Patient-Specific Measurable Residual Disease Markers Predict Outcome in Patients With Myelodysplastic Syndrome and Related Diseases After Hematopoietic Stem-Cell Transplantation.

PURPOSE: Clinical relapse is the major threat for patients with myelodysplastic syndrome (MDS) undergoing hematopoietic stem-cell transplantation (HSCT). Early detection of measurable residual disease (MRD) would enable preemptive treatment and potentially reduced relapse risk. METHODS: Patients with MDS planned for HSCT were enrolled in a prospective, observational study evaluating the association between MRD and clinical outcome. We collected bone marrow (BM) and peripheral blood samples until relapse, death, or end of study 24 months after HSCT. Patient-specific mutations were identified with targeted next-generation sequencing (NGS) panel and traced using droplet digital polymerase chain reaction (ddPCR). RESULTS: Of 266 included patients, estimated relapse-free survival (RFS) and overall survival (OS) rates 3 years after HSCT were 59% and 64%, respectively. MRD results were available for 221 patients. Relapse was preceded by positive BM MRD in 42/44 relapses with complete MRD data, by a median of 71 (23-283) days. Of 137 patients in continuous complete remission, 93 were consistently MRD-negative, 39 reverted from MRD+ to MRD-, and five were MRD+ at last sampling. Estimated 1 year-RFS after first positive MRD was 49%, 39%, and 30%, using cutoff levels of 0.1%, 0.3%, and 0.5%, respectively. In a multivariate Cox model, MRD (hazard ratio [HR], 7.99), WHO subgroup AML (HR, 4.87), TP53 multi-hit (HR, 2.38), NRAS (HR, 3.55), and acute GVHD grade III-IV (HR, 4.13) were associated with shorter RFS. MRD+ was also independently associated with shorter OS (HR, 2.65). In a subgroup analysis of 100 MRD+ patients, presence of chronic GVHD was associated with longer RFS (HR, 0.32). CONCLUSION: Assessment of individualized MRD using NGS + ddPCR is feasible and can be used for early detection of relapse. Positive MRD is associated with shorter RFS and OS (ClinicalTrials.gov identifier: NCT02872662).

Tumour-loaded α-type 1-polarized dendritic cells from patients with chronic lymphocytic leukaemia produce a superior NK-, NKT- and CD8+ T cell-attracting chemokine profile.

Tumour-loaded dendritic cells (DCs) from patients with chronic lymphocytic leukaemia (CLL) matured using an α-type 1-polarized DC cocktail (IL-1ß/TNF-α/IFN-α/IFN-γ/poly-I:C;αDC1) were recently shown to induce more functional CD8(+) T cells against autologous tumour cells in vitro than DCs matured with the 'standard' cocktail (IL-1ß/TNF-α/IL-6/PGE(2) ;PGE(2) DCs). However, the ability of vaccine DCs to induce a type 1-polarized immune response in vivo probably relies on additional features, including their ability to induce a CXCR3-dependent recruitment of NK cells into vaccine-draining lymph nodes. Moreover, their guiding of rare tumour-specific CD8(+) T cells to sites of DC-CD4(+) T cell interactions by secretion of CCL3 and CCL4 is needed. We therefore analysed the chemokine profile and the lymphocyte-attracting ability in vitro of monocyte-derived PGE(2) DCs and αDC1s from patients with CLL. αDC1s produced much higher levels of CXCR3 ligands (CXCL9/CXCL10/CXCL11) than PGE(2) DCs. Functional studies further demonstrated that αDC1s were superior recruiters of both NK and NKT cells. Moreover, αDC1s produced higher levels of CCL3/CCL4 upon CD40 ligation. These findings suggest that functional αDC1s, derived from patients with CLL, produce a desirable NK-, NKT- and CD8(+) T cell-attracting chemokine profile which may favour a guided and Th1-deviated priming of CD8(+) T cells, supporting the idea that αDC1-based vaccines have a higher immunotherapeutic potential than PGE(2) DCs.

Allogeneic Hematopoietic Stem Cell Transplantation for Chronic Myelomonocytic Leukemia: Clinical and Molecular Genetic Prognostic Factors in a Nordic Population.

Chronic myelomonocytic leukemia (CMML) is an aggressive disease in which survival after allogeneic hematopoietic stem cell transplantation (HCT) remains relatively poor. An assessment of prognostic factors is an important part of treatment decision making and has the potential to be greatly improved by the inclusion of molecular genetics. However, there is a significant knowledge gap in the interpretation of mutational patterns. This study aimed to describe outcomes of allogeneic HCT in patients with CMML in relation to clinical and molecular genetic risk factors. This retrospective study included 64 patients with CMML who underwent allogeneic HCT between 2008 and 2018, with a median follow-up of 5.4 years. Next-generation sequencing using targeted myeloid panels was carried out on saved material from 51 patients from the time of transplantation. Kaplan-Meier and Cox regression were used for analysis of overall survival (OS), and cumulative incidence with competing risks and Fine and Gray models were used for analysis of relapse and nonrelapse mortality (NRM). Mutations were detected in 48 patients (94%), indicating high levels of minimal residual disease (MRD) positivity at transplantation, even among those in complete remission (CR) (n = 14), 86% of whom had detectable mutations. The most frequently mutated genes were ASXL1 (37%), TET2 (37%), RUNX1 (33%), SRSF2 (26%), and NRAS (20%). Risk stratification using the CMML-specific Prognostic Scoring System molecular score (CPSS-Mol) resulted in 45% of patients moving to a higher risk-group compared with risk stratification using the CPSS. High leucocyte count (≥13 × 109/L), transfusion requirement, and previous intensive chemotherapy were associated with higher incidence of relapse. Being in CR was not linked to better outcomes. Neither ASXL1 nor RUNX1 mutation was associated with a difference in OS, relapse, or NRM, despite being high risk in the nontransplantation setting. TET2 mutations were associated with a significantly higher 3-year OS (73% versus 40%; P = .039). Achieving MRD-negative CR was rare in this CMML cohort, which may explain why we did not observe better outcomes for those in CR. This merits further investigation. Our analyses suggest that the negative impact of ASXL1 and RUNX1 mutations can be overcome by allogeneic HCT; however, risk stratification is complex in CMML and requires larger cohorts and multivariate models, presenting an ongoing challenge in this rare disease.

Idelalisib Rescues Natural Killer Cells from Monocyte-Induced Immunosuppression by Inhibiting NOX2-Derived Reactive Oxygen Species.

The phosphatidylinositol-4,5-bisphosphate-3 kinase-δ (PI3Kδ) inhibitor idelalisib, used alone or in combination with anti-CD20, is clinically efficacious in B-cell lymphoma and chronic lymphocytic leukemia (CLL) by promoting apoptosis of malignant B cells. PI3K regulates the formation of reactive oxygen species (ROS) by the myeloid NADPH oxidase NOX2, but the role of PI3Kδ in myeloid cell-induced immunosuppression is unexplored. We assessed the effects of idelalisib on the spontaneous and IgG antibody-induced ROS production by human monocytes, on ROS-induced cell death of human natural killer (NK) cells, and on tumor cell clearance in an NK cell-dependent mouse model of metastasis. Idelalisib potently and efficiently inhibited the formation of NOX2-derived ROS from monocytes and rescued NK cells from ROS-induced cell death. Idelalisib also promoted NK cell cytotoxicity against anti-CD20-coated primary human CLL cells and cultured malignant B cells. Experiments using multiple PI3K inhibitors implicated the PI3Kδ isoform in regulating NOX2-induced ROS formation and immunosuppression. In B6 mice, systemic treatment with idelalisib significantly reduced the formation of lung metastases from intravenously injected melanoma cells but did not affect metastasis in B6.129S6-Cybbtm1Din (Nox2 -/-) mice or in NK cell-deficient mice. Our results imply that idelalisib rescues NK cells from NOX2/ROS-dependent immunosuppression and thus exerts antineoplastic efficacy beyond B-cell inhibition.

The expression of NK cell inhibitory receptors on cytotoxic T cells in B-cell chronic lymphocytic leukaemia (B-CLL).

Immune surveillance of tumours is mediated by cytotoxic T cells (CTL) that recognise tumour antigen. Reduced reactivity of CTL towards tumour cells could thus lead to disease progression and loss of tumour control. In B-cell chronic lymphocytic leukaemia (B-CLL), the function of tumour-reactive CTL seems to correlate inversely to disease stage. Inhibitory NK cell receptors are known to suppress the CTL response upon interaction with major histocompatibility complex (MHC) class I and increased expression of such receptors on CTL may inhibit the anti-tumour response. So, the aim of this study was to investigate the expression of NK cell inhibitory receptors on CTL in B-CLL patients and if such expression correlated to disease stage. CD8+ T cells from B-CLL patients in Binet stage A (n = 26) and stage C (n = 14) and healthy controls (n = 14) were analysed for the expression of killer immunoglobulin-like receptors (KIR) CD158a (KIR2DL1), CD158b (KIR2DL2), CD158e (KIR3DL1) and the C-type lectin receptor CD94, by flow cytometry analysis. Patients with advanced disease (Binet stage C) had a significantly greater percentage of CTL expressing CD158b, CD158e and CD94 than patients with non-progressive disease (Binet stage A) and healthy controls. Stage C patients also had a significantly higher percentage of CTL expressing CD158a than stage A patients. No statistically significant differences were found between Binet A patients and healthy controls. Our results suggest that increased expression of KIR and CD94 on CTL in advanced stage B-CLL may potentially contribute to the impaired anti-tumour immune response in these patients.

NOX2-dependent immunosuppression in chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a myeloproliferative and myelodysplastic neoplasm with few treatment options and dismal prognosis. The role of natural killer (NK) cells and other antileukemic lymphocytes in CMML is largely unknown. We aimed to provide insight into the mechanisms of immune evasion in CMML with a focus on immunosuppressive reactive oxygen species (ROS) formed by the myeloid cell NADPH oxidase-2 (NOX2). The dominant population of primary human CMML cells was found to express membrane-bound NOX2 and to release ROS, which, in turn, triggered extensive PARP-1-dependent cell death in cocultured NK cells, CD8+ T effector memory cells, and CD8+ T effector cells. Inhibitors of ROS formation and scavengers of extracellular ROS prevented CMML cell-induced lymphocyte death and facilitated NK cell degranulation toward Ab-coated, primary CMML cells. In patients with CMML, elevation of immature cell counts (CD34+) in blood was associated with reduced expression of several NK cell-activating receptors. We propose that CMML cells may use extracellular ROS as a targetable mechanism of immune escape.

Evaluation of clinical staging in chronic lymphocytic leukemia- population-based study.

The Rai and Binet staging systems are currently being challenged by the development of new biological methods to characterize the prognosis and management of chronic lymphocytic leukemia (CLL). To evaluate these two systems in recently diagnosed CLL patients, we performed a retrospective population-based study including 344 patients in western Sweden diagnosed between 1995 and 2000. Binet stage A patients had longer median overall survival (OS) (100 months) than stage B (55 months; P < 0.001) and C patients (45 months; P < 0.0005). Median OS for stage B and C could not be separated (P = 0.94). When transferring Rai stages into three groups, a similar pattern was found. Overall response differed only between Binet A and C patients and there was no difference regarding time to next treatment between any of the Binet stages. Finally, in both systems, low stage patients had inferior survival compared to age- and sex-matched controls. Our data emphasize the need for a new risk stratification system for CLL patients.

Reactive oxygen species induced by therapeutic CD20 antibodies inhibit natural killer cell-mediated antibody-dependent cellular cytotoxicity against primary CLL cells.

The antibody-dependent cellular cytotoxicity (ADCC) of natural killer (NK) cells is assumed to contribute to the clinical efficacy of monoclonal antibodies (mAbs) in chronic lymphocytic leukemia (CLL) and other hematopoietic malignancies of B cell origin. We sought to determine whether reactive oxygen species (ROS)-producing monocytes regulate the ADCC of NK cells against primary CLL cells using anti-CD20 as the linking antibody. The monoclonal CD20 antibodies rituximab and ofatumumab were found to trigger substantial release of ROS from monocytes. Antibody-exposed monocytes induced NK cell apoptosis and restricted NK cell-mediated ADCC against autologous CLL cells. The presence of inhibitors of ROS formation and scavengers of ROS preserved NK cell viability and restored NK cell-mediated ADCC against primary CLL cells. We propose that limiting the antibody-induced induction of immunosuppressive ROS may improve the anti-leukemic efficacy of anti-CD20 therapy in CLL.

Role of the ERK pathway for oxidant-induced parthanatos in human lymphocytes.

Reactive oxygen species (ROS) are formed by myeloid cells as a defense strategy against microorganisms. ROS however also trigger poly(ADP-ribose) polymerase 1- (PARP-1) dependent cell death (parthanatos) in adjacent lymphocytes, which has been forwarded as a mechanism of immune escape in several forms of cancer. The present study assessed the role of mitogen-activated protein kinases (MAPKs), in particular the extracellular signal-regulated kinase (ERK), in ROS-induced signal transduction leading to lymphocyte parthanatos. We report that inhibitors of ERK1/2 phosphorylation upheld natural killer (NK) cell-mediated cytotoxicity under conditions of oxidative stress and rescued NK cells and CD8(+) T lymphocytes from cell death induced by ROS-producing monocytes. ERK1/2 phosphorylation inhibition also protected lymphocytes from cell death induced by exogenous hydrogen peroxide (H2O2) and from ROS generated by xanthine oxidase or glucose oxidase. Phosphorylation of ERK1/2 was observed in lymphocytes shortly after exposure to ROS. ROS-generating myeloid cells and exogenous H2O2 triggered PARP 1-dependent accumulation of poly ADP-ribose (PAR), which was prevented by ERK pathway inhibitors. ERK1/2 phosphorylation was induced by ROS independently of PARP-1. Our findings are suggestive of a role for ERK1/2 in ROS-induced lymphocyte parthanatos, and that the ERK axis may provide a therapeutic target for the protection of lymphocytes against oxidative stress.

Improved outcome for very elderly patients with diffuse large B-cell lymphoma in the immunochemotherapy era.

The prognosis of diffuse large B-cell lymphoma (DLBCL) has improved significantly since the introduction of immunochemotherapy (rituximab [R] with cyclophosphamide, doxorubicin, vincristine, prednisone [CHOP]). However, few outcome data are available for very elderly patients (≥ 80 years). Therefore, we compared all patients with DLBCL aged ≥ 80 years diagnosed in the Gothenburg area during two time periods (2006-2009, "post-R" and 1997-2000, "pre-R"). Forty and 30 patients were identified, corresponding to 23.5% and 20.5%, respectively, of the entire population with DLBCL. Estimated 3-year progression-free (PFS) and overall (OS) survival was better post-R than pre-R: 41% vs. 17% (p = 0.015) and 41% vs. 17% (p = 0.01), respectively. Fifty-three percent of post-R patients were treated with curative intent with a moderately reduced R-CHOP regimen (median relative dose intensity: 0.86). At a median follow-up of 29 months, the 3-year PFS and OS were 70% (p = 0.018) and 76% (p = 0.0089), respectively. In conclusion, moderately reduced R-CHOP is tolerable and effective for a considerable number of very elderly patients with DLBCL and high age by itself should not be a reason for excluding a patient with DLBCL from such treatment.