A harmonized approach towards immuno-monitoring of myeloid-derived suppressor cells for prediction of clinical outcomes in melanoma patients treated with ipilimumab

Ipilimumab is a monoclonal antibody against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) that belongs to a new class of immunotherapeutic drugs called immune checkpoint inhibitors (ICI) which prevent the feedback inhibition of activated T cells and hold great promise to treat cancer. Indeed, it has been demonstrated that ipilimumab increases overall survival (OS) of metastatic melanoma patients, but a durable 5-year survival benefit is observed in a proportion of patients ranging from 12% to 49% and, the treatment has mild to severe immune-related adverse events (irADR). In this context, the development of reliable biomarkers is of great importance to select patients with higher possibility to benefit from this treatment. In this respect, immune profiling could play a significant role, and, in particular, the monitoring of myeloid-derived suppressor cells (MDSC), which are predictive of OS and response to chemotherapy in many types of cancer, and thus represent a promising biomarker also for response to ICI. However, the validation of the predictive significance of MDSCs is challenged by the phenotypic complexity of these cells, that lacks an international consensus on the minimal requirements for MDSC monitoring. To meet this request, we organised the first proficiency panel to harmonize human MDSC phenotyping in collaboration with the Cancer Immunoguiding Program. Hence, we proceeded to the first phase, that consisted in phenotyping three batches of PBMC distributed to the 23 participating laboratories. We also analysed data from the 23 laboratories and observed that the quantification of MDSCs across different laboratories was affected by high variance, and we identified some parameters responsible for the high heterogeneity of results. Results of this first step will set the basis for the second step which is expected to reduce inter-laboratory variance. From this experience, we developed a standardized approach to monitor the circulating levels of four MDSC subsets in melanoma patients undergoing ipilimumab treatment. These results were included in a wide dataset together with other tumor-associated and immunological parameters (TIPs) and used to identify early predictors of OS and toxicity through a multivariate non-parametric statistical approach. We followed the variations of TIPs during ipilimumab treatment and identified two immune profiles predictive of OS. The immune profile of patients with better prognosis included: lower baseline levels of IL-6, CRP and VEGF and higher post-treatment frequencies of CD3+ T cells and CD4+/CD279+ T cells. On the other hand, patients with worse prognosis presented higher post-treatment levels of MDSC1 and 2 and of CD8+/PD-1+ T cells. In addition, the development of grade 3 irADR was negatively associated to the levels of CD3+ and CD4+/PD-1+ T cells and of eosinophils, while positively associated to an increased variance of MDSC4. In conclusion, we demonstrated that the monitoring of the immunological correlates has the potential to identify patients with better prognosis following ipilimumab treatment, thus guiding a more rational use of this therap

improving the management of high cost anticancer drugs in a health care system

As a consequence of the rise in cancer prevalence and in the cost of anticancer drugs, global spending for cancer is increasing rapidly. The aim of this work is to identify and assess some effectiv ..

Toward harmonized phenotyping of human myeloid-derived suppressor cells by flow cytometry: results from an interim study

There is an increasing interest for monitoring circulating myeloid-derived suppressor cells (MDSCs) in cancer patients, but there are also divergences in their phenotypic definition. To overcome this obstacle, the Cancer Immunoguiding Program under the umbrella of the Association of Cancer Immunotherapy is coordinating a proficiency panel program that aims at harmonizing MDSC phenotyping. After a consultation period, a two-stage approach was designed to harmonize MDSC phenotype. In the first step, an international consortium of 23 laboratories immunophenotyped 10 putative MDSC subsets on pretested, peripheral blood mononuclear cells of healthy donors to assess the level of concordance and define robust marker combinations for the identification of circulating MDSCs. At this stage, no mandatory requirements to standardize reagents or protocols were introduced. Data analysis revealed a small intra-laboratory, but very high inter-laboratory variance for all MDSC subsets, especially for the granulocytic subsets. In particular, the use of a dead-cell marker altered significantly the reported percentage of granulocytic MDSCs, confirming that these cells are especially sensitive to cryopreservation and/or thawing. Importantly, the gating strategy was heterogeneous and associated with high inter-center variance. Overall, our results document the high variability in MDSC phenotyping in the multicenter setting if no harmonization/standardization measures are applied. Although the observed variability depended on a number of identified parameters, the main parameter associated with variation was the gating strategy. Based on these findings, we propose further efforts to harmonize marker combinations and gating parameters to identify strategies for a robust enumeration of MDSC subsets

Activated T cells sustain myeloid-derived suppressor cell-mediated immune suppression

The expansion of myeloid-derived suppressor cells (MDSCs), a suppressive population able to hamper the immune response against cancer, correlates with tumor progression and overall survival in several cancer types. We have previously shown that MDSCs can be induced in vitro from precursors present in the bone marrow and observed that these cells are able to actively proliferate in the presence of activated T cells, whose activation level is critical to drive the suppressive activity of MDSCs. Here we investigated at molecular level the mechanisms involved in the interplay between MDSCs and activated T cells. We found that activated T cells secrete IL-10 following interaction with MDSCs which, in turn, activates STAT3 phosphorylation on MDSCs then leading to B7-H1 expression. We also demonstrated that B7-H1+ MDSCs are responsible for immune suppression through a mechanism involving ARG-1 and IDO expression. Finally, we show that the expression of ligands B7-H1 and MHC class II both on in vitro-induced MDSCs and on MDSCs in the tumor microenvironment of cancer patients is paralleled by an increased expression of their respective receptors PD-1 and LAG-3 on T cells, two inhibitory molecules associated with T cell dysfunction. These findings highlight key molecules and interactions responsible for the extensive cross-talk between MDSCs and activated T cells that are at the basis of immune suppression

A harmonized approach towards immuno-monitoring of myeloid-derived suppressor cells for prediction of clinical outcomes in melanoma patients treated with ipilimumab

Ipilimumab is a monoclonal antibody against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) that belongs to a new class of immunotherapeutic drugs called immune checkpoint inhibitors (ICI) which prevent the feedback inhibition of activated T cells and hold great promise to treat cancer. Indeed, it has been demonstrated that ipilimumab increases overall survival (OS) of metastatic melanoma patients, but a durable 5-year survival benefit is observed in a proportion of patients ranging from 12% to 49% and, the treatment has mild to severe immune-related adverse events (irADR). In this context, the development of reliable biomarkers is of great importance to select patients with higher possibility to benefit from this treatment. In this respect, immune profiling could play a significant role, and, in particular, the monitoring of myeloid-derived suppressor cells (MDSC), which are predictive of OS and response to chemotherapy in many types of cancer, and thus represent a promising biomarker also for response to ICI. However, the validation of the predictive significance of MDSCs is challenged by the phenotypic complexity of these cells, that lacks an international consensus on the minimal requirements for MDSC monitoring. To meet this request, we organised the first proficiency panel to harmonize human MDSC phenotyping in collaboration with the Cancer Immunoguiding Program. Hence, we proceeded to the first phase, that consisted in phenotyping three batches of PBMC distributed to the 23 participating laboratories. We also analysed data from the 23 laboratories and observed that the quantification of MDSCs across different laboratories was affected by high variance, and we identified some parameters responsible for the high heterogeneity of results. Results of this first step will set the basis for the second step which is expected to reduce inter-laboratory variance. From this experience, we developed a standardized approach to monitor the circulating levels of four MDSC subsets in melanoma patients undergoing ipilimumab treatment. These results were included in a wide dataset together with other tumor-associated and immunological parameters (TIPs) and used to identify early predictors of OS and toxicity through a multivariate non-parametric statistical approach. We followed the variations of TIPs during ipilimumab treatment and identified two immune profiles predictive of OS. The immune profile of patients with better prognosis included: lower baseline levels of IL-6, CRP and VEGF and higher post-treatment frequencies of CD3+ T cells and CD4+/CD279+ T cells. On the other hand, patients with worse prognosis presented higher post-treatment levels of MDSC1 and 2 and of CD8+/PD-1+ T cells. In addition, the development of grade 3 irADR was negatively associated to the levels of CD3+ and CD4+/PD-1+ T cells and of eosinophils, while positively associated to an increased variance of MDSC4. In conclusion, we demonstrated that the monitoring of the immunological correlates has the potential to identify patients with better prognosis following ipilimumab treatment, thus guiding a more rational use of this therapyIpilimumab è un anticorpo monoclonale diretto contro cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) e appartiene a una nuova classe di farmaci immunoterapeutici chiamati immune checkpoint inhibitors (ICI). Questi farmaci prevengono la fisiologica inibizione dell’attivazione dei linfociti T e costituiscono una promettente terapia per la cura del cancro. Infatti è stato dimostrato che ipilimumab è in grado di aumentare la sopravvivenza dei pazienti affetti da melanoma metastatico. Tuttavia il farmaco produce una sopravvivenza stabile a 5 anni in una parte variabile dei pazienti trattati (12-49%) e inoltre può comportare effetti collaterali con eziologia di tipo immunologico (irADR) di grado medio o alto. Risulta quindi importante identificare biomarcatori che siano in grado di discriminare i pazienti con più alta probabilità di trarre beneficio da questo tipo di trattamento. A tal fine può essere importante tracciare un profilo immunologico del paziente, e in particolare monitorare i livelli circolanti di cellule soppressorie di derivazione mieloide (MDSC). Infatti i livelli circolanti di queste cellule sono stati associati a una minor sopravvivenza e risposta a trattamenti chemioterapici in diversi tipi di cancro, e quindi possono avere un ruolo potenzialmente predittivo anche nei confronti della risposta agli ICI. Tuttavia la validazione del potere predittivo delle MDSC si deve scontrare con la complessità fenotipica di queste cellule e con la mancanza di un consenso internazionale circa la definizione da applicare per il loro monitoraggio. Per cercare di dare una risposta a questi problemi, abbiamo organizzato in collaborazione con il Cancer Immunoguiding Program, un pannello internazionale di armonizzazione del fenotipo delle MDSC. Attualmente è stata completata la prima fase che consisteva nel fenotipizzare tre campioni di cellule mononucleate da sangue periferico da parte dei 23 laboratori partecipanti. Analizzando i dati provenienti da questi 23 laboratori abbiamo osservato che la quantificazione delle MDSC presentava un’alta varianza e abbiamo identificato alcuni dei parametri responsabili di tale eterogeneità. I risultati di questa prima fase sono le fondamenta per la progettazione della seconda fase da cui ci aspettiamo una riduzione della varianza tra i diversi laboratori. L’esperienza maturata con il pannello di armonizzazione ci è servita per sviluppare un metodo standardizzato per il monitoraggio di quattro sottopopolazioni di MDSC circolanti in pazienti affetti da melanoma e in trattamento con ipilimumab. Questi risultati assieme ai valori di altri parametri, associati al tumore o al profilo immunologico del paziente (TIPs), sono stati elaborati con metodi statistici multivariati e non-parametrici atti ad individuare dei marcatori precoci di sopravvivenza e tossicità in risposta al trattamento con ipilimumab. Grazie al monitoraggio dei livelli di questi parametri durante la terapia con ipilimumab, è stato possibile individuare due profili predittivi di sopravvivenza. I pazienti con migliore prognosi hanno minori livelli basali di IL-6, CRP e VEGF e maggiori livelli di linfociti T, in particolare della sottopopolazione CD4+/CD279+ al termine della terapia. Al contrario, una ridotta sopravvivenza si accompagna a maggiori livelli di MDSC1-2 e di linfociti T CD8+/PD-1+ post-trattamento. Inoltre lo sviluppo di irADR di grado 3 è inversamente correlato con i livelli di linfociti T (CD3+ e CD4+/PD-1+) e di eosinofili, mentre è direttamente correlato con un aumento nella varianza dei valori di MDSC4. In conclusione questo studio ha dimostrato che le modificazioni del profilo immunologico associate al trattamento con ipilimumab possono dare informazioni sulla prognosi del paziente e quindi indirizzare le scelte cliniche verso un uso più razionale di questo tipo di terapi