Immunogenicity in humans of a transdermal multipeptide melanoma vaccine administered with or without a TLR7 agonist.

BACKGROUND: Experimental cancer vaccines are traditionally administered by injection in subcutaneous tissue or muscle, commonly with adjuvants that create chronic inflammatory depots. Injection of melanoma-derived peptides induces T cell responses; however, the depots that form following injection may inhibit optimization of the immune response. In skin, epidermal Langerhans cells (LC) are a dominant source of professional antigen presenting cells. We hypothesized that: (1) applying melanoma-derived peptides topically, in proximity to LC, could be immunogenic and safe, with low vaccine-site toxicity and (2) topical toll-like receptor 7 (TLR7) agonist would increase immunogenicity of the peptide vaccine. METHODS: Twelve melanoma peptides plus a tetanus helper peptide were combined with granulocyte macrophage colony stimulating factor (GM-CSF) and were administered topically on days 1, 8, and 15, to 28 patients randomized to one of four adjuvant preparations: (1) incomplete Freund's adjuvant (IFA); (2) IFA plus a TLR7 agonist (imiquimod) administered on days 0, 7, 14; (3) dimethyl sulfoxide (DMSO) or (4) DMSO+ imiquimod administered on day 0, 7, 14. Every 3 weeks thereafter (x 6), the peptides were combined with GM-CSF and were injected into the dermis and subcutis in an emulsion with IFA. Toxicities were recorded and immune responses assayed by ELIspot. RESULTS: CD8+ T cell responses to transdermal vaccination in DMSO occurred in 83% of participants in group 3 and 86% in group 4, and responses to vaccination in IFA were observed in 29% of participants in group 1 and 14% in group 2. Overall, 61% of participants had CD4+ T cell immune responses to the tetanus peptide, with large, durable responses in groups 3 and 4. Five of seven participants in group 4 had a severe rash, one that was dose limiting. Ten-year overall survival was 67% and disease-free survival was 44%. CONCLUSIONS: These data provide proof of principle for immunogenicity in humans of transdermal immunization using peptides in DMSO. Further study is warranted into the pharmacokinetics and immunobiology of TLR agonists as vaccine adjuvants during transcutaneous application. Overall survival is high, supporting further investigation of this immunization approach.

Declining Operative Experience for Junior-Level Residents: Is This an Unintended Consequence of Minimally Invasive Surgery?

INTRODUCTION: Our group has previously demonstrated an upward shift from junior to senior resident participation in common general surgery operations, traditionally performed by junior-level residents. The objective of this study was to evaluate if this trend would correct over time. We hypothesized that junior resident case volume would improve. METHODS: A sample of essential laparoscopic and open general surgery procedures (appendectomy, inguinal herniorrhaphy, cholecystectomy, and partial colectomy) was chosen for analysis. The American College of Surgeons National Surgical Quality Improvement Program Participant Use Files were queried for these procedures between 2005 and 2012. Cases were stratified by participating resident post-graduate year with "junior resident" defined as post-graduate year1-3. Logistic regression was performed to determine change in junior resident participation for each type of procedure over time. RESULTS: A total of 185,335 cases were included in the study. For 3 of the operations we considered, the prevalence of laparoscopic surgery increased from 2005-2012 (all p < 0.001). Cholecystectomy was an exception, which showed an unchanged proportion of cases performed laparoscopically across the study period (p = 0.119). Junior resident participation decreased by 4.5%/y (p < 0.001) for laparoscopic procedures and by 6.2%/y (p < 0.001) for open procedures. The proportion of laparoscopic surgeries performed by junior-level residents decreased for appendectomy by 2.6%/y (p < 0.001) and cholecystectomy by 6.1%/y (p < 0.001), whereas it was unchanged for inguinal herniorrhaphy (p = 0.75) and increased for partial colectomy by 3.9%/y (p = 0.003). A decline in junior resident participation was seen for all open surgeries, with appendectomy decreasing by 9.4%/y (p < 0.001), cholecystectomy by 4.1%/y (p < 0.002), inguinal herniorrhaphy by 10%/y (p < 0.001) and partial colectomy by 2.9%/y (p < 0.004). CONCLUSIONS: Along with the proliferation of laparoscopy for common general surgical procedures there has been a concomitant reduction in the participation of junior-level residents. As previously thought, familiarity with laparoscopy has not translated to redistribution of basic operations from senior to junior residents. This trend has significant implications for general surgery resident education.

Human melanomas and ovarian cancers overexpressing mechanical barrier molecule genes lack immune signatures and have increased patient mortality risk.

We have identified eight genes whose expression in human melanoma metastases and ovarian cancers is associated with a lack of Th1 immune signatures. They encode molecules with mechanical barrier function in the skin and other normal tissues and include filaggrin (FLG), tumor-associated calcium signal transducer 2 (TACSTD2), and six desmosomal proteins (DST, DSC3, DSP, PPL, PKP3, and JUP). This association has been validated in an independent series of 114 melanoma metastases. In these, DST expression alone is sufficient to identify melanomas without immune signatures, while FLG and the other six putative barrier molecules are overexpressed in a different subset of melanomas lacking immune signatures. Similar associations have been identified in a set of 186 ovarian cancers. RNA-seq data from 471 melanomas and 307 ovarian cancers in the TCGA database further support these findings and also reveal that overexpression of barrier molecules is strongly associated with early patient mortality for melanoma (p = 0.0002) and for ovarian cancer (p < 0.01). Interestingly, this association persists for FLG for melanoma (p = 0.012) and ovarian cancer (p = 0.006), whereas DST overexpression is negatively associated with CD8+ gene expression, but not with patient survival. Thus, overexpression of FLG or DST identifies two distinct patient populations with low immune cell infiltration in these cancers, but with different prognostic implications for each. These data raise the possibility that molecules with mechanical barrier function in skin and other tissues may be used by cancer cells to protect them from immune cell infiltration and immune-mediated destruction.

T cells in the human metastatic melanoma microenvironment express site-specific homing receptors and retention integrins.

T-cell infiltration into the metastatic melanoma microenvironment (MME) correlates with improved patient survival. However, diffuse infiltration into tumor occurs in only 8% of melanoma metastases. Little is known about mechanisms governing T-cell infiltration into human melanoma metastases or about how those mechanisms may be altered therapeutically. We hypothesized that T cells in the MME would be enriched for chemokine receptors CCR4, CCR5, CXCR3 and homing receptors relevant to the tissue site. Viably cryopreserved single cell suspensions from nineteen melanoma metastases representing three metastatic sites (tumor-infiltrated lymph node, skin and small bowel) were evaluated by multiparameter flow cytometry and compared to benign lymph nodes and peripheral blood mononuclear cells from patients with Stage IIB-IV melanoma. T cells in the melanoma metastases contained large effector memory populations, high proportions of activated, moderately differentiated cells and few regulatory T cells. Site-specific homing was suggested in bowel, with high expression of CCR9. We neither encounter the anticipated enrichment of integrin α4ß7 in bowel, cutaneous leukocyte antigen (CLA) in skin, nor integrin α4ß1 or receptor CXCR3 in metastatic sites. Retention integrins αEß7, α1ß1 and α2ß1 were significantly elevated in metastases. These data suggest limited tissue site-specific homing to human melanoma metastases, but a significant role for retention integrins in maintaining intratumoral T cells. Our findings also raise the possibility that T-cell homing, infiltration, and retention in melanoma metastases may be increased by increasing expression of ligands for CLA, α4ß1 and CXCR3 on intratumoral endothelium.

Activation, dysfunction and retention of T cells in vaccine sites after injection of incomplete Freund's adjuvant, with or without peptide.

We conducted a randomized clinical trial in 45 patients with resected AJCC stage IIB-IV melanoma to characterize cellular and molecular events at sites of immunization with incomplete Freund's adjuvant (IFA) alone, or a melanoma vaccine in IFA. At a primary vaccine site, all patients received a multi-peptide melanoma vaccine in IFA. At a replicate vaccine site, which was biopsied, group 1 received IFA only; group 2 received vaccine in IFA. Lymphocytes isolated from replicate vaccine site microenvironments (VSME) were compared to time-matched peripheral blood mononuclear cells (PBMC) in ELISpot and flow cytometry assays. Compared to PBMC, the VSME had fewer naïve and greater proportions of effector memory CD8(+) T cells (TCD8). The vast majority of TCD8 within the VSME were activated (CD69(+)), with a concentration of antigen-specific (tetramer(pos)) cells in the VSME, particularly in vaccine sites with peptide (group 2). CXCR3(+) lymphocytes were concentrated in the VSME of all patients, suggesting IFA-induced chemokine recruitment. TCD8 expression of retention integrins αEß7 and α1ß1 was elevated in VSME, with the highest levels observed in antigen-specific cells in VSME containing peptide (group 2). TCD8 retained in the VSME of both groups were strikingly dysfunctional, with minimal IFN-γ production in response to peptide stimulation and few tetramer(pos) cells producing IFN-γ. These data suggest that vaccine-induced selective retention and dysfunction of antigen-specific TCD8 within VSME may represent a significant mechanism underlying transient immune responses and low clinical response rates to peptide vaccines administered in IFA.