Early CAR- CD4+ T-lymphocytes recovery following CAR-T cell infusion: A worse outcome in diffuse large B cell lymphoma.

CAR- CD4+ T cell lymphopenia is an emerging issue following CAR-T cell therapy. We analyzed the determinants of CD4+ T cell recovery and a possible association with survival in 31 consecutive patients treated with commercial CAR-T for diffuse large B-cell (DLBCL) or mantle cell lymphoma. Circulating immune subpopulations were characterized through multiparametric-flow cytometry. Six-month cumulative incidence of CAR- CD4+ T cell recovery (≥200 cells/µL) was 0.43 (95% confidence interval [CI]: 0.28-0.65). Among possible determinants of CD4+ T cell recovery, we recognized infusion of a 4-1BB product (tisagenlecleucel, TSA) in comparison with a CD28 (axicabtagene/brexucabtagene, AXI/BRX) (hazard ratio [HR] [95% CI]: 5.79 [1.16-24.12] p = 0.016). Higher CD4+ T cell counts resulted with TSA at month-1, -2 and -3. Moderate-to-severe infections were registered with prolonged CD4+ T cell lymphopenia. Early, month-1 CD4+ T cell recovery was associated with a worse outcome in the DLBCL cohort, upheld in a multivariate regression model for overall survival (HR: 4.46 [95% CI: 1.12-17.71], p = 0.03). We conclude that a faster CAR- CD4+ T cell recovery is associated with TSA as compared to AXI/BRX. Month-1 CAR- CD4+ T cell subset recovery could represent a "red flag" for CAR-T cell therapy failure in DLBCL patients.

Donor selection for adoptive immunotherapy with NK cells in AML patients: Comparison between analysis of lytic NK cell clones and phenotypical identification of alloreactive NK cell repertoire.

Natural killer (NK) cell-based adoptive immunotherapy in leukemia patients is an emerging field of interest based on clinical evidence of efficacy and safety. Elderly acute myeloid leukemia (AML) patients have been successfully treated with NK cells from HLA-haploidentical donors, especially when high amounts of alloreactive NK cells were infused. The aim of this study was comparing two approaches to define the size of alloreactive NK cells in haploidentical donors for AML patients recruited in two clinical trials with the acronym "NK-AML" (NCT03955848), and "MRD-NK". The standard methodology was based on the frequency of NK cell clones capable of lysing the related patient-derived cells. The alternative approach consisted of the phenotypic identification of freshly derived NK cells expressing, as inhibitory receptors, only the inhibitory KIR(s) specific for the mismatched KIR-Ligand(s) (HLA-C1, HLA-C2, HLA-Bw4). However, in KIR2DS2+ donors and HLA-C1+ patients, the unavailability of reagents staining only the inhibitory counterpart (KIR2DL2/L3) may lead to an underestimated identification of the alloreactive NK cell subset. Conversely, in the case of HLA-C1 mismatch, the alloreactive NK cell subset could be overestimated due to the ability of KIR2DL2/L3 to recognize with low-affinity also HLA-C2. Especially in this context, the additional exclusion of LIR1-expressing cells might be relevant to refine the size of the alloreactive NK cell subset. We could also associate degranulation assays, using as effector cells IL-2 activated donor peripheral blood mononuclear cells (PBMC) or NK cells upon co-culture with the related patient target cells. The donor alloreactive NK cell subset always displayed the highest functional activity, confirming its identification accuracy by flow cytometry. Despite the phenotypic limitations and considering the proposed corrective actions, a good correlation was shown by the comparison of the two investigated approaches. In addition, the characterization of receptor expression on a fraction of NK cell clones revealed expected but also few unexpected patterns. Thus, in most instances, the quantification of phenotypically defined alloreactive NK cells from PBMC can provide data similar to the analysis of lytic clones, with several advantages, such as a shorter time to achieve the results and, perhaps, higher reproducibility/feasibility in many laboratories.

CD19-Targeted Immunotherapies for Diffuse Large B-Cell Lymphoma.

Surgical resection, chemotherapy and radiotherapy were, for many years, the only available cancer treatments. Recently, the use of immune checkpoint inhibitors and adoptive cell therapies has emerged as promising alternative. These cancer immunotherapies are aimed to support or harness the patient's immune system to recognize and destroy cancer cells. Preclinical and clinical studies, based on the use of T cells and more recently NK cells genetically modified with chimeric antigen receptors retargeting the adoptive cell therapy towards tumor cells, have already shown remarkable results. In this review, we outline the latest highlights and progress in immunotherapies for the treatment of Diffuse Large B-cell Lymphoma (DLBCL) patients, focusing on CD19-targeted immunotherapies. We also discuss current clinical trials and opportunities of using immunotherapies to treat DLBCL patients.

Post-Transplant Nivolumab Plus Unselected Autologous Lymphocytes in Refractory Hodgkin Lymphoma: A Feasible and Promising Salvage Therapy Associated With Expansion and Maturation of NK Cells.

Immune checkpoint inhibitors (CI) have demonstrated clinical activity in Hodgkin Lymphoma (HL) patients relapsing after autologous stem cell transplantation (ASCT), although only 20% complete response (CR) rate was observed. The efficacy of CI is strictly related to the host immune competence, which is impaired in heavily pre-treated HL patients. Here, we aimed to enhance the activity of early post-ASCT CI (nivolumab) administration with the infusion of autologous lymphocytes (ALI). Twelve patients with relapse/refractory (R/R) HL (median age 28.5 years; range 18-65), underwent lymphocyte apheresis after first line chemotherapy and then proceeded to salvage therapy. Subsequently, 9 patients with progressive disease at ASCT received early post-transplant CI supported with four ALI, whereas 3 responding patients received ALI alone, as a control cohort. No severe adverse events were recorded. HL-treated patients achieved negative PET scan CR and 8 are alive and disease-free after a median follow-up of 28 months. Four patients underwent subsequent allogeneic SCT. Phenotypic analysis of circulating cells showed a faster expansion of highly differentiated NK cells in ALI plus nivolumab-treated patients as compared to control patients. Our data show anti-tumor activity with good tolerability of ALI + CI for R/R HL and suggest that this setting may accelerate NK cell development/maturation and favor the expansion of the "adaptive" NK cell compartment in patients with HCMV seropositivity, in the absence of HCMV reactivation.

EZH1/2 Inhibitors Favor ILC3 Development from Human HSPC-CD34+ Cells.

The dysregulation of epigenetic modifications has a well-established role in the development and progression of hematological malignancies and of solid tumors. In this context, EZH1/2 inhibitors have been designed to interfere with EZH1/2 enzymes involved in histone methylation (e.g., H3K27me3), leading to tumor growth arrest or the restoration of tumor suppressor gene transcription. However, these compounds also affect normal hematopoiesis, interfering with self-renewal and differentiation of CD34+-Hematopoietic Stem/Progenitor Cells (HSPC), and, in turn, could modulate the generation of potential anti-tumor effector lymphocytes. Given the important role of NK cells in the immune surveillance of tumors, it would be useful to understand whether epigenetic drugs can modulate NK cell differentiation and functional maturation. CD34+-HSPC were cultured in the absence or in the presence of the EZH1/2 inhibitor UNC1999 and EZH2 inhibitor GSK126. Our results show that UNC1999 and GSK126 increased CD56+ cell proliferation compared to the control condition. However, UNC1999 and GSK 126 favored the proliferation of no-cytotoxic CD56+ILC3, according to the early expression of the AHR and ROR-γt transcription factors. Our results describe novel epigenetic mechanisms involved in the modulation of NK cell maturation that may provide new tools for designing NK cell-based immunotherapy.

Clinical Effects of Driver Somatic Mutations on the Outcomes of Patients With Myelodysplastic Syndromes Treated With Allogeneic Hematopoietic Stem-Cell Transplantation.

PURPOSE: The genetic basis of myelodysplastic syndromes (MDS) is heterogeneous, and various combinations of somatic mutations are associated with different clinical phenotypes and outcomes. Whether the genetic basis of MDS influences the outcome of allogeneic hematopoietic stem-cell transplantation (HSCT) is unclear. PATIENTS AND METHODS: We studied 401 patients with MDS or acute myeloid leukemia (AML) evolving from MDS (MDS/AML). We used massively parallel sequencing to examine tumor samples collected before HSCT for somatic mutations in 34 recurrently mutated genes in myeloid neoplasms. We then analyzed the impact of mutations on the outcome of HSCT. RESULTS: Overall, 87% of patients carried one or more oncogenic mutations. Somatic mutations of ASXL1, RUNX1, and TP53 were independent predictors of relapse and overall survival after HSCT in both patients with MDS and patients with MDS/AML (P values ranging from .003 to .035). In patients with MDS/AML, gene ontology (ie, secondary-type AML carrying mutations in genes of RNA splicing machinery, TP53-mutated AML, or de novo AML) was an independent predictor of posttransplantation outcome (P = .013). The impact of ASXL1, RUNX1, and TP53 mutations on posttransplantation survival was independent of the revised International Prognostic Scoring System (IPSS-R). Combining somatic mutations and IPSS-R risk improved the ability to stratify patients by capturing more prognostic information at an individual level. Accounting for various combinations of IPSS-R risk and somatic mutations, the 5-year probability of survival after HSCT ranged from 0% to 73%. CONCLUSION: Somatic mutation in ASXL1, RUNX1, or TP53 is independently associated with unfavorable outcomes and shorter survival after allogeneic HSCT for patients with MDS and MDS/AML. Accounting for these genetic lesions may improve the prognostication precision in clinical practice and in designing clinical trials.

Wilms Tumor 1 Expression and Pre-emptive Immunotherapy in Patients with Acute Myeloid Leukemia Undergoing an Allogeneic Hemopoietic Stem Cell Transplantation.

Minimal residual disease (MRD) was monitored by Wilms tumor 1 (WT1) expression in 207 patients with acute myeloid leukemia (AML) after an allogeneic hemopoietic stem cell transplantation (HSCT) as a trigger to initiate pre-emptive immunotherapy (IT) with cyclosporin discontinuation and/or donor lymphocyte infusion. The trigger for IT was WT1 ≥ 180 copies/10(4) Abelson cells in marrow cells in the first group of 122 patients (WT1-180) and ≥ 100 copies in a subsequent group of 85 patients (WT1-100). Forty patients received IT. The cumulative incidence (CI) of relapse was 76% in WT1-180 (n = 17) versus 29% in WT1-100 patients (n = 23) receiving IT (P = .006); the leukemia-free survival from MRD positivity was 23% versus 74%, respectively (P = .003). We then looked at the entire AML patient population (n = 207). WT1-180 and WT1-100 patients were comparable for disease phase and age. The overall 4-year CI of transplantation-related mortality was 13% in both groups; the CI of leukemia relapse was 38% in the WT1-180 and 28% in the WT1-100 patients (P = .05) and leukemia-free survival was 56% versus 48%, respectively (P = .07). In conclusion, we suggests that WT1-based pre-emptive immunotherapy is feasible in patients with undergoing an allogeneic HSCT. The protective effect on relapse is greater when IT is triggered at lower levels of WT1.

Improved Outcome of Alternative Donor Transplantations in Patients with Myelofibrosis: From Unrelated to Haploidentical Family Donors.

This is a retrospective analysis of 95 patients with myelofibrosis who were allografted between 2001 and 2014. The aims of the study were to assess whether the outcome of alternative donor grafts has improved with time and how this compares with the outcome of identical sibling grafts. Patients were studied in 2 time intervals: 2000 to 2010 (n = 58) and 2011 to 2014 (n = 37). The Dynamic International Prognostic Scoring System score was comparable in the 2 time periods, but differences in the most recent group included older age (58 versus 53 years, P = .004), more family haploidentical donors (54% versus 5%, P < .0001), and the introduction of the thiotepa-fludarabine-busulfan conditioning regimen (70% of patients versus 2%, P < .0001). Acute and chronic graft-versus-host disease were comparable in the 2 time periods. The 3-year transplantation-related mortality (TRM) in the 2011 to 2014 period versus the 2000 to 2010 period is 16% versus 32% (P = .10), the relapse rate 16% versus 40% (P = .06), and actuarial survival 70% versus 39% (P = .08). Improved survival was most pronounced in alternative donor grafts (69% versus 21%, P = .02), compared with matched sibling grafts (72% versus 45%, P = .40). In conclusion, the outcome of allografts in patients with myelofibrosis has improved in recent years because of a reduction of both TRM and relapse. Improvement is most significant in alternative donor transplantations, with modifications in donor type and conditioning regimen.

CD34 selected cells for the treatment of poor graft function after allogeneic stem cell transplantation.

Poor graft function (PGF) is characterized by pancytopenia and a hypoplastic marrow, with complete donor chimerism, usually without severe graft-versus-host disease (GVHD). We report 41 patients with PGF, treated with granulocyte colony-stimulating factor-mobilized CD34 selected cells, at a median interval from transplant of 140 days, without conditioning and without GVHD prophylaxis. Donors were HLA matched siblings (n = 12), unrelated donors (n = 18), or mismatched family members (n = 11). The median number of infused CD34(+) cells was 3.4 × 10(6)/kg. The rate of trilineage recovery was 75%: 83% for HLA matched siblings and 72% for unrelated and mismatched family members (P = .3). The cumulative incidence of acute grade II GVHD was 15%, and no patient developed de novo chronic GVHD. The actuarial 3-year survival is 63%: 76% and 25% for patients with or without trilineage recovery. These data confirm the role of CD34(+) selected cells from the same donor in the treatment of PGF and warrant the request for a second donation also when the donor is unrelated.

Leukaemia relapse after allogeneic transplants for acute myeloid leukaemia: predictive role of WT1 expression.

We assessed WT1 expression (expressed as messenger copies/10(4) ABL1) from marrow cells of 122 patients with acute myeloid leukaemia (AML), before and after an unmanipulated allogeneic haemopoietic stem cell transplant (HSCT). The median age was 44 years (15-69), 59% were in first remission, 74% received a myeloablative conditioning regimen and the median follow up was 865 d (34-2833). Relapse was higher in 67 patients with WT1 expression, at any time post-HSCT, exceeding 100 copies (54%), as compared to 16%, for 55 patients with post-HSCT WT1 expression <100 copies (P < 0·0001). Similarly, actuarial 5-year survival (OS) was 40% vs. 63%, respectively (P = 0·03). In multivariate Cox analysis, WT1 expression post-HSCT was the strongest predictor of relapse (Hazard Ratio [HR] 4·5, P = 0·0001), independent of disease phase (HR 2·3, P = 0·002). Donor lymphocyte infusions (DLI) were given to 17 patients because of increasing WT1 levels: their OS was 44%, vs. 14% for 21 patients with increasing WT1 expression who did not receive DLI (P = 0·004). In conclusion, WT1 expression post-HSCT is a strong predictor of leukaemia relapse and survival in AML; WT1 may be used as a marker for early interventional therapy.

Unmanipulated haploidentical bone marrow transplantation and posttransplantation cyclophosphamide for hematologic malignancies after myeloablative conditioning.

Fifty patients with high-risk hematologic malignancies, underwent an unmanipulated haploidentical bone marrow transplantation (BMT), followed by posttransplantation high-dose cyclophosphamide (PT-CY): the myeloablative (MA) conditioning consisted of thiotepa, busulfan, fludarabine (n = 35), or total body irradiation (TBI), fludarabine (n = 15). The median age was 42 years (range, 18-66 years); 23 patients were in remission, 27 had active disease, and 10 patients were receiving a second allograft. Graft-versus-host disease (GVHD) prophylaxis consisted in PT-CY on day +3 and +5, cyclosporine (from day 0), and mycophenolate (from day +1). Three patients died before engraftment, and 2 patients had autologous recovery: 45 patients (90%) had full-donor chimerism on day +30. The median day for neutrophil engraftment was day +18 (range, 13-30 days). The cumulative incidence of grade II-III acute GVHD (aGVHD) was 12%, and of moderate chronic GVHD (cGVHD) 10%. With a median follow-up for surviving patients of 333 days (range, 149-623 days), the cumulative incidence of transplantation-related mortality (TRM) was 18%, and the rate of relapse was 26%. The actuarial 22-month disease-free survival (DFS) rate was 68% for patients in remission and 37% for patients with active disease (P < .001). Causes of death were pneumonia (n = 3), hemorrhage (n = 3), sepsis (n = 3), and relapse (n = 7). In conclusion, an MA conditioning regimen followed by haploidentical BMT with PT-CY results in a low risk of aGVHD and cGVHD and encouraging rates of TRM and DFS.

T-cell suppression mediated by mesenchymal stem cells is deficient in patients with severe aplastic anemia.

OBJECTIVE: To compare the suppressive effect of mesenchymal stem cells (MSC), derived from normal individuals or severe aplastic anemia patients (SAA), on T-cell activation. PATIENTS AND METHODS: We studied bone marrow MSC from 19 healthy donors and 23 SAA patients in different phases of the disease: at diagnosis (n = 3), following immunosuppressive therapy (IS) (n = 16), or after a bone marrow transplant (BMT) (n = 4). MSC were tested for T-cell suppression in the following assays: mixed lymphocyte reaction (MLR), phytohemaglutinin (PHA)-primed cultures, activation surface markers, gamma-IFN production, hematopoietic colony formation (CFC), production of cyclic ADP-ribose (cADPR). RESULTS: The abnormalities of SAA MSC included: 1) significantly lower suppression of T-cell proliferation induced by alloantigens (p = 0.009) or PHA (p = 0.006); 2) impaired capacity to suppress CD38 expression on PHA-primed T cells (p = 0.001); 3) impaired ability to suppress gamma-IFN production in PHA cultures, resulting in an 11-fold higher gamma-IFN concentration; 4) no preventive effect on T cell-mediated inhibition of CFC; and 5) significantly reduced (p = 0.009) production of cADPR, a universal calcium mobilizer. MSC-mediated suppression of PHA-induced T-cell proliferation was restored to control levels in 3 of 4 patients post-BMT. CONCLUSION: The ability of MSC to downregulate T-cell priming, proliferation, and cytokine release is deficient in patients with SAA, persists indefinitely after immunosuppressive therapy, but seems to be restored after BMT. Whether these abnormalities are relevant to the pathogenesis of aplastic anemia remains to be determined.