Previous studies have suggested that elevated neutrophil-to-lymphocyte ratio (NLR) is prognostic for worse outcomes in patients with a variety of solid cancers, including those treated with immune ...checkpoint inhibitors.
This was a retrospective analysis of 97 consecutive patients with stage IV melanoma who were treated with nivolumab. Baseline NLR and derived (d) NLR were calculated and, along with other characteristics, correlated with progression-free survival (PFS) and overall survival (OS) in univariate and multivariate analyses. The best cutoff values for NLR and dNLR were derived using Cutoff Finder software based on an R routine which optimized the significance of the split between Kaplan-Meier survival curves.
In univariate analysis, increasing absolute neutrophil count (ANC), NLR, dNLR and lactate dehydrogenase (LDH) (continuous variables) were all significantly associated with OS. Only NLR (hazard ratio HR = 2.85; 95% CI 1.60-5.08; p < 0.0001) and LDH (HR = 2.51; 95% CI 1.36-4.64; p < 0.0001) maintained a significant association with OS in multivariate analysis. Patients with baseline NLR ≥5 had significantly worse OS and PFS than patients with NLR < 5, as did patients with baseline dNLR ≥3 versus < 3. Optimal cut-off values were ≥ 4.7 for NLR and ≥ 3.8 for dNLR. Using this ≥4.7 cut-off for NLR, the values for OS and PFS were overlapping to the canonical cut-off for values, and dNLR< 3.8 was also associated with better OS and PFS.
Both Neutrophil-to-lymphocyte ratio (NLR) and derived (d) NLR were associated with improved survival when baseline levels were lower than cut-off values. NLR and dNLR are simple, inexpensive and readily available biomarkers that could be used to help predict response to immunotherapy in patients with advanced melanoma.
Immunotherapy is effective in metastatic melanoma (MM) but most studies failed in discovering a biomarker predictive of clinical response. Exosomes (Exo) from melanoma cells are detectable in sera of ...MM patients similarly to those produced by immune cells that control the tumor progression. Here, we investigated by flow-cytometry the levels of Exo from both T-cells and dendritic cells (DCs) in 59 patients with MM treated with IPI and the relative expression of PD-1, CD28 and ICOS as well as CD80 and CD86. We found a significant increment of PD-1 and CD28 expression in patients achieving a clinical response reflected by improvement of both PFS and OS. Furthermore, MM patients receiving IPI who showed extended PFS underwent increased expression of CD80 and CD86 on DC-derived Exo at the end of treatment. These results suggest a possible association of both PD-1 and CD28 up-regulation on immune cell-derived Exo in patients with better clinical response to IPI.
Immunotherapy with checkpoint inhibitors (CPIs) strongly improved the outcome of metastatic melanoma patients. However, not all the patients respond to treatment and identification of prognostic ...biomarkers able to select responding patients is currently of outmost importance. Considering that development of vitiligo-like depigmentation in melanoma patients represents both an adverse event of CPIs and a favorable prognostic factor, we analyzed soluble biomarkers of vitiligo to validate them as early indicators of response to CPIs. Fifty-seven metastatic melanoma patients receiving CPIs were enrolled and divided according to the best overall response to treatment. Patient sera were evaluated at pre-treatment and after 1 and 3 months of therapy. We found that basal CD25 serum levels were higher in stable and responding patients and remained higher during the first 3 months of CPI therapy compared to non-responders. CXCL9 was absent in non-responding patients before therapy beginning. Moreover, an increase of CXCL9 levels was observed at 1 and 3 months of therapy for all patients, although higher CXCL9 amounts were present in stable and responding compared to non-responding patients. Variations in circulating immune cell subsets was also analyzed, revealing a reduced number of regulatory T lymphocytes in responding patients. Altogether, our data indicate that a pre-existing and maintained activation of the immune system could be an indication of response to CPI treatment in melanoma patients.
Nivolumab is an anti-PD1 checkpoint inhibitor active in patients with advanced melanoma and as adjuvant therapy in high-risk metastatic melanoma patients.
In this single-center retrospective ...analysis, we investigated the CD73 enzyme activity in patients with metastatic melanoma stage IV and its correlation with the response to nivolumab. The soluble CD73 (sCD73) enzyme activity was measured in the serum of 37 melanoma patients before receiving nivolumab and the Harrel's C index was used to find the best cut-off for this biomarker. The multivariate Cox proportional hazard model was used to evaluate the prognostic value of CD73 enzyme activity for survival and progression-free survival.
Our results show that high levels of sCD73 enzyme activity were significantly associated with poor overall survival and progression-free survival in patients with metastatic melanoma. The median progression-free survival was 2.6 months 95% confidence interval (CI) 1.9-3.3 in patients with high sCD73 enzyme activity (> 27.8 pmol/min/mg protein), and 14.2 months (95% CI 4.6-23.8) in patients with lower CD73 enzyme activity, when patients were follow-up for a median of 24 months range. The median overall survival was not reached in patients with low sCD73 activity (< 27.8 pmol/min/mg protein) compared with 6.1 months (95% CI 0-14.8) in patients with higher sCD73 activity. In multivariate analyses, the sCD73 enzyme activity emerged as the strongest prognostic factor for overall survival and progression-free survival. Elevated basal levels of sCD73 enzyme activity, before starting nivolumab treatment, were associated with lower response rates to therapy.
We observed a significant association between the activity of sCD73 in the blood and clinical outcomes in patients with metastatic melanoma stage IV, receiving nivolumab. Although our results need to be confirmed and validated, we suggest that sCD73 might be used as serologic prognostic biomarker. Potentially evaluating sCD73 enzyme activity in the peripheral blood before treatment could help to estimate the response to nivolumab.
In the context of precision medicine with immunotherapies there is an increasing need for companion diagnostic tests to identify potential therapy responders and avoid treatment coming along with ...severe adverse events for non-responders. Here, we present a retrospective case study to discover image-based signatures for developing a potential companion diagnostic test for ipilimumab (IPI) in malignant melanoma. Signature discovery is based on digital pathology and fully automatic quantitative image analysis using virtual multiplexing as well as machine learning and deep learning on whole-slide images. We systematically correlated the patient outcome data with potentially relevant local image features using a Tissue Phenomics approach with a sound cross validation procedure for reliable performance evaluation. Besides uni-variate models we also studied combinations of signatures in several multi-variate models. The most robust and best performing model was a decision tree model based on relative densities of CD8+ tumor infiltrating lymphocytes in the intra-tumoral infiltration region. Our results are well in agreement with observations described in previously published studies regarding the predictive value of the immune contexture, and thus, provide predictive potential for future development of a companion diagnostic test.
BackgroundCD73 is an ectonucleotidase producing the immunosuppressor mediator adenosine. Elevated levels of circulating CD73 in patients with cancer have been associated with disease progression and ...poor response to immunotherapy. Immunosuppressive pathways associated with exosomes can affect T-cell function and the therapeutic efficacy of anti-programmed cell-death protein 1 (anti-PD-1) therapy. Here, we conducted a retrospective pilot study to evaluate levels of exosomal CD73 before and early during treatment with anti-PD-1 agents in patients with melanoma and its potential contribution to affect T-cell functions and to influence the clinical outcomes of anti-PD-1 monotherapy.MethodsExosomes were isolated by mini size exclusion chromatography from serum of patients with melanoma (n=41) receiving nivolumab or pembrolizumab monotherapy. Expression of CD73 and programmed death-ligand 1 (PD-L1) were evaluated on exosomes enriched for CD63 by on-bead flow cytometry. The CD73 AMPase activity was evaluated by mass spectrometry, also in the presence of selective inhibitors of CD73. Interferon (IFN)-γ production and granzyme B expression were measured in CD3/28 activated T cells incubated with exosomes in presence of the CD73 substrate AMP. Levels of CD73 and PD-L1 on exosomes were correlated with therapy response. Exosomes isolated from healthy subjects were used as control.ResultsIsolated exosomes carried CD73 on their surface, which is enzymatically active in producing adenosine. Incubation of exosomes with CD3/28 activated T cells in the presence of AMP resulted in a significant reduction of IFN-γ release, which was reversed by the CD73 inhibitor APCP or by the selective A2A adenosine receptor antagonist ZM241385. Expression levels of exosomal CD73 from serum of patients with melanoma were not significantly different from those in healthy subjects. Early on-treatment, expression levels of both CD73 and PD-L1 on exosomes isolated from patients receiving pembrolizumab or nivolumab monotherapy were significantly increased compared with baseline. Early during therapy exosomal PD-L1 increased in responders, while exosomal CD73 resulted significantly increased in non-responders.ConclusionsCD73 expressed on exosomes from serum of patients with melanoma produces adenosine and contributes to suppress T-cell functions. Early on-treatment, elevated expression levels of exosomal CD73 might affect the response to anti-PD-1 agents in patients with melanoma who failed to respond to therapy.
The
BRAF
V600E
mutation is associated with melanoma development and its detection in circulating-free DNA cannot be observed in all melanoma patients harboring this mutation in tumor specimens. ...Beside the circulating-free DNA
BRAF
V600E
mutation, other markers of therapeutic response should be identified. Matrix metalloproteinase-9 (MMP-9) could be one of them as its role as indicator of invasiveness in melanoma have been explored. In this study, MMP-9 was evaluated in melanoma cells after treatment with dabrafenib.
In vitro
data were validated in 26 melanoma patients, of which 14 treated with BRAF inhibitor alone and 12 treated with both BRAF and MEK inhibitors, by ELISA assay and droplet digital PCR for measuring MMP-9 serum levels and circulating-free DNA
BRAF
V600E
mutation, respectively. Statistical analyses were performed to evaluate the prognostic significance of MMP-9, progression-free survival (PFS) and overall survival (OS) according to the
BRAF
V600E
mutation and MMP-9 levels. The performed analyses showed that MMP-9 and pEKR1-2 were statistically down-regulated in melanoma cells after treatment with dabrafenib. Circulating-free DNA
BRAF
V600E
mutation was detected in 11 out of 26 melanoma patients showing higher levels of MMP-9 compared to those with undetectable
BRAF
V600E
mutation. Furthermore, higher levels of MMP-9 and circulating-free DNA
BRAF
V600E
mutation were associated with lower PFS and OS. Finally, the monitoring of therapy showed that MMP-9 significantly decreased at T1 and T2, but not at T-last, for the patients with detectable circulating-free DNA
BRAF
V600E
mutation. In conclusion, high levels of MMP-9 and circulating-free DNA
BRAF
V600E
mutation are associated with poor PFS and OS. MMP-9 may represent a promising indicator of response to BRAF inhibitors in combination with the detection of
BRAF
V600E
mutation.
The RAS/MAP kinase pathway has attracted attention because activating mutations of the BRAF serine/threonine kinase was described in over 50% of melanomas. Very recently, selective and potent BRAF ...inhibitors have been developed. Several other signal transduction pathways have been found to be constitutively active or mutated in other subsets of melanoma tumors that are potentially targetable with new agents. Among these, NFκB is another pathway that melanoma tumors use to achieve survival, proliferation and resistance to apoptosis. Inhibition of NF-κB activation appears to be a very promising option for anti-cancer therapies.
The innate immune system, composed of neutrophils, basophils, eosinophils, myeloid-derived suppressor cells (MDSCs), macrophages, dendritic cells (DCs), mast cells (MCs), and innate lymphoid cells ...(ILCs), is the first line of defense. Growing evidence demonstrates the crucial role of innate immunity in tumor initiation and progression. Several studies support the idea that innate immunity, through the release of pro- and/or anti-inflammatory cytokines and tumor growth factors, plays a significant role in the pathogenesis, progression, and prognosis of cutaneous malignant melanoma (MM). Cutaneous melanoma is the most common skin cancer, with an incidence that rapidly increased in recent decades. Melanoma is a highly immunogenic tumor, due to its high mutational burden. The metastatic form retains a high mortality. The advent of immunotherapy revolutionized the therapeutic approach to this tumor and significantly ameliorated the patients’ clinical outcome. In this review, we will recapitulate the multiple roles of innate immune cells in melanoma and the related implications for immunotherapy.
PD-1 blocking agents, such as nivolumab, have demonstrated clear anti-tumor effects and clinical benefits in a subset of patients with advanced malignancies. Nonetheless, more efforts are needed to ...identify reliable biomarkers for outcome, to correctly select patients who will benefit from anti-PD-1 treatment. The aim of this study was to investigate the role of peripheral CD8+T cells expressing CD73, involved in the generation of the immune suppressive molecule adenosine, in predicting outcome after nivolumab treatment in advanced melanoma patients.
PBMCs from 100 melanoma patients treated with nivolumab were collected at National Cancer Institute "G. Pascale" of Naples. Frequencies of CD8+ lymphocytes phenotypes were assessed by flow cytometry at baseline before nivolumab treatment, along with clinical characteristics and blood count parameters. Healthy controls (n = 20) were also analysed. Percentages of baseline T cells expressing PD-1 and CD73 were correlated with outcome after nivolumab treatment.
Melanoma patients presented a lower frequency of total circulating CD8+ lymphocytes than control subjects (p = 0.008). Patients with low baseline percentage of circulating CD8+PD-1+CD73+ lymphocytes (< 2.3%) had better survival (22.4 months vs 6.9 months, p = 0.001). Patients (39%) with clinical benefit from nivolumab therapy presented a significantly lower frequency of circulating CD8+PD-1+CD73+ lymphocytes than patients who progressed to nivolumab treatment (p = 0.02).
Our observations suggest that baseline CD73 expression on circulating CD8+PD-1+ lymphocytes appear a promising biomarker of response to anti-PD-1 treatment in melanoma patients. Further investigations are needed for validation and for clarifying its role as prognostic or predictive marker.
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