumab (ipi) at 3 or 10 mg/kg in combination with high dose IFNα2b (HDI) for locally/regionally advanced or recurrent melanoma may improve the clinical outcomes of t...