Correlation of Baseline Tumor Burden with Clinical Outcome in Melanoma Patients Treated with Ipilimumab.

INTRODUCTION: Tumor burden is a frequently mentioned parameter; however, a commonly accepted definition is still lacking. METHODS: In this double-center prospective and retrospective study, 76 patients with unresectable stage III or stage IV melanoma treated with ipilimumab were included. We defined the baseline tumor burden (BTB) as the global sum of all metastases' longest diameters before treatment started and correlated the calculated BTB with disease control rate (DCR), progression-free survival (PFS), overall survival (OS), and with the baseline levels of LDH, S100B, and sULPB2. RESULTS: BTB correlated significantly with DCR (p = 0.009), PFS (p = 0.002), OS (p = 0.032), and the occurrence of NRAS mutation (p = 0.006). BTB was also correlated to baseline serum levels of LDH (p = 0.011), S100B (p = 0.027), and SULBP (p < 0.0001). Multivariate analysis revealed that BPB and LDH were independently correlated with PFS and OS. With increasing BTB, disease control was less likely; no patient with a BTB >200 mm achieved disease control. For patients with brain metastasis, no correlation of BTB with DCR (p = 0.251), PFS (p = 0.059), or OS (p = 0.981) was observed. CONCLUSION: Calculated BTB is an independent prognostic factor for patients with metastatic melanoma treated with ipilimumab. Using calculated BTB as a definition of tumor burden may help increase comparability of outcome of therapies in future studies.

Putative Cancer Stem Cell Markers are Frequently Expressed by Melanoma Cells in Vitro and in Situ but are also Present in Benign Differentiated Cells.

BACKGROUND: Currently, there remains an incomplete view of cancer stem cells (CSCs) in solid tumours. METHODS: We studied a panel of putative CSC surface markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) in 40 established melanoma cell lines and four early-passage melanoma strains by flow cytometry. We additionally examined 40 formalin-fixed paraffin-embedded melanoma tissues using immunofluorescence microscopy. This was compared with their expression in healthy skin, normal differentiated melanocytes and fibroblasts. RESULTS: Most of the putative CSC markers were expressed by both melanoma cell lines and tissues. When present, these proteins were expressed by the majority of cells in the population. However, the expression of these markers by cells in healthy skin sections, normal differentiated melanocytes, and fibroblasts revealed that differentiated non-malignant cells also expressed CSC markers indicating that they lack of specificity for CSCs. Culturing cell lines under conditions more characteristic of the tumour microenvironment upregulated CSC marker expressions in a proportion of cell lines, which correlated with improved cell growth and viability. CONCLUSIONS: The testing of melanoma cell lines (n = 40), early-passage cell strains (n = 4), and melanoma tissues (n = 40) showed that several putative CSC markers (ALDH1A1, ABCG2, CD44v7/8, CD44v10, CD133, CD271, and Nestin) are commonly present in a large proportion of melanoma cells in vitro and in situ. Further, we showed that these putative markers lack specificity for CSCs because they are also expressed in differentiated non-malignant cell types (melanocytes, fibroblasts, and skin), which could limit their use as therapeutic targets. These data are consistent with the emerging notion of CSC plasticity and phenotype switching within cancer cell populations.

Early decrease of blood myeloid-derived suppressor cells during checkpoint inhibition is a favorable biomarker in metastatic melanoma.

BACKGROUND: The need for reliable clinical biomarkers to predict which patients with melanoma will benefit from immune checkpoint blockade (ICB) remains unmet. Several different parameters have been considered in the past, including routine differential blood counts, T cell subset distribution patterns and quantification of peripheral myeloid-derived suppressor cells (MDSC), but none has yet achieved sufficient accuracy for clinical utility. METHODS: Here, we investigated potential cellular biomarkers from clinical routine blood counts as well as several myeloid and T cell subsets, using flow cytometry, in two independent cohorts of a total of 141 patients with stage IV M1c melanoma before and during ICB. RESULTS: Elevated baseline frequencies of monocytic MDSCs (M-MDSC) in the blood were confirmed to predict shorter overall survival (OS) (HR 2.086, p=0.030) and progression-free survival (HR 2.425, p=0.001) in the whole patient cohort. However, we identified a subgroup of patients with highly elevated baseline M-MDSC frequencies that fell below a defined cut-off during therapy and found that these patients had a longer OS that was similar to that of patients with low baseline M-MDSC frequencies. Importantly, patients with high M-MDSC frequencies exhibited a skewed baseline distribution of certain other immune cells but these did not influence patient survival, illustrating the paramount utility of MDSC assessment. CONCLUSION: We confirmed that in general, highly elevated frequencies of peripheral M-MDSC are associated with poorer outcomes of ICB in metastatic melanoma. However, one reason for an imperfect correlation between high baseline MDSCs and outcome for individual patients may be the subgroup of patients identified here, with rapidly decreasing M-MDSCs on therapy, in whom the negative effect of high M-MDSC frequencies was lost. These findings might contribute to developing more reliable predictors of late-stage melanoma response to ICB at the individual patient level. A multifactorial model seeking such markers yielded only MDSC behavior and serum lactate dehydrogenase as predictors of treatment outcome.

Dynamics of Melanoma-Associated Epitope-Specific CD8+ T Cells in the Blood Correlate With Clinical Outcome Under PD-1 Blockade.

Immune checkpoint blockade (ICB) is standard-of-care for patients with metastatic melanoma. It may re-invigorate T cells recognizing tumors, and several tumor antigens have been identified as potential targets. However, little is known about the dynamics of tumor antigen-specific T cells in the circulation, which might provide valuable information on ICB responses in a minimally invasive manner. Here, we investigated individual signatures composed of up to 167 different melanoma-associated epitope (MAE)-specific CD8+ T cells in the blood of stage IV melanoma patients before and during anti-PD-1 treatment, using a peptide-loaded multimer-based high-throughput approach. Additionally, checkpoint receptor expression patterns on T cell subsets and frequencies of myeloid-derived suppressor cells and regulatory T cells were quantified by flow cytometry. Regression analysis using the MAE-specific CD8+ T cell populations was applied to identify those that correlated with overall survival (OS). The abundance of MAE-specific CD8+ T cell populations, as well as their dynamics under therapy, varied between patients. Those with a dominant increase of these T cell populations during PD-1 ICB had a longer OS and progression-free survival than those with decreasing or balanced signatures. Patients with a dominantly increased MAE-specific CD8+ T cell signature also exhibited an increase in TIM-3+ and LAG-3+ T cells. From these results, we created a model predicting improved/reduced OS by combining data on dynamics of the three most informative MAE-specific CD8+ T cell populations. Our results provide insights into the dynamics of circulating MAE-specific CD8+ T cell populations during ICB, and should contribute to a better understanding of biomarkers of response and anti-cancer mechanisms.

Chemotherapy after immune checkpoint inhibitor failure in metastatic melanoma: a retrospective multicentre analysis.

INTRODUCTION: Despite remarkably improved outcomes with immune checkpoint inhibition, many patients with metastatic melanoma will eventually require further therapy. Chemotherapy has limited activity when used first-line but can alter the tumour microenvironment and does improve efficacy when used in combination with immunotherapy in lung cancer. Whether chemotherapy after checkpoint inhibitor failure has relevant activity in patients with metastatic melanoma is unknown. METHODS: Patients with metastatic melanoma treated with chemotherapy after progression on immunotherapy with checkpoint inhibitors were identified retrospectively from 24 melanoma centres. Objective response rate (ORR), progression-free survival (PFS), overall survival (OS) and safety were examined. RESULTS: In total, 463 patients were treated between 2007 and 2017. Fifty-six per cent had received PD-1-based therapy before chemotherapy. Chemotherapy regimens included carboplatin + paclitaxel (32%), dacarbazine (25%), temozolomide (15%), taxanes (9%, nab-paclitaxel 4%), fotemustine (6%) and others (13%). Median duration of therapy was 7.9 weeks (0-108). Responses included 0.4% complete response (CR), 12% partial response (PR), 21% stable disease (SD) and 67% progressive disease (PD). Median PFS was 2.6 months (2.2, 3.0), and median PFS in responders was 8.7 months (6.3, 16.3), respectively. Twelve-month PFS was 12% (95% CI 2-15%). In patients who had received anti-PD-1 before chemotherapy, the ORR was 11%, and median PFS was 2.5 months (2.1, 2.8). The highest activity was achieved with single-agent taxanes (N = 40), with ORR 25% and median PFS 3.9 months (2.1, 6.2). Median OS from chemotherapy start was 7.1 months (6.5, 8.0). Subsequent treatment with checkpoint inhibitors achieved a response rate of 16% with a median PFS of 19.1 months (2.0-43.1 months). No unexpected toxicities were observed. CONCLUSION: Chemotherapy has a low response rate and short PFS in patients with metastatic melanoma who have failed checkpoint inhibitor therapy, although activity varied between regimens. Chemotherapy has a limited role in the management of metastatic melanoma.

Early disappearance of tumor antigen-reactive T cells from peripheral blood correlates with superior clinical outcomes in melanoma under anti-PD-1 therapy.

BACKGROUND: Anti-programmed cell death protein 1 (PD-1) antibodies are now routinely administered for metastatic melanoma and for increasing numbers of other cancers, but still only a fraction of patients respond. Better understanding of the modes of action and predictive biomarkers for clinical outcome is urgently required. Cancer rejection is mostly T cell-mediated. We previously showed that the presence of NY-ESO-1-reactive and/or Melan-A-reactive T cells in the blood correlated with prolonged overall survival (OS) of patients with melanoma with a heterogeneous treatment background. Here, we investigated whether such reactive T cells can also be informative for clinical outcomes in metastatic melanoma under PD-1 immune-checkpoint blockade (ICB). METHODS: Peripheral blood T cell stimulation by NY-ESO-1 and Melan-A overlapping peptide libraries was assessed before and during ICB in two independent cohorts of a total of 111 patients with stage IV melanoma. In certain cases, tumor-infiltrating lymphocytes could also be assessed for such responses. These were characterized using intracellular cytokine staining for interferon gamma (IFN-γ), tumor negrosis factor (TNF) and CD107a. Digital pathology analysis was performed to quantify NY-ESO-1 and Melan-A expression by tumors. Endpoints were OS and progression-free survival (PFS). RESULTS: The initial presence in the circulation of NY-ESO-1- or Melan-A-reactive T cells which became no longer detectable during ICB correlated with validated, prolonged PFS (HR:0.1; p>0.0001) and OS (HR:0.2; p=0.021). An evaluation of melanoma tissue from selected cases suggested a correlation between tumor-resident NY-ESO-1- and Melan-A-reactive T cells and disease control, supporting the notion of a therapy-associated sequestration of cells from the periphery to the tumor predominantly in those patients benefitting from ICB. CONCLUSIONS: Our findings suggest a PD-1 blockade-dependent infiltration of melanoma-reactive T cells from the periphery into the tumor and imply that this seminally contributes to effective treatment.

Pretreatment metastatic growth rate determines clinical outcome of advanced melanoma patients treated with anti-PD-1 antibodies: a multicenter cohort study.

BACKGROUND: Checkpoint inhibitors revolutionized the treatment of metastatic melanoma patients. Although tumor burden and lactate dehydrogenase (LDH) are associated with overall survival (OS), the impact of tumor growth kinetics remains elusive and in part contradictory. The aims of this study were to develop a novel simple and rapid method that estimates pretreatment metastatic growth rate (MGR) and to investigate its prognostic impact in melanoma patients treated with antiprogrammed death receptor-1 (PD-1) antibodies. METHODS: MGR was assessed in three independent cohorts of a total of 337 unselected consecutive metastasized stage IIIB-IV melanoma patients (discovery cohort: n=53, confirmation cohort: n=126, independent multicenter validation cohort: n=158). MGR was computed during the pretreatment period before initiation of therapy with anti-PD-1 antibodies nivolumab or pembrolizumab by measuring the increase of the longest diameter of the largest target lesion. Tumor doubling time served as quality control. Kaplan-Meier analysis and univariable as well as multivariable Cox regression were used to examine the prognostic impact of MGR. RESULTS: Pretreatment MGR >3.9 mm/month was associated with impaired OS in the discovery cohort (HR 6.19, 95% CI 2.92 to 13.10, p<0.0001), in the confirmation cohort (HR 3.62, 95% CI 2.19 to 5.98, p<0.0001) and in the independent validation cohort (HR 2.57, 95% CI 1.56 to 4.25, p=0.00023). Prior lines of systemic treatment did not influence the significance of MGR. Importantly, the prognostic impact of MGR was independent of total tumor burden, diameter of the largest metastasis, number of prior lines of systemic treatment, LDH, as well as liver and brain metastasis (discovery and confirmation cohorts: both p<0.0001). Superiority of MGR compared with these variables was confirmed in the independent multicenter validation cohort (HR 2.92, 95% CI 1.62 to 5.26, p=0.00036). CONCLUSIONS: High pretreatment MGR is an independent strong prognostic biomarker associated with unfavorable survival of melanoma patients receiving anti-PD-1 antibodies. Further investigations are warranted to assess the predictive impact of MGR in distinct systemic therapeutic regimens.

Serum S100B and LDH at Baseline and During Therapy Predict the Outcome of Metastatic Melanoma Patients Treated with BRAF Inhibitors.

BACKGROUND: Despite impressive response rates, most patients with advanced melanoma ultimately progress following therapy with B-Raf proto-oncogene (BRAF) inhibitors (BRAFi). Therefore, frequent radiologic assessments are necessary, and reliable serum biomarkers would be beneficial for disease monitoring. OBJECTIVE: This study investigated the ability of lactate dehydrogenase (LDH) and S100 calcium-binding protein B (S100B) to detect response and disease progression during treatment with BRAFi. PATIENTS AND METHODS: Baseline levels of LDH and S100B and repeated measurements during therapy were recorded retrospectively in 191 patients with metastatic melanoma. LDH and S100B levels were compared between distinct time points (baseline, first follow-up visit [FV], best objective response [BR], and progressive disease [PD]). The prognostic ability of the serum biomarkers in relation to disease-specific survival (DSS) was assessed with univariable and multivariable Cox regression analysis. RESULTS: Elevated baseline LDH and S100B correlated with impaired DSS. In contrast with LDH (P = 0.12), S100B levels at FV correlated with response (P = 0.0030). Both markers significantly decreased during the first weeks of BRAFi treatment (LDH, P = 0.00034; S100B, P < 0.0001) and increased between BR and PD (LDH, P = 0.016; S100B, P < 0.0001). Patients with elevated S100B (P = 0.00062) but not with elevated LDH (P = 0.067) at the time point of radiologically confirmed PD showed significantly impaired DSS after PD. Interestingly, DSS after PD differed significantly according to S100B levels determined as early as 8 weeks (median) before PD (P = 0.0024). CONCLUSIONS: LDH and S100B are suitable serum biomarkers during therapy with BRAFi. S100B shows stronger correlation with response and exhibits more accuracy in predicting PD. Close biomarker monitoring with S100B is recommended during treatment with BRAFi to detect PD early.

Prognostic role of gamma-glutamyl transferase in metastatic melanoma patients treated with immune checkpoint inhibitors.

BACKGROUND: Hepatic immune-related adverse events (irAE) including elevated liver function tests (transaminases) occur in 1.4-22.3% of melanoma patients receiving immune checkpoint inhibitors (ICPI) and constitute a potentially serious toxicity that is challenging to treat. In contrast to the liver transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST), only little is known about the frequency and impact of gamma-glutamyl transferase (GGT) elevations. METHODS: GGT determined prior to and during therapy of metastatic melanoma patients treated with ICPI were retrospectively assessed in two independent cohorts (PD-1: n = 218, Ipi + Nivo: n = 148). Overall survival (OS) and best objective response were analyzed according to baseline and immune-related GGT (irGGT) elevations during treatment. RESULTS: In multivariate analysis, OS was reduced in patients with elevated baseline GGT (PD-1 group: hazard ratio [HR] 1.76, p = .0073; Ipi + Nivo group: HR 1.77, p = .032). Immune-related GGT elevation was recorded in 17% (PD-1 group) and 38.5% (Ipi + Nivo group). Of these patients, the majority (81 and 68%, respectively) had normal ALT and AST and showed no clinical signs of hepatotoxicity. Patients who experienced irGGT elevation had superior response (PD-1 group: odds ratio [OR] 3.57, p = .00072; Ipi + Nivo group: OR 1.74, p = .12) and OS (PD-1 group: HR 0.37, p = .0016; Ipi + Nivo group: HR 0.33, p = .00050). CONCLUSIONS: The frequency of hepatic irAE is currently underestimated. The addition of the sensitive enzyme GGT to the laboratory panel before and during therapy with ICPI allows to detect two to three times more patients developing hepatic or hepatobiliary toxicity than known so far. Immune-related GGT elevations correlate with response and favorable survival. Precis for use in the Table of Contents The frequency of hepatotoxicity under immune checkpoint blockade is currently underestimated. We suggest the addition of gamma-glutamyl transferase to the laboratory panel in checkpoint inhibitor patients for the detection of hepatobiliary toxicity.

Serum CD73 is a prognostic factor in patients with metastatic melanoma and is associated with response to anti-PD-1 therapy.

BACKGROUND: Inhibitors of immune checkpoint programmed cell death protein 1 (PD-1) receptor on T cells have shown remarkable clinical outcomes in metastatic melanoma. However, most patients are resistant to therapy. Production of extracellular adenosine, via CD73-mediated catabolism of AMP, contributes to suppress T-cell-mediated responses against cancer. In this study, we analyzed the expression and activity of soluble CD73 in sera of patients with melanoma undergoing anti-PD-1± cytotoxic T-lymphocyte-associated antigen 4 therapy. METHODS: Soluble CD73 expression and activity were retrospectively analyzed in serum of a total of 546 patients with melanoma from different centers before starting treatment (baseline) with anti-PD-1 agents, nivolumab or pembrolizumab, and compared with those of 96 healthy subjects. The CD73 activity was correlated with therapy response and survival of patients. RESULTS: Patients with melanoma show significantly higher CD73 activity and expression than those observed in healthy donors (p<0.0001). Elevated pretreatment levels of CD73 activity were associated with non-response to therapy with nivolumab or pembrolizumab. During treatment, levels of soluble CD73 activity remain unchanged from baseline and still stratify clinical responders from non-responders. High levels of serum CD73 enzymatic activity associate with reduced overall survival (OS; HR=1.36, 95% CI 1.03 to 1.78; p=0.03) as well as progression-free survival (PFS; HR=1.42, 95% CI 1.13 to 1.79, p=0.003). Further, the multivariate Cox regression analysis indicates that serum CD73 activity is an independent prognostic factor besides serum lactate dehydrogenase levels and the presence of brain metastases for both OS (p=0.009) and PFS (p=0.001). CONCLUSION: Our data indicate the relevance of serum CD73 in patients with advanced melanoma receiving anti-PD-1 therapy and support further investigation on targeting CD73 in combination with anti-PD-1 antibodies.

Application of Circulating Cell-Free Tumor DNA Profiles for Therapeutic Monitoring and Outcome Prediction in Genetically Heterogeneous Metastatic Melanoma.

PURPOSE: Circulating cell-free tumor DNA (ctDNA) reflects the heterogeneous spectrum of tumor-specific mutations, especially in systemic disease. We validated plasma-based assays that allow the dynamic quantitative detection of ctDNA as a prognostic biomarker for tumor load and prediction of therapy response in melanoma. MATERIALS AND METHODS: We analyzed plasma-derived ctDNA from a large training cohort (n = 96) of patients with advanced-stage melanoma, with assays for the BRAF V600E and NRAS Q61 driver mutations as well as TERT C250T and TERT C228T promoter mutations. An independent patient cohort (n = 35) was used to validate the utility of ctDNA monitoring under mitogen-activated protein kinase-targeted or immune checkpoint therapies. RESULTS: Elevated plasma ctDNA level at baseline was an independent prognostic factor of disease progression when compared with serum S100 and lactate dehydrogenase levels in multivariable analyses (hazard ratio [HR], 7.43; 95% CI, 1.01 to 55.19; P = .05). The change in ctDNA levels during therapy correlated with treatment response, where increasing ctDNA was predictive for shorter progression-free survival (eg, for BRAF V600E ctDNA, HR, 3.70; 95% CI, 1.86 to 7.34; P < .001). Increasing ctDNA levels predicted disease progression significantly earlier than did routine radiologic scans (P < .05), with a mean lead time of 3.5 months. NRAS-mutant ctDNA was detected in a significant proportion of patients with BRAF-mutant tumors under therapy, but unexpectedly also at baseline. In vitro sensitivity studies suggested that this represents higher-than-expected intratumoral heterogeneity. The detection of NRAS Q61 ctDNA in baseline samples of patients with BRAF V600E mutation who were treated with mitogen-activated protein kinase inhibitors significantly correlated with shorter progression-free survival (HR, 3.18; 95% CI, 1.31 to 7.68; P = .03) and shorter overall survival (HR, 4.08; 95% CI, 1.57 to 10.58; P = .01). CONCLUSION: Our results show the potential role of ctDNA measurement as a sensitive monitoring and prediction tool for the early assessment of disease progression and therapeutic response in patients with metastatic melanoma.

Is there a link between very early changes of primary and secondary lymphoid organs in 18F-FDG-PET/MRI and treatment response to checkpoint inhibitor therapy?

Response assessment or prediction to checkpoint inhibitor therapy (CIT) is an unsolved problem in current routine diagnostics of patients with melanoma. Here, we evaluated very early changes of primary and secondary lymphoid organs under CIT in multiparametric [18F]-labeled fluorodeoxyglucose-positron emission tomography (18F-FDG-PET)/MRI as possible predictors of treatment response and investigated their correlation with baseline blood immune biomarkers. Between October 2014 and November 2017, 17 patients with unresectable melanoma (8 females; 65±11 years) undergoing CIT were prospectively evaluated using whole-body 18F-FDG-PET/MRI before CIT start (t0), 2 weeks (t1) and 3 months after CIT initiation (t2). At each time point, the volume, the 18F-FDG-uptake and the mean apparent diffusion coefficient (ADC) of the spleen as well as the 18F-FDG uptake of the bone marrow were assessed. Relative lymphocyte count (RLC), relative eosinophil count (REC) and neutrophil-lymphocyte ratio (NLR) were assessed at baseline. Response Evaluation Criteria in Solid Tumours modified for immune-based therapeutics (iRECIST) and decisions from an interdisciplinary tumor board were used for treatment response evaluation at t2 iRECIST was compared with PET response criteria in solid tumors for image-based response evaluation at different time points. Comparative analysis was conducted with Mann-Whitney U test with false discovery rate correction for multiple testing and correlation coefficients were computed. In lymphoid organs, significant differences (p<0.05) between responders (9/17) and non-responders were found for the 18F-FDG-uptake in the spleen at t1 and the increase of the uptake t1-t0 (responders/non-responders: standardized uptake value lean body mass 1.19/0.93; +49%/-1%). The best correlation coefficients to baseline biomarkers were found for the 18F-FDG-uptake in the spleen at t1: NLR, r=-0.46; RLC, r=0.43; REC, r=0.58 (p<0.05), respectively. Compared with the non-responder group, the responder group showed marked increases also in the volume of the spleen (+22%/+10%), the 18F-FDG-uptake of bone marrow (+31%/-9%) at t1 and the ADCmean at t2 (+46%/+15%) compared with t0, however, not reaching significance. Our findings indicate that an effective systemic immune response in patients undergoing CIT can be detected as a significantly increased spleen activity in 18F-FDG-PET as early as 2 weeks after treatment initiation. TRIAL REGISTRATION NUMBER: NCT03132090, DRKS00013925.

Dermatofluoroscopy diagnostics in different pigmented skin lesions: Strengths and weaknesses.

BACKGROUND: The melanin fluorescence of skin lesions is measurable with two-photon excitation, a process termed dermatofluoroscopy, which has shown a shift from the green spectra in benign melanocytic lesions to the red spectra in melanoma. This study addressed the question as to which kind of pigmented lesions can be correctly diagnosed as melanin-bearing malignant tumors. METHODS: 476 pigmented lesions including 101 cutaneous melanomas were analyzed with dermatofluoroscopy, measuring the melanin fluorescence in a grid-like fashion with a separation of measurement points of 0.2 mm. The results of the dermatofluoroscopy are presented as a diagnostic score with a cut-off score of ≥ 28 for the diagnosis of melanin-bearing malignant tumors, and were compared to the gold standard of histopathology. RESULTS: A highly significant difference (p < 0.0001) between the diagnostic scores of different skin tumors was found. Dermatofluoroscopy scores showed the highest sensitivity for melanomas (92.1 %). Interestingly, most pigmented basal cell carcinomas (BCCs, 88.9 %) were diagnosed as melanin-bearing malignant tumors. A higher sensitivity for the correct diagnosis was observed in older patients (≥ 53 years, p = 0.003), in patients with skin tanning (p = 0.025), and in patients with freckles during childhood (p = 0.046). CONCLUSIONS: Two-photon fluorescence is an innovative technique for the diagnosis of pigmented skin lesions, and shows a high sensitivity for detection of melanomas and pigmented BCCs.

Immune Signatures and Survival of Patients With Metastatic Melanoma, Renal Cancer, and Breast Cancer.

Despite remarkable recent progress in treating solid cancers, especially the success of immunomodulatory antibody therapies for numerous different cancer types, it remains the case that many patients fail to respond to treatment. It is therefore of immense importance to identify biomarkers predicting clinical responses to treatment and patient survival, which would not only assist in targeting treatments to patients most likely to benefit, but might also provide mechanistic insights into the reasons for success or failure of the therapy. Several peripheral blood or tumor tissue diagnostic and predictive biomarkers known to be informative for cancer patient survival may be applicable for this purpose. The use of peripheral blood ("liquid biopsy") offers numerous advantages not only for predicting treatment responses at baseline but also for monitoring patients on-therapy. Assessment of the tumor microenvironment and infiltrating immune cells also delivers important information on cancer-host interactions but the requirement for tumor tissues makes this more challenging, especially for monitoring sequential changes in the individual patient. In this contribution, we will review our findings on immune signatures potentially informative for clinical outcome in melanoma, breast cancer and renal cell carcinoma, particularly the outcome of checkpoint blockade, by applying multiparametric flow cytometry and mass cytometry, routine clinical monitoring and functional testing for predicting and following individual patient responses to therapy.

Author Correction: Integrative molecular and clinical modeling of clinical outcomes to PD1 blockade in patients with metastatic melanoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Impact of a preceding radiotherapy on the outcome of immune checkpoint inhibition in metastatic melanoma: a multicenter retrospective cohort study of the DeCOG.

BACKGROUND: Immune checkpoint inhibition (ICI) is an essential treatment option in melanoma. Its outcome may be improved by a preceding radiation of metastases. This study aimed to investigate the impact of a preceding radiotherapy on the clinical outcome of ICI treatment. METHODS: This multicenter retrospective cohort study included patients who received anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or anti-programmed cell death protein 1 (PD-1) ICI with or without preceding radiotherapy for unresectable metastatic melanoma. ICI therapy outcome was measured as best overall response (BOR), progression-free (PFS) and overall survival (OS). Response and survival analyses were adjusted for confounders identified by directed acyclic graphs. Adjusted survival curves were calculated using inverse probability treatment weighting. RESULTS: 835 patients who received ICI (anti-CTLA-4, n=596; anti-PD-1, n=239) at 16 centers were analyzed, whereof 235 received a preceding radiotherapy of metastatic lesions in stage IV disease. The most frequent organ sites irradiated prior to ICI therapy were brain (51.1%), lymph nodes (17.9%) and bone (17.9%). After multivariable adjustment for confounders, no relevant differences in ICI therapy outcome were observed between cohorts with and without preceding radiotherapy. BOR was 8.7% vs 13.0% for anti-CTLA-4 (adjusted relative risk (RR)=1.47; 95% CI=0.81 to 2.65; p=0.20), and 16.5% vs 25.3% for anti-PD-1 (RR=0.93; 95% CI=0.49 to 1.77; p=0.82). Survival probabilities were similar for cohorts with and without preceding radiotherapy, for anti-CTLA-4 (PFS, adjusted HR=1.02, 95% CI=0.86 to 1.25, p=0.74; OS, HR=1.08, 95% CI=0.81 to 1.44, p=0.61) and for anti-PD-1 (PFS, HR=0.84, 95% CI=0.57 to 1.26, p=0.41; OS, HR=0.73, 95% CI=0.43 to 1.25, p=0.26). Patients who received radiation last before ICI (n=137) revealed no better survival than those who had one or more treatment lines between radiation and start of ICI (n=86). In 223 patients with brain metastases, we found no relevant survival differences on ICI with and without preceding radiotherapy. CONCLUSIONS: This study detected no evidence for a relevant favorable impact of a preceding radiotherapy on anti-CTLA-4 or anti-PD-1 ICI treatment outcome in metastatic melanoma.

Pitfalls in the characterization of circulating and tissue-resident human γδ T cells.

Dissection of the role and function of human γδ T cells and their heterogeneous subsets in cancer, inflammation, and auto-immune diseases is a growing and dynamic research field of increasing interest to the scientific community. Therefore, harmonization and standardization of techniques for the characterization of peripheral and tissue-resident γδ T cells is crucial to facilitate comparability between published and emerging research. The application of commercially available reagents to classify γδ T cells, in particular the combination of multiple Abs, is not always trouble-free, posing major demands on researchers entering this field. Occasionally, even entire γδ T cell subsets may remain undetected when certain Abs are combined in flow cytometric analysis with multicolor Ab panels, or might be lost during cell isolation procedures. Here, based on the recent literature and our own experience, we provide an overview of methods commonly employed for the phenotypic and functional characterization of human γδ T cells including advanced polychromatic flow cytometry, mass cytometry, immunohistochemistry, and magnetic cell isolation. We highlight potential pitfalls and discuss how to circumvent these obstacles.

Cancer immunotherapy is accompanied by distinct metabolic patterns in primary and secondary lymphoid organs observed by non-invasive in vivo18F-FDG-PET.

Purpose: Cancer immunotherapy depends on a systemic immune response, but the basic underlying mechanisms are still largely unknown. Despite the very successful and widespread use of checkpoint inhibitors in the clinic, the majority of cancer patients do not benefit from this type of treatment. In this translational study, we investigated whether noninvasive in vivo positron emission tomography (PET) imaging using 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) is capable of detecting immunotherapy-associated metabolic changes in the primary and secondary lymphoid organs and whether this detection enables the prediction of a successful anti-cancer immune response. Methods: RIP1-Tag2 mice with progressed endogenous insular cell carcinomas underwent a combined cancer immunotherapy consisting of CD4+ T cells plus monoclonal antibodies (mAbs) against programmed death ligand-1 (PD-L1) and lymphocyte activation gene-3 (LAG-3) or a sham treatment after radiation-mediated immune cell depletion. A second cohort of RIP1-Tag2 mice underwent exclusive checkpoint inhibitor therapy (CIT) using anti-PD-L1/LAG-3 mAbs or sham treatment without initial immune cell depletion to mimic the clinical situation. All mice were monitored by 18F-FDG-PET combined with anatomical magnetic resonance imaging (MRI). In addition, we retrospectively analyzed PET / computed tomography (CT) scans (PET/CT) regarding 18F-FDG uptake of CIT-treated metastatic melanoma patients in the spleen (n=23) and bone marrow (BM; n=20) as well as blood parameters (n=17-21). Results: RIP1-Tag2 mice with advanced insular cell carcinomas treated with combination immunotherapy exhibited significantly increased 18F-FDG uptake in the spleen compared to sham-treated mice. Histopathology of the spleens from treated mice revealed atrophy of the white pulp with fewer germinal centers and an expanded red pulp with hyperplasia of neutrophils than those of sham-treated mice. Immunohistochemistry and flow cytometry analyses of the spleens revealed a lower number of T cells and a higher number of neutrophils compared to those in the spleens of sham-treated mice. Flow cytometry of the BM showed enhanced activation of T cells following the treatment schemes that included checkpoint inhibitors. The ratio of 18F-FDG uptake at baseline to the uptake at follow-up in the spleens of exclusively CIT-treated RIP1-Tag2 mice was significantly enhanced, but the ratio was not enhanced in the spleens of the sham-treated littermates. Flow cytometry analysis confirmed a reduced number of T cells in the spleens of exclusively CIT-treated mice compared to that of sham-treated mice. A retrospective analysis of clinical 18F-FDG-PET/CT scans revealed enhanced 18F-FDG uptake in the spleens of some successfully CIT-treated patients with metastatic melanoma, but there were no significant differences between responders and non-responders. The analysis of the BM in clinical 18F-FDG-PET/CT scans with a computational segmentation tool revealed significantly higher baseline 18F-FDG uptake in patients who responded to CIT than in non-responders, and this relationship was independent of bone metastasis, even in the baseline scan. Conclusions: Thus, we are presenting the first translational study of solid tumors focusing on the metabolic patterns of primary and secondary lymphoid organs induced by the systemic immune response after CIT. We demonstrate that the widely available 18F-FDG-PET modality is an applicable translational tool that has high potential to stratify patients at an early time point.

Integrative molecular and clinical modeling of clinical outcomes to PD1 blockade in patients with metastatic melanoma.

Immune-checkpoint blockade (ICB) has demonstrated efficacy in many tumor types, but predictors of responsiveness to anti-PD1 ICB are incompletely characterized. In this study, we analyzed a clinically annotated cohort of patients with melanoma (n = 144) treated with anti-PD1 ICB, with whole-exome and whole-transcriptome sequencing of pre-treatment tumors. We found that tumor mutational burden as a predictor of response was confounded by melanoma subtype, whereas multiple novel genomic and transcriptomic features predicted selective response, including features associated with MHC-I and MHC-II antigen presentation. Furthermore, previous anti-CTLA4 ICB exposure was associated with different predictors of response compared to tumors that were naive to ICB, suggesting selective immune effects of previous exposure to anti-CTLA4 ICB. Finally, we developed parsimonious models integrating clinical, genomic and transcriptomic features to predict intrinsic resistance to anti-PD1 ICB in individual tumors, with validation in smaller independent cohorts limited by the availability of comprehensive data. Broadly, we present a framework to discover predictive features and build models of ICB therapeutic response.