Case report: Is severe toxicity the price to pay for high sensitivity to checkpoint inhibitors immunotherapy in desmoplastic melanoma?

Background: Desmoplastic melanoma (DM) is a rare subtype of melanoma characterized by high immunogenicity which makes it particularly suitable for immune checkpoint inhibitors (ICIs) treatment. Case presentation: We report the case of a 53-year-old man with metastatic DM successfully treated with the combination of anti-CTLA-4 and anti-PD-1 antibodies, who developed serious immune-related adverse events (irAEs). The primary tumor was characterized by absent PD-L1 expression and no-brisk lymphocytes infiltration. NGS showed absence of BRAF mutation, a high tumor mutational burden, and an UV-induced DNA damage signature. Metastatic lesions regressed rapidly after few cycles of ICIs until complete response, however the patient developed serious irAEs including hypothyroidism, adrenal deficiency, and acute interstitial nephritis which led to the definitive suspension of treatment. Currently, the patient has normal renal functionality and no disease relapse after 26 months from starting immunotherapy, and after 9 months from its definitive suspension. Conclusion: Efficacy and toxicity are two sides of the same coin of high sensitivity to ICIs in DM. For this reason, these patients should be closely monitored during ICIs therapy to promptly identify serious side effects and to correctly manage them.

Circulating extracellular vesicles are monitoring biomarkers of anti-PD1 response and enhancer of tumor progression and immunosuppression in metastatic melanoma.

BACKGROUND: Clinical drawback in checkpoint inhibitors immunotherapy (ICI) of metastatic melanoma (MM) is monitoring clinical benefit. Soluble forms of PD1(sPD1) and PD-L1(sPD-L1) and extracellular vesicles (EVs) expressing PD1 and PD-L1 have recently emerged as predictive biomarkers of response. As factors released in the blood, EVs and soluble forms could be relevant in monitoring treatment efficacy and adaptive resistance to ICI. METHODS: We used pre-therapy plasma samples of 110 MM patients and longitudinal samples of 46 patients. Elisa assay and flow cytometry (FCM) were used to measure sPD-L1 and sPD1 concentrations and the percentage of PD1+ EVs and PD-L1+ EVs, released from tumor and immune cells in patients subsets. Transwell assays were conducted to investigate the impact of EVs of each patient subset on MM cells invasion and interaction between tumor cells and macrophages or dendritic cells. Viability assays were performed to assess EVs effect on MM cells and organoids sensitivity to anti-PD1. FCM was used to investigate immunosuppressive markers in EVs and immune cells. RESULTS: The concentrations of sPD1 and sPD-L1 in pre-treatment and longitudinal samples did not correlate with anti-PD1 response, instead only tumor-derived PD1+ EVs decreased in long responders while increased during disease progression in responders. Notably, we observed reduction of T cell derived EVs expressing LAG3+ and PD1+ in long responders and their increase in responders experiencing progression. By investigating the impact of EVs on disease progression, we found that those isolated from non-responders and from patients with progression disease accelerated tumor cells invasiveness and migration towards macrophages, while EVs of long responders reduced the metastatic potential of MM cells and neo-angiogenesis. Additionally, the EVs of non-responders and of progression disease patients subset reduced the sensitivity of MM cells and organoids of responder to anti-PD1 and the recruitment of dendritic cells, while the EVs of progression disease subset skewed macrophages to express higher level of PDL-1. CONCLUSION: Collectively, we suggest that the detection of tumor-derived PD1 + EVs may represent a useful tool for monitoring the response to anti-PD1 and a role for EVs shed by tumor and immune cells in promoting tumor progression and immune dysfunction.

Italian nivolumab Expanded Access Programme in melanoma adjuvant setting: patient outcomes and safety profile.

INTRODUCTION: The CheckMate 238 randomised study demonstrated the relevant benefit in terms of recurrence-free survival (RFS) of nivolumab versus ipilimumab in resected stage IIIB-C or IV melanoma patients with a tolerable safety profile. MATERIALS AND METHODS: From November 2018 to June 2019, 611 patients with stage III and IV resected melanoma were enroled to receive nivolumab as part of an Italian Expanded Access Programme (EAP). According to stages, 77% were stage III while 141 (23%) were stage IV with no evidence of disease (NED). Among stage III, 121 patients had IIIA (19.8%). RESULTS: After a median follow-up of 23 months, the RFS in the Intention-to-Treat (ITT) population was 76.6% at 1 year and 59.6% at 2 years; 1- and 2-year distant metastasis-free survival were 83.7% and 71.2%, respectively. The overall survival rate in the ITT population was 93.8% at 1 year and 85.5% at 2 years. No significant differences in RFS were observed according to BRAF status. Treatment-related adverse events of grades 3-4 occurred in 11.5% of patients. CONCLUSION: This paper reports the results of the Italian Nivolumab EAP in the adjuvant setting of stage III and IV NED melanoma patients. Our results confirm in a real-life setting the clinical activity and safety of nivolumab reported in the CheckMate238 registrative/pivotal. The enroled cohort of 611 patients highlights the relevant clinical need in this setting, also confirmed by the very short accrual time, representing one of the largest series reported as adjuvant EAP with the longest follow-up.

Cervical cancer benefits from trabectedin combination with the ß-blocker propranolol: in vitro and ex vivo evaluations in patient-derived organoids.

Background: Cervical cancer (CC) is characterized by genomic alterations in DNA repair genes, which could favor treatment with agents causing DNA double-strand breaks (DSBs), such as trabectedin. Hence, we evaluated the capability of trabectedin to inhibit CC viability and used ovarian cancer (OC) models as a reference. Since chronic stress may promote gynecological cancer and may hinder the efficacy of therapy, we investigated the potential of targeting ß-adrenergic receptors with propranolol to enhance trabectedin efficacy and change tumor immunogenicity. Methods: OC cell lines, Caov-3 and SK-OV-3, CC cell lines, HeLa and OV2008, and patient-derived organoids were used as study models. MTT and 3D cell viability assays were used for drug(s) IC50 determination. The analysis of apoptosis, JC-1 mitochondrial membrane depolarization, cell cycle, and protein expression was performed by flow cytometry. Cell target modulation analyses were carried out by gene expression, Western blotting, immunofluorescence, and immunocytochemistry. Results: Trabectedin reduced the proliferation of both CC and OC cell lines and notably of CC patient-derived organoids. Mechanistically, trabectedin caused DNA DSBs and S-phase cell cycle arrest. Despite DNA DSBs, cells failed the formation of nuclear RAD51 foci and underwent apoptosis. Under norepinephrine stimulation, propranolol enhanced trabectedin efficacy, further inducing apoptosis through the involvement of mitochondria, Erk1/2 activation, and the increase of inducible COX-2. Notably, trabectedin and propranolol affected the expression of PD1 in both CC and OC cell lines. Conclusion: Overall, our results show that CC is responsive to trabectedin and provide translational evidence that could benefit CC treatment options. Our study pointed out that combined treatment offset trabectedin resistance caused by ß-adrenergic receptor activation in both ovarian and cervical cancer models.

Discontinuation of anti-PD1 in advanced melanoma: an observational retrospective study from the Italian Melanoma Intergroup.

BACKGROUND: Immunotherapy has improved the survival of patients with stage IV melanoma. In responders, clinical benefits may be long-lasting and persist even after treatment discontinuation. The optimal duration of anti-PD1 (anti-Programmed cell death-1) therapy in metastatic melanoma patients remains to be elucidated. Moreover, limited data are available on clinical outcomes of patients that discontinued anti-PD1 immunotherapy in a real-life setting. The aim of this study was to evaluate the progression-free survival (PFS) in patients with metastatic melanoma who interrupted anti-PD-1 treatment in the in the absence of disease progression. METHODS: We retrospectively reviewed patients with advanced/metastatic melanoma treated with anti-PD1 immunotherapy at 23 Italian Melanoma Intergroup (IMI) centres. The study investigated the risk of relapse in patients who stopped anti-PD1 therapy due to CR (Complete response), treatment-related toxicity, or by their own choice after a long period of treatment. Clinical and biological factors associated with or without recurrence were evaluated. RESULTS: The study population included 237 patients. The median age of patients was 68.9 years (standard deviation: 13; range 33-95). The median time on treatment was 33 months (standard deviation: 18, 7; range 1-98). Among the 237 patients, 128 (54%) interrupted the anti-PD1 for CR, 74 patients (31.2%) for adverse events (37 patients in CR, 27 patients in partial response (PR), ten patients in stable disease (SD), and 35 patients (14.8%) by their own choice (12 patients in CR, 17 patients in PR, and 6 patients in SD). After a mean follow-up of 21 months (range 1-81), PFS after anti-PD1 discontinuation was 85.7%. Thirty-four patients (14.3%) developed disease progression after a median of 12 months (range 1-35): ten patients (29.4%) after discontinuation in CR, 17 patients (50%) after discontinuation for treatment-related toxicity (seven in CR, five in PR, five in SD), and seven (20.6%) after discontinuation due to the patient's decision (two in CR, four in PR, one in SD). Only 7.8% of patients who interrupted in CR (10/128), along with 23% of patients who interrupted for limiting toxicity (17/74) and 20% of patients who interrupted by their own choice (7/35), developed recurrence. Regarding patients who discontinued therapy because of CR, we observed a negative association between recurrence and site of primary melanoma, especially mucosal sites (p = <0.05, HR (Hazard ratio) 15.57 IC (confidence interval) 95% 2.64-91.73). Moreover, M1b patients who achieved a CR showed a lower number of relapses (p = <0.05, HR 3.84 IC 95% 1.40-8.48). CONCLUSIONS: This study shows in a real-life setting that, with anti-PD-1 therapy, long-lasting responses, can be maintained after anti-PD1 interruption. In 70.6% of cases, recurrences were observed among patients who did not obtain a CR at treatment discontinuation.

Melanoma Brain Metastases: A Retrospective Analysis of Prognostic Factors and Efficacy of Multimodal Therapies.

Brain metastasis in cutaneous melanoma (CM) has historically been considered to be a dismal prognostic feature, although recent evidence has highlighted the intracranial activity of combined immunotherapy (IT). Herein, we completed a retrospective study to investigate the impact of clinical-pathological features and multimodal therapies on the overall survival (OS) of CM patients with brain metastases. A total of 105 patients were evaluated. Nearly half of the patients developed neurological symptoms leading to a negative prognosis (p = 0.0374). Both symptomatic and asymptomatic patients benefited from encephalic radiotherapy (eRT) (p = 0.0234 and p = 0.011). Lactate dehydrogenase (LDH) levels two times higher than the upper limit normal (ULN) at the time of brain metastasis onset was associated with poor prognosis (p = 0.0452) and identified those patients who did not benefit from eRT. Additionally, the poor prognostic role of LDH levels was confirmed in patients treated with targeted therapy (TT) (p = 0.0015) concerning those who received immunotherapy (IT) (p = 0.16). Based on these results, LDH levels higher than two times the ULN at the time of the encephalic progression identify those patients with a poor prognosis who did not benefit from eRT. The negative prognostic role of LDH levels on eRT observed in our study will require prospective evaluations.

A deep learning model based on whole slide images to predict disease-free survival in cutaneous melanoma patients.

The application of deep learning on whole-slide histological images (WSIs) can reveal insights for clinical and basic tumor science investigations. Finding quantitative imaging biomarkers from WSIs directly for the prediction of disease-free survival (DFS) in stage I-III melanoma patients is crucial to optimize patient management. In this study, we designed a deep learning-based model with the aim of learning prognostic biomarkers from WSIs to predict 1-year DFS in cutaneous melanoma patients. First, WSIs referred to a cohort of 43 patients (31 DF cases, 12 non-DF cases) from the Clinical Proteomic Tumor Analysis Consortium Cutaneous Melanoma (CPTAC-CM) public database were firstly annotated by our expert pathologists and then automatically split into crops, which were later employed to train and validate the proposed model using a fivefold cross-validation scheme for 5 rounds. Then, the model was further validated on WSIs related to an independent test, i.e. a validation cohort of 11 melanoma patients (8 DF cases, 3 non-DF cases), whose data were collected from Istituto Tumori 'Giovanni Paolo II' in Bari, Italy. The quantitative imaging biomarkers extracted by the proposed model showed prognostic power, achieving a median AUC value of 69.5% and a median accuracy of 72.7% on the public cohort of patients. These results remained comparable on the validation cohort of patients with an AUC value of 66.7% and an accuracy value of 72.7%, respectively. This work is contributing to the recently undertaken investigation on how treat features extracted from raw WSIs to fulfil prognostic tasks involving melanoma patients. The promising results make this study as a valuable basis for future research investigation on wider cohorts of patients referred to our Institute.

BRAFV600E;K601Q metastatic melanoma patient-derived organoids and docking analysis to predict the response to targeted therapy.

The V600E mutation in BRAF is associated with increased phosphorylation of Erk1/2 and high sensitivity to BRAFi/MEKi combination in metastatic melanoma. In very few patients, a tandem mutation in BRAF, V600 and K601, causes a different response to BRAFi/MEKi combination. BRAFV600E;K601Q patient-derived organoids (PDOs) were generated to investigate targeted therapy efficacy and docking analysis was used to assess BRAFV600E;K601Q interactions with Vemurafenib. PDOs were not sensitive to Vemurafenib and Cobimetinib given alone and sensitive to their combination, although not as responsive as BRAFV600E PDOs. The docking analysis justified such a result showing that the tandem mutation in BRAF reduced the affinity for Vemurafenib. Tumor analysis showed that BRAFV600E;K601Q displayed both increased phosphorylation of Erk1/2 at cytoplasmic level and activation of Notch resistance signaling. This prompted us to inhibit Notch signaling with Nirogacestat, achieving a greater antitumor response and providing PDOs-based evaluation of treatment efficacy in such rare metastatic melanoma.

Basal and one-month differed neutrophil, lymphocyte and platelet values and their ratios strongly predict the efficacy of checkpoint inhibitors immunotherapy in patients with advanced BRAF wild-type melanoma.

BACKGROUND: To evaluate the capability of basal and one-month differed white blood cells (WBC), neutrophil, lymphocyte and platelet values and their ratios (neutrophils-to-lymphocytes ratio, NLR, and platelets-to-lymphocytes ratio, PLR) in predicting the response to immune checkpoint inhibitors (ICI) in metastatic melanoma (MM). METHODS: We performed a retrospective study of 272 BRAF wild-type MM patients treated with first line ICI. Bivariable analysis was used to correlate patient/tumor characteristics with clinical outcomes. Variations between time 1 and time 0 (Δ) of blood parameters were also calculated and dichotomized using cut-off values assessed by ROC curve. RESULTS: At baseline, higher neutrophils and NLR negatively correlated with PFS, OS and disease control rate (DCR). Higher PLR was also associated with worse OS. In multivariable analysis, neutrophils (p = 0.003), WBC (p = 0.069) and LDH (p = 0.07) maintained their impact on PFS, while OS was affected by LDH (p < 0.001), neutrophils (p < 0.001) and PLR (p = 0.022), while DCR by LDH (p = 0.03) and neutrophils (p = 0.004). In the longitudinal analysis, PFS negatively correlated with higher Δplatelets (p = 0.039), ΔWBC (p < 0.001), and Δneutrophils (p = 0.020), and with lower Δlymphocytes (p < 0.001). Moreover, higher ΔNLR and ΔPLR identified patients with worse PFS, OS and DCR. In the multivariable model, only ΔNLR influenced PFS (p = 0.004), while OS resulted affected by higher ΔWBC (p < 0.001) and lower Δlymphocytes (p = 0.038). Higher ΔWBC also affected the DCR (p = 0.003). When clustering patients in 4 categories using basal LDH and ΔNLR, normal LDH/lower ΔNLR showed a higher PFS than high LDH/higher ΔNLR (20 vs 5 months). Moreover, normal LDH/higher Δlymphocytes had a higher OS than high LDH/lower Δlymphocytes (50 vs. 10 months). CONCLUSIONS: Baseline and early variations of blood cells, together with basal LDH, strongly predict the efficacy of ICI in MM. Our findings propose simple, inexpensive biomarkers for a better selection of patient treatments. Prospective multicenter studies are warranted to confirm these data.

Circulating extracellular vesicles expressing PD1 and PD-L1 predict response and mediate resistance to checkpoint inhibitors immunotherapy in metastatic melanoma.

BACKGROUND: The immunotherapy with immune checkpoints inhibitors (ICI) has changed the life expectancy in metastatic melanoma (MM) patients. Nevertheless, several patients do not respond hence, the identification and validation of novel biomarkers of response to ICI is of crucial importance. Circulating extracellular vesicles (EVs) such as PD-L1+ EV mediate resistance to anti-PD1, instead the role of PD1+ EV is not fully understood. METHODS: We isolated the circulating EVs from the plasma of an observational cohort study of 71 metastatic melanoma patients and correlated the amount of PD-L1+ EVs and PD1+ EVs with the response to ICI. The analysis was performed according to the origin of EVs from the tumor and the immune cells. Subsequently, we analysed the data in a validation cohort of 22 MM patients to assess the reliability of identified EV-based biomarkers. Additionally we assessed the involvement of PD1+ EVs in the seizure of nivolumab and in the perturbation of immune cells-mediated killing of melanoma spheroids. RESULTS: The level of PD-L1+ EVs released from melanoma and CD8+ T cells and that of PD1+ EVs irrespective of the cellular origin were higher in non-responders. The Kaplan-Meier curves indicated that higher levels of PD1+ EVs were significantly correlated with poorer progression-free survival (PFS) and overall survival (OS). Significant correlations were found for PD-L1+ EVs only when released from melanoma and T cells. The multivariate analysis showed that high level of PD1+ EVs, from T cells and B cells, and high level of PD-L1+ EVs from melanoma cells, are independent biomarkers of response. The reliability of PD-L1+ EVs from melanoma and PD1+ EVs from T cells in predicting PFS was confirmed in the validation cohort through the univariate Cox-hazard regression analysis. Moreover we discovered that the circulating EVs captured nivolumab and reduced the T cells trafficking and tumor spheroids killing. CONCLUSION: Our study identified circulating PD1+ EVs as driver of resistance to anti-PD1, and highlighted that the analysis of single EV population by liquid biopsy is a promising tool to stratify MM patients for immunotherapy.

Cemiplimab in an Elderly Frail Population of Patients With Locally Advanced or Metastatic Cutaneous Squamous Cell Carcinoma: A Single-Center Real-Life Experience From Italy.

BACKGROUND: Cutaneous squamous cell carcinoma (CSCC) is the second most common skin cancer whose incidence is growing parallel to the lengthening of the average lifespan. Cemiplimab, an antiPD-1 monoclonal antibody, is the first approved immunotherapy for patients with locally advanced CSCC (laCSCC) or metastatic CSCC (mCSCC) thanks to phase I and II studies showing high antitumor activity and good tolerability. Nevertheless, at present, very few data are available regarding cemiplimab in real-life experience and in frail, elderly, and immunosuppressed patients as well as regarding biomarkers able to predict response so as to guide therapeutic choices. PATIENTS AND METHODS: We built a retroprospective cohort study including 30 non-selected patients with laCSCC (25) and mCSCC (five) treated with cemiplimab from August 2019 to November 2020. Clinical outcomes, toxicity profile, and correlations with disease, patients, and peripheral blood parameters are explored. RESULTS: The median age was 81 years (range, 36-95), with 24 males and five patients having an immunosuppressive condition, while the frailty prevalence was 83% based on index derived from age, Eastern Cooperative Oncology Group performance status, and Charlson Comorbidity Index. We reported 23 responses (76.7%) with nine complete responses (30%). A statistically significant higher response rate was observed in head and neck primary tumors and in patients with hemoglobin level >12 g/dl. No difference was observed with respect to frailty, median age, sex, and body mass index. The baseline low neuthophil/lymphocyte ratio and low platelet/lymphocyte ratio resulted to be also correlated with a better response. Moreover, lymphocyte, neutrophil, and monocyte behaviors had an opposite trend in responders and non-responders. An overall response was reported in four of five immunosuppressed patients. Seventeen patients (57.6%) have an ongoing response and are still alive. Six responders had interrupted treatment (two for toxicity and four for personal choice) but maintained their response. The treatment was well tolerated by the majority of patients. The most common adverse events were fatigue in seven patients (23.3%) and skin toxicity in 10 patients (33.3%), including pruritus in six patients, rash in three patients, and bullous erythema in one patient. CONCLUSIONS: In our real-life experience, cemiplimab showed a high antitumor activity with acceptable safety profile similar to those in trials with selected patients. Moreover, its antitumor activity resulted to be not impaired in very elderly patients and in those with immunocompromised status.

Electrochemotherapy as a Trigger to Overcome Primary Resistance to Anti-PD-1 Treatment: A Case Report of Melanoma of the Scalp.

BACKGROUND: Immunotherapy with immune checkpoint inhibitors is one of the main therapies for advanced melanoma. Nevertheless, albeit remarkable, immunotherapy results are still unsatisfactory as more than half of patients progress, and resistance to treatment still has a dramatic impact on clinical outcomes. Local treatments such as radiotherapy or electrochemotherapy (ECT), in addition to local control with palliative intent, have been shown to release tumoral neoantigens that can stimulate a robust systemic antitumor immune response. CASE PRESENTATION: We report the case of a patient with multiple nodular melanoma lesions of the scalp initially treated with local ECT. Soon after the procedure, multiple new lesions appeared close to the treated ones, therefore the patient started a systemic treatment with the anti-PD-1 nivolumab. The lesions of the scalp did not respond to immunotherapy, presenting a loco-regional spreading. To control the bleeding and painful lesions, we performed a second ECT, while continuing systemic immunotherapy. The treated lesions responded to the second procedure, while the other lesions continued progressing in number and dimension. Unexpectedly, after 2 months from the second ECT, the patient presented a progressive shrinkage of both treated and untreated lesions until complete remission. Concomitantly, he developed immune-related adverse events including grade 4 thyroid toxicity, grade 2 vitiligo-like depigmentation and grade 2 pemphigoid. At present, after 18 months from the first ECT and 14 months from the starting of anti-PD-1 immunotherapy, the patient is in good clinical condition and complete remission of disease still persists. CONCLUSION: This case highlights the potential role of ECT in increasing tumor immunogenicity and consequently in inducing a powerful immune response overcoming primary resistance to checkpoint inhibitor immunotherapy.

Case Report: Autoimmune Pemphigus Vulgaris in a Patient Treated With Cemiplimab for Multiple Locally Advanced Cutaneous Squamous Cell Carcinoma.

BACKGROUND: Pemphigus vulgaris (PV) is a rare and severe autoimmune blistering disorder affecting the skin and mucous membranes, characterized by the production of autoantibodies against two desmosomal adhesion proteins, desmoglein 1 and 3. In patients with advanced squamous cell carcinoma of the skin unfit for surgery and radiotherapy, immune check-point inhibitors, including the anti-Programmed Death-1 (PD-1) agent cemiplimab have been successfully employed proving relevant clinical outcomes. Cemiplimab is a monoclonal antibody capable of inhibiting PD-1 signalling that has recently been approved for the treatment of patients with metastatic or locally advanced cutaneous squamous cell carcinoma. Although the peculiar setting of advanced CSCC involving elderly patients, rare and unusual skin immune-related adverse events such as PV could be observed in cemiplimab treated patients. CASE REPORT: A 95-year-old man without a history of autoimmune disease was treated with cemiplimab for multiple and advanced squamous cell carcinomas of the head obtaining a complete response to therapy. After seven cycles of cemiplimab administered every 21 days, the patient developed a mucocutaneous blistering eruption. Clinical diagnosis of PV was suspected on the basis of the diffuse involvement of trunk and extremities with large blisters and necrotic eschar. It was carried out an ELISA test, that showed high level of circulating antibodies against desmoglein 1, thus confirming the diagnosis of PV. For this reason, cemiplimab infusion was discontinued and complete resolution of skin lesions was obtained using oral prednisone 0,8 mg/kg/daily for four weeks. Once remission was achieved, a maintenance dose of 10 mg/day was administered, observing a good control of bullous disease and low value of desmoglein 1. Response to CSCC persisted also during cemiplimab discontinuation, until obtaining a complete remission still persisting at 9 months after the last cycle of therapy. CONCLUSION: The case we observed is the first description of PV revealed from cemiplimab therapy, thus suggesting that cemiplimab could allow the arise of underlying autoimmune PV, through a mechanism both T and B-cell-mediated.

uPAR+ extracellular vesicles: a robust biomarker of resistance to checkpoint inhibitor immunotherapy in metastatic melanoma patients.

BACKGROUND: Emerging evidence has highlighted the importance of extracellular vesicle (EV)-based biomarkers of resistance to immunotherapy with checkpoint inhibitors in metastatic melanoma. Considering the tumor-promoting implications of urokinase-type plasminogen activator receptor (uPAR) signaling, this study aimed to assess uPAR expression in the plasma-derived EVs of patients with metastatic melanoma to determine its potential correlation with clinical outcomes. METHODS: Blood samples from 71 patients with metastatic melanoma were collected before initiating immunotherapy. Tumor-derived and immune cell-derived EVs were isolated and analyzed to assess the relative percentage of uPAR+ EVs. The associations between uPAR and clinical outcomes, sex, BRAF status, baseline lactate dehydrogenase levels and number of metastatic sites were assessed. RESULTS: Responders had a significantly lower percentage of tumor-derived, dendritic cell (DC)-derived and CD8+ T cell-derived uPAR +EVs at baseline than non-responders. The Kaplan-Meier survival curves for the uPAR+EV quartiles indicated that higher levels of melanoma-derived uPAR+ EVs were strongly correlated with poorer progression-free survival (p<0.0001) and overall survival (p<0.0001). We also found a statistically significant correlation between lower levels of uPAR+ EVs from both CD8+ T cells and DCs and better survival. CONCLUSIONS: Our results indicate that higher levels of tumor-derived, DC-derived and CD8+ T cell-derived uPAR+ EVs in non-responders may represent a new biomarker of innate resistance to immunotherapy with checkpoint inhibitors. Moreover, uPAR+ EVs represent a new potential target for future therapeutic approaches.

Examining the Relationship between Circulating CD4- CD8- Double-Negative T Cells and Outcomes of Immuno-Checkpoint Inhibitor Therapy-Looking for Biomarkers and Therapeutic Targets in Metastatic Melanoma.

BACKGROUND: The role of circulating CD4-/CD8- double-negative T cells (DNTs) in the immune response to melanoma is poorly understood, as are the effects of checkpoint inhibitors on T cell subpopulations. METHODS: We performed a basal and longitudinal assessment of circulating immune cells, including DNTs, in metastatic melanoma patients treated with checkpoint blockade in a single-center cohort, and examined the correlations levels of immune cells with clinical features and therapy outcomes. RESULTS: Sixty-eight patients (48 ipilimumab, 20 PD1 inhibitors) were enrolled in the study. Our analysis indicated that better outcomes were associated with normal LDH, fewer than three metastatic sites, an ECOG performance status of 0, M1a stage, lower WBC and a higher lymphocyte count. The increase in lymphocyte count and decrease of DNTs were significantly associated with the achievement of an overall response. The median value of DNT decreased while the CD4+ and NK cells increased in patients that responded to treatment compare to those who did not respond to treatment. CONCLUSIONS: DNT cells change during treatment with checkpoint inhibitors and may be adept at sensing the immune response to melanoma. The complementary variation of DNT cells with respect to CD4+ and other immune actors may improve the reliability of lymphocyte assessment. Further investigation of DNT as a potential target in checkpoint inhibitor resistant melanoma is warranted.

No Impact of NRAS Mutation on Features of Primary and Metastatic Melanoma or on Outcomes of Checkpoint Inhibitor Immunotherapy: An Italian Melanoma Intergroup (IMI) Study.

AIMS: It is debated whether the NRAS-mutant melanoma is more aggressive than NRAS wildtype. It is equally controversial whether NRAS-mutant metastatic melanoma (MM) is more responsive to checkpoint inhibitor immunotherapy (CII). 331 patients treated with CII as first-line were retrospectively recruited: 162 NRAS-mutant/BRAF wild-type (mut/wt) and 169 wt/wt. We compared the two cohorts regarding the characteristics of primary and metastatic disease, disease-free interval (DFI) and outcome to CII. No substantial differences were observed between the two groups at melanoma onset, except for a more frequent ulceration in the wt/wt group (p = 0.03). Also, the DFI was very similar in the two cohorts. In advanced disease, we only found lung and brain progression more frequent in the wt/wt group. Regarding the outcomes to CII, no significant differences were reported in overall response rate (ORR), disease control rate (DCR), progression free survival (PFS) or overall survival (OS) (42% versus 37%, 60% versus 59%, 12 (95% CI, 7-18) versus 9 months (95% CI, 6-16) and 32 (95% CI, 23-49) versus 27 months (95% CI, 16-35), respectively). Irrespectively of mutational status, a longer OS was significantly associated with normal LDH, <3 metastatic sites, lower white blood cell and platelet count, lower neutrophil-to-lymphocyte (N/L) ratio. Our data do not show increased aggressiveness and higher responsiveness to CII in NRAS-mutant MM.

Cellular analysis of bronchoalveolar lavage fluid to narrow differential diagnosis of checkpoint inhibitor-related pneumonitis in metastatic melanoma.

BACKGROUND: The diagnosis of check-point inhibitor-related pneumonitis (CIP) relies on radiological and clinical patterns which are not specific and can mimic other conditions (cancer progression, infectious diseases or interstitial pneumonitis). Cell pattern analysis of bronchoalveolar lavage (BAL) is well-known to support the diagnosis of interstitial lung disease; nevertheless, this analysis is somewhat performed and not required by immune-toxicity management guidelines for CIP. METHODS: We performed BAL analysis in 5 metastatic melanoma (MM) patients who developed CIP among 112 patients treated with checkpoint inhibitors. We also correlated the BAL features with the computed tomography (CT) scan patterns and with various peripheral blood parameters to better define the profile of this patient population. RESULTS: BAL flow cytometer and cytopathology analyses showed typical and homogeneous features with increased lymphoid population, prevalent CD8 + T cells and inversion of the CD4/CD8 ratio. Moreover, the extent of activated CD3 + HLA-DR + T cells was related to the grading of adverse events. Blood leucocytosis, hypoxemia, normal values for procalcitonin and lactate dehydrogenase were also found together with a cryptogenic organizing pneumonia-like radiologic pattern. In all our patients, CIP was associated with partial or complete response. CONCLUSIONS: Identification of a specific BAL cellular pattern allows clinicians to place this investigation in the appropriate position of CIP diagnosis and management to avoid misdiagnosis or considering this condition as progressive disease and delaying proper treatment.

Case Report: Lymphocytosis Associated With Fatal Hepatitis in a Thymoma Patient Treated With Anti-PD1: New Insight Into the Immune-Related Storm.

Recent advances in tumor immunotherapy have made it possible to efficiently unleash immune effectors, reacting against neoplastic cells. Although these approaches primarily aim to eradicate malignancy, immune-related adverse events (irAEs) often influence patients' prognosis, constituting a new spectrum of side effects. Taking into account the typical microenvironment and the intricate equilibrium between the anti-tumor response and the immune cells, the thymoma constitutes a unicum in the immune-oncology field. We report a fatal immune-mediated adverse events' storm in a thymoma patient treated with Pembrolizumab, leading to hepatotoxicity accompanied by lymphocytosis, thrombocytopenia, and thyroid dysfunction, unveiling a novel potential pathophysiological effect of immunotherapy. The clinical proficiency of the immune checkpoint inhibitors in thymoma patients warrants timely prevention and management of off-target consequences in order to optimize this promising therapeutic option. This case report describes a unique consequence of irAEs, emerging as a red flag warranting a multidisciplinary approach.

An Italian Retrospective Survey on Bone Metastasis in Melanoma: Impact of Immunotherapy and Radiotherapy on Survival.

BACKGROUND: We performed a multicenter retrospective observational study to investigate the impact of clinical-pathological features and therapeutic strategies on both the complications and survival of patients with bone metastases (BMs) from malignant melanoma. PATIENTS AND METHODS: A total of 305 patients with melanoma and radiological evidence of BMs were retrospectively enrolled from 19 Italian centers. All patients received conventional treatments in accordance with each own treating physician's practice. Both univariate and multivariate models were used to explore the impact of melanoma features, including skeletal-related events (SREs), and different treatments on both overall survival (OS) and time-to-SREs. The chi-squared test evaluated the suitability of several parameters to predict the occurrence of SREs. RESULTS: Eighty-three percent of patients had metachronous BMs. The prevalent (90%) bone metastatic site was the spine, while 45% had involvement of the appendicular skeleton. Forty-seven percent experienced at least one SRE, including palliative radiotherapy (RT) in 37% of cases. No melanoma-associated factor was predictive of the development of SREs, although patients receiving early treatment with bone-targeted agents showed 62% lower risk and delayed time of SRE occurrence. Median OS from the diagnosis of bone metastasis was 10.7 months. The multivariate analysis revealed as independent prognostic factors the number of BMs, number of metastatic organs, baseline lactate dehydrogenase levels, and treatment with targeted therapy or immunotherapy. Subgroup analyses showed the best OS (median = 16.5 months) in the subset of patients receiving both immunotherapy and palliative RT. CONCLUSION: Based on our results, patients undergoing immunotherapy and palliative RT showed an OS benefit suggestive of a possible additive effect. The apparent protective role of bone targeting agent use on SREs observed in our analysis should deserve prospective evaluation.

The ß-adrenergic receptor antagonist propranolol offsets resistance mechanisms to chemotherapeutics in diverse sarcoma subtypes: a pilot study.

Standard chemotherapy for soft tissue sarcomas has shown limited efficacy. Here, we sought to evaluate whether ß-adrenergic receptor (ß-AR) signalling contributed to the progression of sarcomas and therapy resistance. To assess the translational potential of ß-adrenergic receptors, we performed immunohistochemical detection of ß1-AR, ß2-AR and ß3-AR in leiomyosarcoma, liposarcoma and angiosarcoma tissue specimens, reporting the results scored for the intensity. By using established and patient-derived sarcoma cells, we demonstrated the antitumour potential of the pharmacological targeting of ß-ARs with the nonselective ß-blocker propranolol in such sarcomas. Of note, pharmacological ß-AR inhibition synergized with doxorubicin in inhibiting the cell viability of liposarcoma and leiomyosarcoma cells and increased the response to docetaxel in angiosarcoma- and solitary fibrous tumour (SFT)-patient-derived cells. Notably, the SFT patient was treated with the combination of propranolol and docetaxel, reporting prolonged disease control. Mechanistically, we found that propranolol reduced the activity of the multidrug resistance efflux pump P-gp, thereby increasing the intracellular doxorubicin concentration and antitumour activity. In addition, propranolol attenuated the Akt-dependent survival signal induced by doxorubicin and strongly reduced the activation of the NF-kB/COX-2 pathway, increasing cell sensitivity to docetaxel. Overall, our study highlighted the therapeutic potential of propranolol, alone or in rational combination therapies, for sarcoma treatment.