Nobuhiko Imahashi

Target Antigen Density Governs the Efficacy of Anti–CD20-CD28-CD3 ζ Chimeric Antigen Receptor–Modified Effector CD8+ T Cells

The effectiveness of chimeric Ag receptor (CAR)–transduced T (CAR-T) cells has been attributed to supraphysiological signaling through CARs. Second- and later-generation CARs simultaneously transmit costimulatory signals with CD3ζ signals upon ligation, but may lead to severe adverse effects owing to the recognition of minimal Ag expression outside the target tumor. Currently, the threshold target Ag density for CAR-T cell lysis and further activation, including cytokine production, has not yet been investigated in detail. Therefore, we determined the threshold target Ag density required to induce CAR-T cell responses using novel anti-CD20 CAR-T cells with a CD28 intracellular domain and a CD20-transduced CEM cell model. The newly developed CD20CAR–T cells demonstrated Ag-specific lysis and cytokine secretion, which was a reasonable level as a second-generation CAR. For lytic activity, the threshold Ag density was determined to be ∼200 molecules per target cell, whereas the Ag density required for cytokine production of CAR-T cells was ∼10-fold higher, at a few thousand per target cell. CD20CAR–T cells responded efficiently to CD20-downregulated lymphoma and leukemia targets, including rituximab- or ofatumumab-refractory primary chronic lymphocytic leukemia cells. Despite the potential influence of the structure, localization, and binding affinity of the CAR/Ag, the threshold determined may be used for target Ag selection. An Ag density below the threshold may not result in adverse effects, whereas that above the threshold may be sufficient for practical effectiveness. CD20CAR–T cells also demonstrated significant lytic activity against CD20-downregulated tumor cells and may exhibit effectiveness for CD20-positive lymphoid malignancies.

Impact of macrophage infiltration of skin lesions on survival after allogeneic stem cell transplantation: a clue to refractory graft-versus-host disease

We retrospectively reviewed 104 biopsy specimens of previously untreated skin acute graft-versus-host disease (GVHD) within 100 days after allogeneic stem cell transplantation, and analyzed the relationship between types of infiltrating cells and clinical outcomes. Counting the total number of CD8(+) T cells, CD163(+) macrophages, and CD1a(+) dendritic cells in 4 fields under original magnification x200, the infiltration of more than 200 cells of CD163(+) macrophages (many macrophages [MM]) was the only significant predictor for refractory GHVD (odds ratio, 3.79; 95% confidence interval, 1.22-11.8; P = .02). In 46 patients given steroid treatments, MM was the only significant predictor for refractory acute GVHD (odds ratio, 5.05; 95% confidence interval, 1.19-21.3; P = .03). Overall survival of patients with MM was significantly lower than that of those with an infiltration of less than 200 cells of CD163(+) macrophages. Macrophage infiltration of skin lesions could be a significant predictive factor for refractory GVHD and a poor prognosis.

Specific combinations of donor and recipient KIR-HLA genotypes predict for large differences in outcome after cord blood transplantation

The ability of cord blood transplantation (CBT) to prevent relapse depends partly on donor natural killer (NK) cell alloreactivity. NK effector function depends on specific killer-cell immunoglobulin-like receptors (KIR) and HLA interactions. Thus, it is important to identify optimal combinations of KIR-HLA genotypes in donors and recipients that could improve CBT outcome. We studied clinical data, KIR and HLA genotypes, and NK-cell reconstitution in CBT patients (n = 110). Results were validated in an independent cohort (n = 94). HLA-KIR genotyping of recipient germline and transplanted cord blood (CB) grafts predicted for large differences in outcome. Patients homozygous for HLA-C2 group alleles had higher 1-year relapse rate and worse survival after CBT than did HLA-C1/C1 or HLA-C1/C2 (HLA-C1/x) patients: 67.8% vs 26.0% and 15.0% vs 52.9%, respectively. This inferior outcome was associated with delayed posttransplant recovery of NK cells expressing the HLA-C2-specific KIR2DL1/S1 receptors. HLA-C1/x patients receiving a CB graft with the combined HLA-C1-KIR2DL2/L3/S2 genotype had lower 1-year relapse rate (6.7% vs 40.1%) and superior survival (74.2% vs 41.3%) compared with recipients of grafts lacking KIR2DS2 or HLA-C1 HLA-C2/C2 patients had lower relapse rate (44.7% vs 93.4%) and better survival (30.1% vs 0%) if they received a graft with the combined HLA-C2-KIR2DL1/S1 genotype. Relapsed/refractory disease at CBT, recipient HLA-C2/C2 genotype, and donor HLA-KIR genotype were independent predictors of outcome. Thus, we propose the inclusion of KIR genotyping in graft selection criteria for CBT. HLA-C1/x patients should receive an HLA-C1-KIR2DL2/L3/S2 CB graft, while HLA-C2/C2 patients may benefit from an HLA-C2-KIR2DL1/S1 graft.

Eosinophilia predicts better overall survival after acute graft-versus-host-disease

The prognostic significance of eosinophilia after allogeneic hematopoietic SCT (HSCT) and the relationship between eosinophilia and acute GVHD are not well studied. We retrospectively analyzed 201 adult patients who underwent their first allogeneic HSCT. Seventy-three (36%) patients developed eosinophilia within the first 100 days after HSCT. Eosinophilia was observed more frequently among those patients with acute GVHD than those without it (48 vs 25%, P=0.009). However, it was associated with milder acute GVHD and lower incidence of gut and liver acute GVHD. Among patients with acute GVHD, the 3-year OS for patients with and without eosinophilia was 63.4 and 47.2% (P=0.02), respectively, and 3-year nonrelapse mortality (NRM) was 20.2 and 37.5% (P=0.01), respectively. Multivariate analysis confirmed that eosinophilia was associated with a better OS (P=0.03) and lower NRM (P=0.046) in patients with acute GVHD, whereas it was not associated with a higher relapse rate (P=0.45). In contrast, eosinophilia was not associated with outcomes in those patients without acute GVHD. In conclusion, eosinophilia was associated with milder acute GVHD and better prognosis among patients with acute GVHD. The pathophysiology behind eosinophilia after allogeneic HSCT remains to be investigated.

Efficacy of Continuous, Daily, Oral, Ultra-low-dose 200 mg Acyclovir to Prevent Herpes Zoster Events Among Bortezomib-treated Patients: A Report From Retrospective Study

OBJECTIVE Herpes zoster is the most common infection in patients treated with bortezomib-containing regimens for multiple myeloma. Some clinical trials have reported on the use of acyclovir prophylaxis to decrease the incidence of herpes zoster. However, the appropriate acyclovir dose and duration of prophylaxis remain unclear. The primary objective of this study was to evaluate the efficacy of continuous oral 200 mg/day acyclovir prophylaxis and the secondary objective was to determine the risk factors for developing herpes zoster. METHODS We collected medical information from consecutive patients who received bortezomib with or without acyclovir prophylaxis for relapsed or refractory multiple myeloma at our hospital and retrospectively analyzed the efficacy of acyclovir prophylaxis and the parameters for predicting the risk factors for developing herpes zoster. The definition of acyclovir prophylaxis was oral continuous administration of 200 mg of once daily, without cessation, during the entire period of bortezomib treatment. RESULTS Six of the 33 patients in the study developed herpes zoster during bortezomib treatment. No varicella-zoster virus reactivation was observed in the 19 patients in the acyclovir prophylaxis group. The incidence of herpes zoster was significantly higher in the group that did not receive acyclovir prophylaxis (43%, 6 of 14 patients) than in the group that did (0%, 0 of 19; P = 0.003). The predictive factors for varicella-zoster virus reactivation were male sex (P = 0.035) and the use of acyclovir (P = 0.003). CONCLUSIONS Continuous prophylaxis by oral 200 mg/day acyclovir in multiple myeloma patients receiving bortezomib treatment is effective and sufficient in preventing herpes zoster.

Combinations of cytogenetics and international scoring system can predict poor prognosis in multiple myeloma after high-dose chemotherapy and autologous stem cell transplantation

High‐dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a standard therapy for newly diagnosed multiple myeloma. Combinations of recently proposed prognostic factors such as cytogenetics and international scoring system (ISS) may be useful to predict prognosis after ASCT. This study evaluated 60 consecutive patients who underwent ASCT in four institutes. The median age of patients was 57 years old. Cytogenetic analyses of bone marrow at diagnosis detected metaphase abnormalities in 9 of 51 patients and interphase abnormalities in six of 35 patients (17p13 deletion, t(4;14) and t(14;16)). Seventeen patients had ISS stage 3 at diagnosis. Twenty‐five patients who had any of these risk factors were defined as high risk. All patients were conditioned with high‐dose melphalan. With a median follow‐up of 3.4 years, overall survival and event‐free survival at 3 years were significantly worse in high‐risk patients (48% vs. 97%; P = 0.0005 and 16% vs. 37%; P = 0.038, respectively) despite the higher CR plus VGPR rate among high‐risk patients. In addition, survival at 1 year after progression was significantly worse in high‐risk patients despite salvage chemotherapy containing thalidomide (32% vs. 100%, P = 0.0001). Combinations of cytogenetics and ISS could readily predict prognosis. Quality of response is a poor surrogate marker for ultimate outcome. High‐risk patients may need more effective treatment. Am. J. Hematol. 2009.

Dexamethasone palmitate successfully attenuates hemophagocytic syndrome after allogeneic stem cell transplantation: macrophage-targeted steroid therapy

Hemophagocytic syndrome (HPS) induced by uncontrolled macrophage activation and subsequent graft failure is a frequent and prominent complication after allogeneic stem cell transplantation (allo-SCT), a cause of severe morbidity and death, and a therapeutic challenge. Liposome-incorporated dexamethasone, dexamethasone palmitate (DP), shows greater efficacy against macrophages as compared to dexamethasone sodium phosphate (DSP). Based on our findings that DP achieves significantly larger decrease than DSP on the viability of primary human macrophages compared in vitro, we tested the effects of DP in patients with HPS. A decrease in number of macrophages in the bone marrow and prevention of engraftment failure were observed in all patients without any severe complications. In conclusion, these data provide a rationale for testing DP as a first-line treatment for patients with HPS after allo-SCT.

Dexamethasone Palmitate Ameliorates Macrophages-Rich Graft-versus-Host Disease by Inhibiting Macrophage Functions

Macrophage infiltration of skin GVHD lesions correlates directly with disease severity, but the mechanisms underlying this relationship remain unclear and GVHD with many macrophages is a therapeutic challenge. Here, we characterize the macrophages involved in GVHD and report that dexamethasone palmitate (DP), a liposteroid, can ameliorate such GVHD by inhibiting macrophage functions. We found that host-derived macrophages could exacerbate GVHD in a mouse model through expression of higher levels of pro-inflammatory TNF-α and IFN-γ, and lower levels of anti-inflammatory IL-10 than resident macrophages in mice without GVHD. DP significantly decreased the viability and migration capacity of primary mouse macrophages compared to conventional dexamethasone in vitro. DP treatment on day 7 and day 14 decreased macrophage number, and attenuated GVHD score and subsequent mortality in a murine model. This is the first study to provide evidence that therapy for GVHD should be changed on the basis of infiltrating cell type.

Clinical significance of hemophagocytosis in BM clot sections during the peri-engraftment period following allogeneic hematopoietic SCT

The effects of macrophage activation on the outcome of allogeneic hematopoietic SCT (allo-HSCT) have yet to be fully examined. A total of 70 adult patients who received a first allo-HSCT for hematological diseases were studied. We counted the number of hemophagocytic cells in BM clot sections on day +14±7, and analyzed its impact on subsequent outcome. In all, 23 patients were diagnosed as having increased numbers of hemophagocytic cells (HP group), whereas 47 were not (non-HP group). The HP group was not associated with an increased incidence of acute or chronic GVHD, but was associated with worse hematopoietic recovery than the non-HP group. The 2-year OS for the HP group and the non-HP group was 30 and 65% (P<0.01), respectively, and 2-year non-relapse mortality was 48% and 27% (P<0.01), respectively. Multivariate analysis confirmed that the HP group was associated with a lower OS (hazard ratio (HR)=2.3; 95% confidence interval (CI), 1.0–5.4; P=0.048) and higher non-relapse mortality (HR=4.0; 95% CI, 1.6–9.9; P<0.01). The HP group had higher incidences of death due to graft failure (P<0.01) and endothelial complications, such as sinusoidal obstruction syndrome and transplant-associated microangiopathy (P=0.01). Macrophage activation is a previously unrecognized complication with negative impact on outcome of allo-HSCT.

Cytotoxic T-lymphocyte antigen 4 haplotype correlates with relapse and survival after allogeneic hematopoietic SCT.

CTLA-4 is a negative regulator of activated T cells and the association of CTLA-4 polymorphisms with autoimmune diseases and transplant outcome has been reported. We evaluated the effect of donor CTLA-4 polymorphisms on outcome after allogeneic hematopoietic SCT (HSCT). We analyzed 147 Japanese HLA-matched sibling recipients and their donors who had undergone allogeneic HSCT. Genotyping of three single-nucleotide polymorphisms in CTLA-4 (−318, +49, CT60) was performed using TaqMan-PCR. According to the international HapMap database, only these three CTLA-4 haplotypes, classified as C-G-G, C-A-A and T-A-G, are present in the Japanese population. In this study, percentage expression of the C-G-G, C-A-A and T-A-G haplotypes was 59.5, 30.6 and 9.9%, respectively. Recipients of the C-A-A haplotype donor showed a significantly lower risk of relapse (HR: 0.54, 95% CI: 0.30–0.97, P=0.040) and a trend toward higher OS (HR: 0.61, 95% CI: 0.36–1.0, P=0.054) than did recipients of a donor without the C-A-A haplotype. The presence or absence of the C-A-A haplotype did not affect GVHD or non-relapse mortality. As the presence of the C-A-A haplotype reduced relapse risk and improved survival after allogeneic HSCT, this CTLA-4 haplotype may provide useful information for donor selection.