BackgroundIpilimumab improves overall survival in a subset of patients with metastatic melanoma. Peripheral blood T cell receptor (TCR) repertoire diversity has been associated with favorable outcomes in patients with cancer, but its relevance as a biomarker for ipilimumab outcomes remains unknown.FindingsIn this pilot study, we analyzed the pre-treatment peripheral blood TCR repertoire in 12 patients with metastatic melanoma who received ipilimumab at 3 mg/kg (clinical benefit, n = 4; no clinical benefit, n = 8). TCR diversity was evaluated using a polymerase chain reaction assay which measures TCR combinatorial diversity between V and J genes from genomic DNA. TCR repertoire diversity was studied through richness (observed V-J rearrangements) and evenness (similarity between the frequencies of specific V-J rearrangements). The Wilcoxon rank sum test was used to compare patients with clinical benefit and those without. Association with benefit in a dichotomized analysis was assessed through a Fisher’s exact test. Overall survival was studied through log-rank analysis.There was a significant difference in richness (p = 0.033) and evenness (p = 0.028) between patients with and without clinical benefit. Dichotomized analysis showed that none of the patients with low richness (n = 0/5, p = 0.081) nor low evenness (n = 0/7, p = 0.01) achieved clinical benefit. There were no significant differences in overall survival.ConclusionsIn this small group of patients, baseline TCR diversity in the peripheral blood was associated with clinical outcomes. Further investigation is ongoing in larger cohorts of patients to explore these preliminary findings and determine whether TCR diversity can be used as a predictive biomarker in cancer immunotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-015-0070-4) contains supplementary material, which is available to authorized users.

The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase III␣, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase III␣ but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase III␣-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADPribose) polymer.Three human genes, LIG1, LIG3, and LIG4, that encode DNA ligases have been identified (30). Unlike the LIG1 and LIG4 genes, which appear to be conserved among all eukaryotes, the LIG3 gene has been found only in the genomes of mammals and of the amphibian Xenopus laevis (6,22,32). Intriguingly, the LIG3 gene is more closely related to poxvirus DNA ligase genes than to those for the other eukaryotic DNA ligases (6, 10). Furthermore, the LIG3 gene is more complex than the other LIG genes in that it encodes multiple products that appear to have distinct biological functions.Alternative splicing of the LIG3 gene transcript generates two species of mRNA, designated ␣ and ␤, that encode polypeptides with different C termini (17,22). DNA ligase III␣ mRNA is ubiquitously expressed, whereas DNA ligase III␤ mRNA has been detected only in germ cells (17,22). The unique C terminus of DNA ligase III␣, which exhibits homology with the BRCT motif initially identified in the product of the breast cancer susceptibility gene BRCA1 (5, 11), mediates formation of a stable complex with the DNA repair protein XRCC1 (3,4,17,21,29). In contrast, no protein partner or biochemical activity has been ascribed to the unique C terminus of DNA ligase III␤. Further heterogeneity of products from the LIG3 gene is generated by translation initiation at different ATG codons within DNA ligase III mRNA, generating mitochondrial and nuclear forms of DNA ligase III (14,15,22).A unique feature of the DNA ligases encoded by the LIG3 gene is the zinc finger motif situated at the N termini of these polypeptides (32). Interestingly, this motif is closely related to the two tandem-arrayed zinc fingers that constitute the DNA binding domain of poly(ADP-ribose) polymerase 1 (PARP-1), a nuclea...

BackgroundWhile immune checkpoint blockade has greatly improved clinical outcomes in diseases such as melanoma, there remains a need for predictive biomarkers to determine who will likely benefit most from which therapy. To date, most biomarkers of response have been identified in the tumors themselves. Biomarkers that could be assessed from peripheral blood would be even more desirable, because of ease of access and reproducibility of sampling.MethodsWe used mass cytometry (CyTOF) to comprehensively profile peripheral blood of melanoma patients, in order to find predictive biomarkers of response to anti-CTLA-4 or anti-PD-1 therapy. Using a panel of ~ 40 surface and intracellular markers, we performed in-depth phenotypic and functional immune profiling to identify potential predictive biomarker candidates.ResultsImmune profiling of baseline peripheral blood samples using CyTOF revealed that anti-CTLA-4 and anti-PD-1 therapies have distinct sets of candidate biomarkers. The distribution of CD4+ and CD8+ memory/non-memory cells and other memory subsets was different between responders and non-responders to anti-CTLA-4 therapy. In anti-PD-1 (but not anti-CTLA-4) treated patients, we discovered differences in CD69 and MIP-1β expressing NK cells between responders and non-responders. Finally, multivariate analysis was used to develop a model for the prediction of response.ConclusionsOur results indicate that anti-CTLA-4 and anti-PD-1 have distinct predictive biomarker candidates. CD4+ and CD8+ memory T cell subsets play an important role in response to anti-CTLA-4, and are potential biomarker candidates. For anti-PD-1 therapy, NK cell subsets (but not memory T cell subsets) correlated with clinical response to therapy. These functionally active NK cell subsets likely play a critical role in the anti-tumor response triggered by anti-PD-1.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0328-8) contains supplementary material, which is available to authorized users.

Purpose To assess the safety and tolerability of pre-operative cryoablation-mediated tumor antigen presentation and/or ipilimumab-mediated immune modulation in women with operable breast cancer. Experimental design In this pilot study, 19 women with breast cancer for whom mastectomy was planned were treated with pre-operative tumor cryoablation (n=7), single-dose ipilimumab at 10mg/kg (n=6), or both (n=6). The primary outcome for this pilot study was safety/tolerability as defined as freedom from delays in pre-planned, curative-intent mastectomy. Exploratory studies of immune activation were performed on peripheral blood and tumor. Results Pre-operative cryoablation and/or ipilimumab were safe and tolerable, with no delays in pre-planned surgery. Grade III toxicity was seen in 1/19 (unrelated rash after ipilimumab). Combination therapy was associated with sustained peripheral elevations in: Th1-type cytokines, activated (ICOS+) and proliferating (Ki67+) CD4+ and CD8+ T cells, and post-treatment proliferative T-effector cells relative to T-regulatory cells within tumor. Conclusions Pre-operative cryoablation and single-dose ipilimumab are safe alone or in combination with no surgical delays incurred. Potentially favorable intra-tumoral and systemic immunologic effects were observed with the combination, suggesting the possibility for induced and synergistic anti-tumor immunity with this strategy.

Ipilimumab, an antibody that blocks cytotoxic T lymphocyte antigen 4 (CTLA-4), improves overall survival (OS) for patients with metastatic melanoma. Given its role in angiogenesis and immune evasion, serum vascular endothelial growth factor (VEGF) levels were evaluated for association with clinical benefit in ipilimumab-treated patients. Sera were collected from 176 patients treated at 3 (n=98) or 10 mg/kg (n=68). The VEGF levels before treatment and at induction completion (week 12) were analyzed using the Meso Scale Discovery kit. The association of the levels of VEGF with clinical responses and overall survival (OS) were assessed using the Fisher’s exact and Kaplan-Meier log-rank tests. VEGF as a continuous variable was associated with overall survival (p=0.002). Using 43 pg/ml as the cutoff pre-treatment VEGF value defined by maximally selected log-rank statistics, pretreatment VEGF values correlated with clinical benefit at week 24 (p=0.019; 159 patients evaluable). Pretreatment VEGF ≥ 43 pg/ml was associated with decreased OS (Median OS 6.6 vs. 12.9 months, p=0.006; 7.4 vs. 14.3 months, p=0.037 for 3 mg/kg; 6.2 vs. 10.9 months, p=0.048 for 10 mg/kg). There was no correlation between VEGF changes and clinical outcome. Serum VEGF may be a predictive biomarker for ipilimumab treatment, and is worthy of prospective investigation with various forms of immunologic checkpoint blockade.