Next-generation sequencing of a family with a high penetrance of monoclonal gammopathies for the identification of candidate risk alleles.

BACKGROUND: The authors describe a family with a high penetrance of plasma cell dyscrasias, suggesting inheritance of an autosomal dominant risk allele. METHODS: The authors performed whole-exome sequencing and reported on a combined approach aimed at the identification of causative variants and risk loci, using the wealth of data provided by this approach. RESULTS: The authors identified gene mutations and single-nucleotide polymorphisms of potential significance, and pinpointed a known risk locus for myeloma as a potential area of transmissible risk in the family. CONCLUSIONS: To the authors' knowledge, the current study is the first to provide a whole-exome sequencing approach to such cases, and a framework analysis that could be applied to further understanding of the inherited risk of developing plasma cell dyscrasias. Cancer 2017;123:3701-3708. © 2017 American Cancer Society.

Graft monocytic myeloid-derived suppressor cell content predicts the risk of acute graft-versus-host disease after allogeneic transplantation of granulocyte colony-stimulating factor-mobilized peripheral blood stem cells.

Myeloid-derived suppressor cells (MDSCs) are powerful immunomodulatory cells that in mice play a role in infectious and inflammatory disorders, including acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. Their relevance in clinical acute GVHD is poorly known. We analyzed whether granulocyte colony-stimulating factor (G-CSF) administration, used to mobilize hematopoietic stem cells, affected the frequency of MDSCs in the peripheral blood stem cell grafts of 60 unrelated donors. In addition, we evaluated whether the MDSC content in the peripheral blood stem cell grafts affected the occurrence of acute GVHD in patients undergoing unrelated donor allogeneic stem cell transplantation. Systemic treatment with G-CSF induces an expansion of myeloid cells displaying the phenotype of monocytic MDSCs (Lin(low/neg)HLA-DR(-)CD11b(+)CD33(+)CD14(+)) with the ability to suppress alloreactive T cells in vitro, therefore meeting the definition of MDSCs. Monocytic MDSC dose was the only graft parameter to predict acute GVHD. The cumulative incidence of acute GVHD at 180 days after transplantation for recipients receiving monocytic MDSC doses below and above the median was 63% and 22%, respectively (P = .02). The number of monocytic MDSCs infused did not impact the relapse rate or the transplant-related mortality rate (P > .05). Although further prospective studies involving larger sample size are needed to validate the exact monocytic MDSC graft dose that protects from acute GVHD, our results strongly suggest the modulation of G-CSF might be used to affect monocytic MDSCs graft cell doses for prevention of acute GVHD.

Long-term patterns of humoral and cellular response after vaccination against influenza A (H1N1) in patients with hematologic malignancies.

OBJECTIVES: The efficacy of a novel vaccine against influenza virus A (H1N1) in patients with hematologic malignancies is largely unknown. METHODS: We prospectively evaluated the humoral and cellular immune responses after one injection of monovalent adjuvanted 2009 H1N1 vaccine in 47 adults with hematologic malignancies and 77 controls by hemagglutination-inhibition assay and flow-cytometry analysis on day 0, 28, 50, and 90. RESULTS: On day 28 postvaccination, patients had lower seroprotection (95.2% vs. 75.2%, P < 0.01) and seroconversion (88.7% vs. 51.1%, P < 0.01) rates, as well as geometric mean titer (GMT; 256 vs. 134, P < 0.05), relative to controls. Response to vaccination varied according to the evaluated time point and the patient status: Patients not receiving chemotherapy had seroprotection and GMTs similar to controls in all time points, while patients receiving chemotherapy or allogeneic hematopoietic stem cell transplant (HSCT) had lower seroprotection and seroconversion levels than controls on day 28 and 50. EMEA cutoffs for efficacy were reached from day 28 by patients in follow-up or under treatment and only from day 90 by those with HSCT, especially if still under immunosuppressants. Patients treated with immunomodulatory drugs had higher antibody responses in terms of seroprotection and GMTs. T- and NK cell-mediated responses mounted from day 50 and did not differ between patients and controls. CONCLUSIONS: According to EMEA recommendation, H1N1 vaccination strategy was effective at protecting most of the hematologic patients, but needed to be improved in those more immunocompromised.

Haploidentical stem cell transplantation after a reduced-intensity conditioning regimen for the treatment of advanced hematologic malignancies: posttransplantation CD8-depleted donor lymphocyte infusions contribute to improve T-cell recovery.

Haploidentical hematopoietic stem cell transplantation provides an option for patients with advanced hematologic malignancies lacking a compatible donor. In this prospective phase 1/2 trial, we evaluated the role of reduced-intensity conditioning (RIC) followed by early add-backs of CD8-depleted donor lymphocyte infusions (DLIs). The RIC regimen consisted of thiotepa, fludarabine, cyclophosphamide, and 2 Gy total body irradiation. Twenty-eight patients with advanced lymphoproliferative diseases (n = 24) or acute myeloid leukemia (n = 4) were enrolled. Ex vivo and in vivo T-cell depletion was carried out by CD34(+) cell selection and alemtuzumab treatment. The 2-year cumulative incidence of nonrelapse mortality was 26% and the 2-year overall survival (OS) was 44%, with a better outcome for patients with chemosensitive disease (OS, 75%). Overall, 54 CD8-depleted DLIs were administered to 23 patients (82%) at 3 different dose levels without loss of engraftment or acute toxicities. Overall, 6 of 23 patients (26%) developed grade II-IV graft-versus-host disease, mainly at dose level 2. In conclusion, our RIC regimen allowed a stable engraftment with a rather low nonrelapse mortality in poor-risk patients; OS is encouraging with some long-term remissions in lymphoid malignancies. CD8-depleted DLIs are feasible and promote the immune reconstitution.

Qualitative and quantitative polymerase chain reaction monitoring of minimal residual disease in relapsed chronic lymphocytic leukemia: early assessment can predict long-term outcome after reduced intensity allogeneic transplantation.

BACKGROUND: The graft-versus-leukemia effect is able to induce clinical responses in patients with chronic lymphocytic leukemia treated with a reduced intensity conditioning regimen, followed by allogeneic stem cell transplantation. We investigated whether molecular remissions could be attained after reduced intensity conditioning and allogeneic stem cell transplantation in patients with relapsed chronic lymphocytic leukemia and whether the assessment of minimal residual disease might be used to predict the clinical outcome. DESIGN AND METHODS: Minimal residual disease was monitored by polymerase chain reaction using the immunoglobulin heavy-chain gene rearrangement as a molecular marker in 29 relapsed patients who achieved complete remission following reduced intensity conditioning and allogeneic stem cell transplantation. A nested-polymerase chain reaction with patient-specific primers derived from complementarity determining regions (CDR2 and CDR3) was carried out in all the patients. Real-time polymerase chain reaction was performed in patients whose nested reaction gave positive or mixed results. RESULTS: Three patterns of minimal residual disease were observed: negative (31%), mixed (24%), and always positive (45%). The cumulative incidence of relapse according to the minimal residual disease status at 6 and 12 months after transplantation was significantly different between polymerase chain reaction-negative and -positive patients (p=0.031 and p=0.04, respectively). Two-year disease-free survival was 93% and 46% for polymerase chain reaction-negative and -positive patients at 6 months after transplantation, respectively (p=0.012). Similarly, 2-year disease-free survival was 100% and 57% for polymerase chain reaction-negative and -positive patients at 12 months, respectively (p=0.037). No clinical or biological factors were predictive of the achievement of polymerase chain reaction negativity after allogeneic stem cell transplantation. Graft-versus-host disease was more frequent in patients who did not relapse (p=0.04). Quantitative monitoring of minimal residual disease was able to identify polymerase chain reaction-positive patients with a higher risk of relapse. CONCLUSIONS: These findings demonstrate that relapsed patients can achieve molecular remission after reduced intensity conditioning and allogeneic stem cell transplantation and suggest a minimal residual disease-driven intervention that might be useful to prevent overt hematologic relapse.

Noninvasive Molecular Monitoring in Multiple Myeloma Patients Using Cell-Free Tumor DNA: A Pilot Study.

Novel treatments for multiple myeloma (MM) have increased rates of complete response, raising interest in more accurate methods to evaluate residual disease. Cell-free tumor DNA (cfDNA) analysis may represent a minimally invasive approach complementary to multiparameter flow cytometry (MFC) and molecular methods on bone marrow aspirates. A sequencing approach using the Ion Torrent Personal Genome Machine was applied to identify clonal IGH gene rearrangements in tumor plasma cells (PCs) and in serial plasma samples of 25 patients with MM receiving second-line therapy. The same clonal IGH rearrangement identified in tumor PCs was detected in paired plasma samples, and levels of IGH cfDNA correlated with outcome and mirrored tumor dynamics evaluated using conventional laboratory parameters. In addition, IGH cfDNA levels reflected the number of PCs enumerated by MFC immunophenotyping even in the complete response context. Patients determined by MFC to be free of minimal residual disease were characterized by low frequencies of tumor clonotypes in cfDNA and longer survival. This pilot study supports the clinical applicability of the noninvasive monitoring of tumor levels in plasma samples of patients with MM by IGH sequencing.

Circulating miRNA panel for prediction of acute graft-versus-host disease in lymphoma patients undergoing matched unrelated hematopoietic stem cell transplantation.

Acute graft-versus-host disease (aGVHD) results in significant morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Noninvasive diagnostic and prognostic tests for aGVHD are currently lacking, but would be beneficial in predicting aGVHD and improving the safety of allo-HSCT. Circulating microRNAs exhibit marked stability and may serve as biomarkers in several clinical settings. Here, we evaluated the use of circulating microRNAs as predictive biomarkers of aGVHD in lymphoma patients after allo-HSCT from matched unrelated donors (MUDs). After receiving informed consent, we prospectively collected plasma samples from 24 lymphoma patients before and after unmanipulated MUD allo-HSCT; microRNAs were then isolated. Fourteen patients developed aGVHD symptoms at a median of 48 days (range: 32-90) post-transplantation. Two patients developed intestinal GVHD, eight cutaneous GVHD, and four multiorgan GVHD. The microRNA expression profile was examined using quantitative real-time polymerase chain reaction (qRT-PCR). MicroRNAs 194 and 518f were significantly upregulated in aGVHD samples compared with samples taken from non-aGVHD patients. Remarkably, these upregulated microRNAs could be detected before the onset of aGVHD. Pathway prediction analysis indicated that these microRNAs may regulate critical pathways involved in aGVHD pathogenesis. Considering the noninvasive characteristics of plasma sampling and the feasibility of detecting miRNAs after allo-HSCT using real-time polymerase chain reaction, our results indicate that circulating microRNAs have the potential to enable an earlier aGVHD diagnosis and might assist in individualizing therapeutic strategies after MUD allo-HSCT. Nevertheless, standardization of blood sampling and analysis protocols is mandatory for the introduction of miRNA profiling into routine clinical use.

Pharmacologic Inhibition of JAK1/JAK2 Signaling Reduces Experimental Murine Acute GVHD While Preserving GVT Effects.

PURPOSE: Immune-mediated graft-versus-tumor (GVT) effects can occur after allogeneic hematopoietic stem cell transplantation (HSCT), but GVT is tightly linked to its main complication, graft-versus-host disease (GVHD). Strategies aimed at modulating GVHD, while maintaining the GVT effect, are needed to improve the cure rate of transplant. Given the emerging role of Janus-activated kinase (JAK) signaling in lymphoproliferative and myeloproliferative diseases and its established function at dictating T-cell differentiation, we postulated that JAKs might be potential therapeutic targets through a pharmacologic approach. EXPERIMENTAL DESIGN: We examined the effect of JAK1/JAK2 modulation by ruxolitinib in a mouse model of fully MHC mismatched bone marrow transplant comprising in vivo tumor inoculation. RESULTS: JAK1/JAK2 inhibition by ruxolitinib improved both overall survival (P = 0.03) and acute GVHD pathologic score at target organs (P ≤ 0.001) of treated mice. In addition, treatment with ruxolitinib was associated with a preserved GVT effect, as evidenced by reduction of tumor burden (P = 0.001) and increase of survival time (P = 0.01). JAK1/JAK2 inhibition did not impair the in vivo acquisition of donor T-cell alloreactivity; this observation may account, at least in part, to the preserved GVT effect. Rather, JAK1/JAK2 inhibition of GVHD was associated with the modulation of chemokine receptor expression, which may have been one factor in the reduced infiltration of donor T cells in GVHD target organs. CONCLUSIONS: These data provide further evidence that JAK inhibition represents a new and potentially clinically relevant approach to GVHD prevention.