The rationale for therapeutic targeting of Vδ2+ γδ T cells in breast cancer is strongly supported by in vitro and murine preclinical investigations, characterizing them as potent breast tumor cell killers and source of Th1-related cytokines, backing cytotoxic αβ T cells. Nonetheless, insights regarding Vδ2+ γδ T cell phenotypic alterations in human breast cancers are still lacking. This paucity of information is partly due to the challenging scarcity of these cells in surgical specimens. αβ T cell phenotypic alterations occurring in the tumor bed are detectable in the periphery and correlate with adverse clinical outcomes. Thus, we sought to determine through an exploratory study whether Vδ2+ γδ T cells phenotypic changes can be detected within breast cancer patients’ peripheral blood, along with association with tumor progression. By using mass cytometry, we quantified 130 immune variables from untreated breast cancer patients’ peripheral blood. Supervised analyses and dimensionality reduction algorithms evidenced circulating Vδ2+ γδ T cell phenotypic alterations already established at diagnosis. Foremost, terminally differentiated Vδ2+ γδ T cells displaying phenotypes of exhausted senescent T cells associated with lymph node involvement. Thereby, our results support Vδ2+ γδ T cells implication in breast cancer pathogenesis and progression, besides shedding light on liquid biopsies to monitor surrogate markers of tumor-infiltrating Vδ2+ γδ T cell antitumor activity.

Aims This study sought to clarify the molecular pathways underlying the putative evolution from lymphomatoid papulosis (LyP) to cutaneous anaplastic large‐cell lymphoma (c‐ALCL) and lymph node invasion (LNI). Methods and results We analysed nine sequential tumours from the same patient presenting with parallel evolution of LyP (n = 3) and c‐ALCL (n = 1) with LNI (n = 1), combined with systemic diffuse large B‐cell lymphoma (DLBCL) (n = 4). Clonality analysis showed a common clonal T‐cell origin in the five CD30+ lesions, and a common clonal B‐cell origin in the four DLBCL relapses. Array‐comparative genomic hybridisation and targeted next‐generation sequencing analysis demonstrated relative genomic stability of LyP lesions as compared with clonally related anaplastic large‐cell lymphoma (ALCL) tumours, which showed 4q and 22q13 deletions involving the PRDM8 and TIMP3 tumour suppressor genes, respectively. The three analysed CD30+ lesions showed mostly private (specific to each sample) genetic alterations, suggesting early divergence from a common precursor. In contrast, DLBCL tumours showed progressive accumulation of private alterations, indicating late divergence. Conclusions Sequential cutaneous and nodal CD30+ tumours were clonally related. This suggests that LyP, c‐ALCL and LNI represent a continuous spectrum of clonal evolution emerging from a common precursor of cutaneous CD30+ lymphoproliferations. Therefore, nodal ALCL tumours in the context of LyP should be considered as a form of transformation rather than composite lymphoma.

Purpose Follicular lymphoma (FL) accounting for 20% of non-Hodgkin lymphoma, is currently considered treatable but not curable despite more effective treatment options. Idelalisib, a selective inhibitor of phosphatidylinositol 3-kinase δ (PI3Kδ) that blocks PI3Kδ-AKT signaling and promotes apoptosis, is approved for patients with relapsed/refractory (R/R) FL who have received ≥ 2 prior systemic therapies. Previous studies suggest that somatic tumor mutations identified at relapse may have an impact on the response to targeted therapies (Bartlett N et al., Blood 2018). The goal of this study was to analyze the lymphoma mutational profile to identify the prognostic value of mutations in R/R follicular lymphoma patients treated with idelalisib. Methods We performed a retrospective multicenter study of patients (pts) with relapsed follicular lymphoma and no evidence of transformation, having received at least 2 prior regimens before idelalisib treatment. Patients received idelalisib 150mg BID until progression or toxicity. Next-generation sequencing (NGS) of 51 genes was performed either at FL diagnosis and/or at relapse prior idelalisib therapy. The primary endpoint was to analyze the relationship between the mutational status and the duration of response (DOR) to idelalisib. DOR was measured from the time of initial response until documented lymphoma progression. According to DOR pts were classified as: refractory (response < 1 month), short-responder (1 month ≤ DOR ≤ 12 months) and long-responder (> 12 months). Results 24 pts with R/R FL were enrolled with a median age of 62.5 years (range: 57-86). Patients had received a median of 3 prior treatments (range: 2-7). FL was refractory to rituximab in 16 pts (67%), to alkylating agents in 11 pts (46%) and to 2 or more prior treatments in 11 pts (46%). Twelve (50%) had refractory disease to the last therapy before idelalisib. Pts received idelalisib during a median of 5.5 months (range: 0.5-31). Overall response rate was 83% (n=20) including 3 (15%) complete response and 17 (85%) partial response. The median DOR was 9.5 months (range: 0-28). Eleven pts were short-responders and 9 long-responders. Four pts (17%) had refractory disease to idelalisib. Median progression-free survival and overall survival were 11.5 (range: 1-30) and 16.5 months (range: 1-56) respectively. Three pts (12.5%) are still continuing idelalisib. Twenty-one pts (87.5%) discontinued treatment mostly due to progressive disease (n=14) and adverse events (n=5); 3 pts remained progression-free after idelalisib discontinuation and observation with a median follow-up of 23 months (range: 20-24). All the pts had at least one mutation detectable for one of the targeted genes in both diagnosis (n=17) and relapse (n=20) samples. The median number of targeted genes with non-silent mutations per patient was 7 at diagnosis (range: 2-11) and 6 at relapse (range: 3-28). The most frequent genes found in 10% of patients (≥ 2) or more at diagnosis and at relapse are listed in Figure 1. The mutational profile at diagnosis predominantly included mutations in epigenetics gene family, KTM2D (n=16; 94%), EP300 (n=9; 52%), ARID1A (n=6; 35%), KTM2A (n=5; 29%) and CREBBP (n=4; 23%). The m7-FLIPI score (Pastore A et al., Lancet Oncology 2015) at diagnosis helped identifying pts with low-risk (n=12; 71%) and high-risk (n=5; 29%) of idelalisib treatment failure. The median m7-FLIPI score in the refractory group was 1.25 compared to 0.26 in the short responder group and 0.04 in the long responder group. All long responder patients had low-risk m7-FLIPI. The mutational profile at relapse was significantly enriched in mutations of TNFAIP3 (n=7; 35%) and NFKBIE (n=4; 20%), affecting the NF-kB inhibitor pathway, and mutations of transcription factors, TP53 (n=10; 50%), MEF2B (n=5; 25%), FOXO1, STAT6 and IRF4 (n=4; 20% each). Overall the mutational profile of the 3 sub-groups according to DOR was detailed in Figure 2. The genes more frequently mutated in refractory pts were: EP300 (n=3/4; 75%), B2M (n=2/4; 50%), FBXW7 (n=2/4; 50%), CARD11, CXCR4 and MYD88 (n=1/4; 25% each). Conclusion The m7FLIPI at diagnosis identifies patients with higher risk of treatment failure in patients with R/R FL treated with idelalisib. Patients with idelalisib refractory disease have more frequently mutations of EP300, B2M, FBXW7, which suggests they could be related to resistance to idelalisib. Disclosures No relevant conflicts of interest to declare.

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