Purpose Data suggest that DNA damage by poly (ADP-ribose) polymerase inhibition and/or reduced vascular endothelial growth factor signaling by vascular endothelial growth factor receptor inhibition may complement antitumor activity of immune checkpoint blockade. We hypothesize the programmed death-ligand 1 (PD-L1) inhibitor, durvalumab, olaparib, or cediranib combinations are tolerable and active in recurrent women's cancers. Patients and Methods This phase I study tested durvalumab doublets in parallel 3 + 3 dose escalations. Durvalumab was administered at 10 mg/kg every 2 weeks or 1,500 mg every 4 weeks with either olaparib tablets twice daily or cediranib on two schedules. The primary end point was the recommended phase II dose (RP2D). Response rate and pharmacokinetic analysis were secondary end points. Results Between June 2015 and May 2016, 26 women were enrolled. The RP2D was durvalumab 1,500 mg every 4 weeks with olaparib 300 mg twice a day, or cediranib 20 mg, 5 days on/2 days off. No dose-limiting toxicity was recorded with durvalumab plus olaparib. The cediranib intermittent schedule (n = 6) was examined because of recurrent grade 2 and non-dose-limiting toxicity grade 3 and 4 adverse events (AEs) on the daily schedule (n = 8). Treatment-emergent AEs included hypertension (two of eight), diarrhea (two of eight), pulmonary embolism (two of eight), pulmonary hypertension (one of eight), and lymphopenia (one of eight). Durvalumab plus intermittent cediranib grade 3 and 4 AEs were hypertension (one of six) and fatigue (one of six). Exposure to durvalumab increased cediranib area under the curve and maximum plasma concentration on the daily, but not intermittent, schedules. Two partial responses (≥15 months and ≥ 11 months) and eight stable diseases ≥ 4 months (median, 8 months [4 to 14.5 months]) were seen in patients who received durvalumab plus olaparib, yielding an 83% disease control rate. Six partial responses (≥ 5 to ≥ 8 months) and three stable diseases ≥ 4 months (4 to ≥ 8 months) were seen in 12 evaluable patients who received durvalumab plus cediranib, for a 50% response rate and a 75% disease control rate. Response to therapy was independent of PD-L1 expression. Conclusion To our knowledge, this is the first reported anti-PD-L1 plus olaparib or cediranib combination therapy. The RP2Ds of durvalumab plus olaparib and durvalumab plus intermittent cediranib are tolerable and active. Phase II studies with biomarker evaluation are ongoing.

BackgroundWe recently reported a 56% objective response rate in patients with advanced Merkel cell carcinoma (MCC) receiving pembrolizumab. However, a biomarker predicting clinical response was not identified.MethodsPretreatment FFPE tumor specimens (n = 26) were stained for CD8, PD-L1, and PD-1 by immunohistochemistry/immunofluorescence (IHC/IF), and the density and distribution of positive cells was quantified to determine the associations with anti-PD-1 response. Multiplex IF was used to test a separate cohort of MCC archival specimens (n = 16), to identify cell types expressing PD-1.ResultsTumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ and PD-L1+ cells when compared to non-responders (median cells/mm2, 70.7 vs. 6.7, p = 0.03; and 855.4 vs. 245.0, p = 0.02, respectively). There was no significant association of CD8+ cell density with clinical response. Quantification of PD-1+ cells located within 20 μm of a PD-L1+ cell showed that PD-1/PD-L1 proximity was associated with clinical response (p = 0.03), but CD8/PD-L1 proximity was not. CD4+ and CD8+ cells in the TME expressed similar amounts of PD-1.ConclusionsWhile the binomial presence or absence of PD-L1 expression in the TME was not sufficient to predict response to anti-PD-1 in patients with MCC, we show that quantitative assessments of PD-1+ and PD-L1+ cell densities as well as the geographic interactions between these two cell populations correlate with clinical response. Cell types expressing PD-1 in the TME include CD8+ T-cells, CD4+ T-cells, Tregs, and CD20+ B-cells, supporting the notion that multiple cell types may potentiate tumor regression following PD-1 blockade.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0404-0) contains supplementary material, which is available to authorized users.

PD-L1 expression in the tumor immune microenvironment is recognized as both a prognostic and predictive biomarker in patients with cutaneous melanoma, a finding closely related to its adaptive (IFN-γ-mediated) mechanism of expression. Approximately 35% of cutaneous melanomas express PD-L1, however, the expression patterns, levels, and prevalence in rarer melanoma subtypes are not well described. We performed immunohistochemistry for PD-L1 and CD8 on 200 formalin-fixed paraffin-embedded specimens from patients with acral (n = 16), mucosal (n = 36), uveal (n = 103), and chronic sun-damaged (CSD) (n = 45) melanomas (24 lentigo maligna, 13 ‘mixed’ desmoplastic, and 8 ‘pure’ desmoplastic melanomas). CD8+ tumor-infiltrating lymphocyte (TIL) densities were characterized as mild, moderate, or severe, and their geographic association with PD-L1 expression was evaluated. Discrete lymphoid aggregates, the presence of a spindle cell morphology, and the relationship of these features with PD-L1 expression were assessed. PD-L1 expression was observed in 31% of acral melanomas, 44% of mucosal melanomas, 10% of uveal melanomas, and 62% of CSD melanomas (P<0.0001). Compared to our previously characterized cohort of cutaneous melanomas, the proportion of PD-L1(+) tumors was lower in uveal (P = 0.0002) and higher in CSD (P = 0.0073) melanomas, while PD-L1 expression in the acral and mucosal subtypes was on par. PD-L1 expression in all subtypes correlated with a moderate–severe grade of CD8+ TIL (all, P<0.003), supporting an adaptive mechanism of expression induced during the host antitumor response. The tumor microenvironments observed in CSD melanomas segregated by whether they were the pure desmoplastic subtype, which showed lower levels of PD-L1 expression when compared to other CSD melanomas (P = 0.047). The presence of lymphoid aggregates was not associated with the level of PD-L1 expression, while PD-L1(+) cases with spindle cell morphology demonstrated higher levels of PD-L1 than those with a nested phenotype (P<0.0001). Our findings may underpin the reported clinical response rates for anti-PD-1 monotherapy, which vary by subtype.

Monoclonal antibodies that block immune regulatory proteins such as programmed death-1 (PD-1) have demonstrated remarkable efficacy in controlling the growth of multiple tumor types. Unresectable or metastatic basal cell carcinoma, however, has largely gone untested. Because PD-Ligand-1 (PD-L1) expression in other tumor types has been associated with response to anti-PD-1, we investigated the expression of PD-L1 and its association with PD-1 expression in the basal cell carcinoma tumor microenvironment. Among 40 basal cell carcinoma specimens, 9/40 (22%) demonstrated PD-L1 expression on tumor cells, and 33/40 (82%) demonstrated PD-L1 expression on tumor-infiltrating lymphocytes and associated macrophages. PD-L1 was observed in close geographic association to PD-1+ tumor infiltrating lymphocytes. Additionally, we present, here, the first report of an objective anti-tumor response to pembrolizumab (anti-PD-1) in a patient with metastatic PD-L1 (+) basal cell carcinoma, whose disease had previously progressed through hedgehog pathway-directed therapy. The patient remains in a partial response 14 months after initiation of therapy. Taken together, our findings provide a rationale for testing anti-PD-1 therapy in patients with advanced basal cell carcinoma, either as initial treatment or after acquired resistance to hedgehog pathway inhibition.

Next-generation tissue-based biomarkers for immunotherapy will likely include the simultaneous analysis of multiple cell types and their spatial interactions, as well as distinct expression patterns of immunoregulatory molecules. Here, we introduce a comprehensive platform for multispectral imaging and mapping of multiple parameters in tumor tissue sections with high-fidelity single-cell resolution. Image analysis and data handling components were drawn from the field of astronomy. Using this “AstroPath” whole-slide platform and only six markers, we identified key features in pretreatment melanoma specimens that predicted response to anti–programmed cell death-1 (PD-1)–based therapy, including CD163+PD-L1– myeloid cells and CD8+FoxP3+PD-1low/mid T cells. These features were combined to stratify long-term survival after anti–PD-1 blockade. This signature was validated in an independent cohort of patients with melanoma from a different institution.

Purpose: PD-L1 expression in the pre-treatment tumor microenvironment enriches for response to anti-PD-1/PD-L1 therapies. The purpose of this study was to quantitatively compare the performance of five monoclonal anti-PD-L1 antibodies used in recent landmark publications. Experimental Design: PD-L1 immunohistochemistry (IHC) was performed on thirty-four formalin-fixed paraffin-embedded archival melanoma samples using the 5H1, SP142, 28-8, 22C3 and SP263 clones. The percentage of total cells (including melanocytes and immune cells) demonstrating cell surface PD-L1 staining, as well as intensity measurements/H-scores, were assessed for each melanoma specimen using a computer-assisted platform. Staining properties were compared between antibodies. Results: Strong correlations were observed between the percentage of PD-L1(+) cells across all clones studied (R2=0.81–0.96). When present, discordant results were attributable to geographic heterogeneity of the melanoma tissue section rather than differences in PD-L1 antibody staining characteristics. PD-L1 intensity/H-scores strongly correlated with percentage of PD-L1(+) cells (R2>0.78, all clones). Conclusions: The 5H1, SP142, 28-8, 22C3 and SP263 clones all demonstrated similar performance characteristics when used in a standardized IHC assay on melanoma specimens. Reported differences in PD-L1 IHC assays using these antibodies are thus most likely due to assay characteristics beyond the antibody itself. Our findings also argue against the inclusion of an intensity/H-score in chromogenic PD-L1 IHC assays.

BackgroundThis study evaluated the expression of PD-L1 and markers of immune mediated resistance in human medulloblastoma (MB), the most common malignant pediatric brain tumor.ResultsOverall levels of PD-L1 in human MB were low; however, some cases demonstrated robust focal expression associated with increased immune infiltrates. The case with highest PD-L1 expression was a sonic hedgehog (SHH) MB. In cell lines, SHH MB, which are low-MYC expressing, demonstrated both constitutive and inducible expression of PD-L1 while those in Group 3/4 that expressed high levels of MYC had only inducible expression. In vitro, IFN-γ robustly stimulated the expression of PD-L1 in all cell lines while radiation induced variable expression. Forced high MYC expression did not significantly alter PD-L1.MethodsHuman MB tumor samples were evaluated for expression of PD-L1 and immune cell markers in relation to molecular subgroup assignment. PD-L1 expression was functionally analyzed under conditions of interferon gamma (IFN-γ), radiation, and MYC overexpression.ConclusionsMB expresses low levels of PD-L1 facilitating immune escape. Importantly, TH1 cytokine stimulation appears to be the most potent inducer of PD-L1 expression in vitro suggesting that an inflamed tumor microenvironment is necessary for PD-1 pathway activation in this tumor.

Importance The programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) pathway play an important immunosuppressive role in cancer and chronic viral infection, and have been effectively targeted in cancer therapy. Anal squamous cell carcinoma (SCC) is associated with both human papillomavirus and HIV infection. To date, patients with HIV have been excluded from most trials of immune checkpoint blocking agents, such as anti-PD-1 and anti-PD-L1, because it was assumed that their antitumor immunity was compromised compared with immunocompetent patients. Objective To compare the local tumor immune microenvironment (TME) in anal SCCs from HIV-positive and HIV-negative patients. Design, Setting, and Participants Anal SCC tumor specimens derived from the AIDS and Cancer Specimen Resource (National Cancer Institute) and Johns Hopkins Hospital included specimens. Tumors were subjected to immunohistochemical analysis for immune checkpoints (PD-L1, PD-1, LAG-3) and immune cell (IC) subsets (CD3, CD4, CD8, CD68). Expression profiling for immune-related genes was performed on select HIV-positive and HIV-negative cases in PD-L1+ tumor areas associated with ICs. Main Outcomes and Measures Programmed death-ligand 1 expression on tumor cells and ICs, PD-L1 patterns (adaptive vs constitutive), degree of IC infiltration, quantified densities of IC subsets, and gene expression profiles in anal SCCs from HIV-positive vs HIV-negative patients. Results Approximately half of 40 tumor specimens from 23 HIV-positive and 17 HIV-negative patients (29 men and 11 women; mean [SD] age, 51 [9.9] years) demonstrated tumor cell PD-L1 expression, regardless of HIV status. Median IC densities were not significantly decreased in HIV-associated tumors for any cellular subset studied. Both adaptive (IC-associated) and constitutive PD-L1 expression patterns were observed. Immune cell PD-L1 expression correlated with increasing intensity of IC infiltration (r = 0.52; 95% CI, 0.26-0.78; P < .001) and with CD8+ T-cell density (r = 0.35; 95% CI, 0.11-0.59; P = .03). Gene expression profiling revealed comparable levels of IFNG in the TME of both HIV-positive and HIV-negative patients. A significant increase in IL18 expression levels was observed in HIV-associated anal SCCs (fold change, 12.69; P < .001). Conclusions and Relevance HIV status does not correlate with the degree or composition of IC infiltration or PD-L1 expression in anal SCC. These findings demonstrate an immune-reactive TME in anal SCCs from HIV-positive patients and support clinical investigations of PD-1/PD-L1 checkpoint blockade in anal SCC, irrespective of patient HIV status.