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 ¼ 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 ¼ 0.001) and increase of survival time (P ¼ 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 GVHDwas associated withthe 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.

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.

The online version of this article contains a supplementary appendix. BackgroundThe 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 MethodsMinimal 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. ResultsThree 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. ConclusionsThese 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.Key words: chronic lymphocytic leukemia; minimal residual disease; allogeneic stem cell transplantation. Haematologica 2009; 94:654-662. doi:10.3324/haematol.2008 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

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.

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-