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Cutaneous sclerosis is one of the most common clinical manifestations of chronic graft-versus-host disease (cGVHD). Donor CD4(+) T and B cells play important roles in cGVHD pathogenesis, but the role of antibodies from donor B cells remains unclear. In the current studies, we generated immunoglobulin (Ig)H(µγ1) DBA/2 mice whose B cells have normal antigen-presentation and regulatory functions but cannot secrete antibodies. With a murine cGVHD model using DBA/2 donors and BALB/c recipients, we have shown that wild-type (WT) grafts induce persistent cGVHD with damage in the thymus, peripheral lymphoid organs, and skin, as well as cutaneous T helper 17 cell (Th17) infiltration. In contrast, IgH(µγ1) grafts induced only transient cGVHD with little damage in the thymus or peripheral lymph organs or with little cutaneous Th17 infiltration. Injections of IgG-containing sera from cGVHD recipients given WT grafts but not IgG-deficient sera from recipients given IgH(µγ1) grafts led to deposition of IgG in the thymus and skin, with resulting damage in the thymus and peripheral lymph organs, cutaneous Th17 infiltration, and perpetuation of cGVHD in recipients given IgH(µγ1) grafts. These results indicate that donor B-cell antibodies augment cutaneous cGVHD in part by damaging the thymus and increasing tissue infiltration of pathogenic Th17 cells.

Recipient tissue expression of programmed death-ligand 1 (PD-L1, B7H1) down-regulates graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT), but this information has not been translated clinically. Here we demonstrate a critical role for recipient PD-L1 expression in preventing both acute and chronic GVHD while preserving graft-versus-leukemia (GVL) effects when donor CD4+ T cells are temporarily depleted in vivo early after HCT. Depletion of donor CD4+ T cells increases serum IFN-γ concentrations and enhances recipient tissue expression of PD-L1 and donor CD8+ T cell expression of the PD-L1 receptors CD80 and PD-1. In GVHD target tissues, depletion of CD4+ T cells increases anergy and apoptosis of infiltrating CD8+ T cells in a manner that depends on recipient PD-L1 expression, thereby preventing damage to intestinal Paneth cells and stem cells, hepatocytes, and thymic medullary epithelial cells, and preventing both acute and chronic GVHD. In lymphoid tissues, depletion of CD4+ T cells augments CD8+ T cell proliferation without increasing CD8+ T cell anergy or apoptosis, resulting in expansion of donor CD8+ T cells and strong GVL effects. Therefore, temporary depletion of donor CD4+ T cells early after HCT represents a novel approach for preventing GVHD while preserving GVL effects. This work was supported by NIH R01 AI066008 and Nesvig Lymphoma Fundation (to D.Z.) and by NSFC 81090413, 81270638 (to J.W.). Disclosures Forman: Amgen: Consultancy; Mustang: Research Funding. Martin:Neovii: Research Funding; RegImmune: Research Funding; Enlivex: Consultancy; Janssen: Consultancy; Pfizer: Consultancy; Pharmacyclics: Consultancy.

We recently reported that in a chronic graft versus host disease (GVHD) model of DBA/2 donor to MHC-matched BALB/c recipient, donor CD4+ T and B cell interaction resulted in not only hyperglobulinemia and glomerulonephritis but also scleroderma (J. Immunol. 2012). It is well known that glomerulonephritis is caused by immune complex deposition. However, the role of antibodies from donor B cells in the pathogenesis of scleroderma remains unclear. To address this question, we generated DBA/2 mice whose B cells have APC function but cannot secrete antibodies by backcrossing IgHµg1 mice from Dr. Rajewsky’s lab (JEM 2007). We observed that, while transplanting T-cell-depleted bone marrow (TCD-BM) and spleen cells from littermate control mice induced proteinuria and scleroderma, transplanting BM and spleen cells from IgHµg1 DBA/2 mice induced no proteinuria, but the recipients developed scleroderma ~35 days after HCT. Interestingly, the scleroderma gradually recovered ~55 days after HCT. 40 days after HCT, scleroderma recipients transplanted with WT spleen cells (Rec-WT) or recipients transplanted with IgHµg1 spleen cells (Rec-IgHµg1) both had high percentage (~12%) of IFN-g+ or IL-17+ CD4+ T cells in the peripheral lymph node (PLN) and skin tissues, as compared to that (~3%) of GVHD-free recipients given TCD-BM alone (Rec-TCD). While Rec-WT had severe reduction of CD4+CD8+ thymocytes, the Rec-IgHµg1 had no reduction of the thymocytes, as compared to that of Rec-TCD. By day 60 after HCT, the Rec-WT with ongoing scleroderma still had ~10% IFN-g+ or IL-17+ CD4+ T cells in the PLN and skin tissues; in contrast, although the Rec-IgHµg1 with reversal of scleroderma still had >10% IFN-g+or IL-17+ CD4+ T cells in the PLN, those cells in the skin had reduced to Disclosures No relevant conflicts of interest to declare.

Donor T cell-mediated graft versus leukemia/lymphoma (GVL) effect plays a critical role in preventing tumor relapse in leukemia/lymphoma patients treated with allogeneic hematopoietic cell transplantation (HCT). However, these same donor T cells also induce acute and chronic graft versus host disease (GVHD), which remains a major obstacle for allogeneic HCT as a curative therapy for hematological malignancies. Chronic GVHD is a systemic lupus- and scleroderma-like autoimmune syndrome. We and others reported that donor B cells play important roles in augmenting pathogenesis of chronic GVHD in mouse models and humans. Rituxan (an anti-CD20 mAb) has been used in the clinic for the treatment of ongoing chronic GVHD, but the effect is variable and minimum in some reports, and the mechanisms for this ineffectiveness remains unclear. In the current studies, we evaluated the effect of in vivo administration of a depleting anti-CD20 mAb in preventing and treating autoimmune-like chronic GVHD as well as the impact on GVL effect. With the mouse chronic GVHD model of DBA/2 donor to MHC-matched BALB/c recipient established in our lab (Blood, 2006, J. Immunol 2012), we found that one intravenous injection of anti-CD20 (50mg/kg) immediately after HCT effectively prevented induction of autoimmune-like chronic GVHD. While all (12/12) recipients treated with control IgG developed chronic GVHD with proteinuria and hair-loss and died by 30 days, none (0/12) of the anti-CD20-treated recipients developed proteinuria or hair-loss and all survived for more than 50 days after HCT without signs of chronic GVHD. In addition, the anti-CD20-treated recipients eliminated BCL1 leukemia/lymphoma cells without signs of chronic GVHD. The preventative anti-CD20 treatment had little impact on donor CD8+ T cell activation and expansion in the periphery and allowed for the strong CD8+ T cell-mediated GVL effect. The effective prevention of chronic GVHD by anti-CD20 was associated with significant changes in donor B and CD4+ T cells as well as associated with protection of host thymus. Preventive anti-CD20 treatment depleted IL-6-producing donor B cells and increased IL-10-producing B cells. In addition, the treatment also significantly reduced donor CD4+ T cell expansion, the percentage of IFN-g-producing CD4+ T as well as CD4+ T cells expressing CD8αα, the latter of which has recently been reported to represent over-activated pathogenic CD4+ T cells, such that the host thymus was protected from donor T-cell-mediated damage. Anti-CD20 or control IgG treatment of recipients with ongoing chronic GVHD 15-20 days after HCT did not show any difference in disease progress, and all (8/8) in each group died within 30 days after HCT. The ineffectiveness of anti-CD20 therapy was associated with little reduction of CD19+ B or CD4+ T cells, as the majority of CD19+ B cells became CD20- in the recipients with ongoing chronic GVHD. These results indication that anti-CD20 can effectively prevent induction of autoimmune-like chronic GVHD while preserving the GVL effect, but anti-CD20 is ineffective in treating ongoing chronic GVHD. (This study is supported by the Nesvig Lymphoma Foundation). Disclosures: Chan: Genentech Inc.: Employment.

Acute myeloid leukemia (AML) is generally regarded as a stem cell disease, known as leukemic initiating cells (LIC), which initiate the disease and contribute to relapses. Although the phenotype of these cells remains unclear in most patients, they are enriched within CD34+CD38- compartment. In core binding factor (CBF) AML, the cytogenetic abnormablities are also existed in LIC. The aim of this study was to determine the prognostic power of minimal residual disease measured by fluorescence in situ hybridization (FISH) in flow sorted CD34+CD38- cells (FISH+CD34+CD38- population) at different period during the therapy. Thirty-six patients under 65 years of age with de novo CBF AML and treated with CHAML 2010 protocol were retrospectively included in this study. FISH efficiently identified the LICs (FISH+CD34+CD38-) in the CD34+CD38- population. The last follow-up was March 31, 2013, and the median follow-up was 336 days (range: 74-814 days). 33 patients with complete remission (CR) were eligible for the study, and 23 patients (23/33, 69.7%) with t (8;21) or AML1/ETO, and the remaining (10/33, 30.3%) with inv(16)/t(16;16) or CBFβ/MYH11. Flow-cytometry based FISH (F-FISH) procedure was performed at diagnosis, before every cycle of consolidation therapy, and every 3 months during follow-up. The FISH+ percentage at diagnosis constituting an average of 2.1% (range: 0.01%-27.5%) of the blast cells and 64.6% (range: 14%-87.8%) of the CD34+CD38- cells. Before the consolidation, FISH+CD34+CD38- population was detected in 13/33 (39.4%) patients. At this checkpoint, we have found the existence of FISH+CD34+CD38- population had prognostic value for the end points relapse free survival (RFS, 12% versus 68%, P=.008), and retained prognostic significance for RFS in multivariate analysis. Furthermore, the detection of FISH+CD34+CD38- before consolidation was found to be significantly associated with decreased OS. (11% versus 75%, P=.0005) Minimal residual disease (MRD) detected with F-FISH had a prognostic value at an earlier checkpoint when compared with flow cytometry and RT-PCR. Meanwhile, the concordance of flow cytomety, RT-PCR and F-FISH was investigated in the same patient cohort. 14 (70%) of 20 samples with detectable fusion transcripts by PCR did not have detectable leukemic cells by F-FISH. Therefore, the concordance for PCR and F-FISH was 63.7%. The concordance of FC and F-FISH was 64.3%: in 40 samples MRD was detected by both methods and in 61 samples MRD was ruled out by a negative result with the tests. With further analysis, the discrepancies among MRD detected with different MRD monitoring approaches before consolidation and after the first consolidation therapy contribute to 84% of the disconcordance. In summary, the detection of FISH+CD34+CD38- cells before consolidation therapy was significantly correlated with long-term survival in de novo CBF AML patients. F-FISH might be easily adopted as MRD monitor approach in clinical practice to identify patients at risk of treatment failure from the early stage during therapy. Disclosures: No relevant conflicts of interest to declare.

Great advances have been made in the diagnosis, molecular pathogenesis and treatment for acute lymphoblastic leukemia (ALL) in the past decade. Due to the lack of comprehensive population-based studies, the updated trend of incidence and geographic variations of ALL has not been well documented in Shanghai, China. To better understand the incidence and epidemiological features of ALL in Shanghai, we conducted a survey based on the database in the Center for Disease Control and the medical record in all hospitals in Shanghai, especially 28 major hospitals with hematology department. According to the CDC data, 544 patients with median age of 32y (1.2~89y) were diagnosed as de novo ALL during January 1, 2002 and December 31, 2006 and followed-up until December 31, 2007. The yearly incidence of ALL was (0.81±0.11)/100 000, which is slightly higher in men (0.86/100 000) than in women (0.75/100 000). The age-stratified incidence showed 2.31/100 000 in pts ≤17y, 0.54/100 000 in 18~34y, 0.46/100 000 in 35~59y, and 0.94/100 000 in pts > 60y. There were substantial geographic variations in the incidence of ALL, with 0.57±0.14/100 000 in Chongming county, an island eastern of Shanghai city, much lower than that of other districts in the central or around the central city, such as Qingpu, Baoshan, Hongkou, and Jiading districts (>0.9/100 000, P

Promising efficacy of bortezomib, the first therapeutic proteasome inhibitor, has been demonstrated in the treatment of multiple myeloma (MM). In this study, we evaluate the therapeutic effects and adverse reactions of bortezomib combination with epirubicin, dexamethasone and thalidomide (BADT) for the treatment of multiple myeloma (MM). Using BADT regimen (bortezomib 1.0mg/m2 on days 1, 4, 8 and 11; epirubicin 12mg/m2 on days 1, 4, 8, 11; dexamethasone 20mg on days 1–2, 4–5, 8–9, 11–12; thalidomide 100mg/m2 on days 1–28) for a maximum of eight 4-week cycles. 12 MM patients were treated, including 5 previously untreated patients and 7 previously treated patients. Each patient completed at least 2 cycles with a mean of 4.5 cycles. Of the 12 patients, 9 patients (75%) achieved complete remission (CR)and 2 patient (16.7%) achieved partial remission (PR), with an overall response (OR) of 91.5%. The mean number of cycles to CR was 2.8 (1–6), and the mean number of cycles to PR was 1.3 (1–3). 7 patients achieved PR after one cycle treatment, and 4 patients achieved CR after one cycle treatment. The main adverse reactions included peripheral neuropathy (4/12), thrombocytopenia (3/12), abnormal change of ECG (4/12), neutropenia (5/12), and liver function impairment (3/12), mainly grade ±-II. Infection occurred in three patients with neutropenia, including sepsis in one patient and herpes zoster in another patient. One patient died of diseases progression, another patient died of secondly sarcoma after achievement CR. The median progression-free time was 14 months (2–26) months, including 2 patients who have been in CR for 15 months since discontinuation of the treatment. In Conclusion, the BADT regimen is highly effective with low toxicity in the treatment of MM. It is warranted to conduct further study on this regimen in more cases.

Homoharringtonine (HHT) is a plant alkaloid which has been used in China for the treatment of acute myeloid leukemia (AML) and chronic myeloid leukemia for over 30 years. We present here a retrospective analysis designed to compare the efficacy and toxicity of HHT with daunorubicin (DNR) and idarubicin for the treatment of AML in elderly patients. Fifty-three patients over 60 years with newly diagnosed non-M3 AML between January 1998 and December 2007 were treated with cytarabine (Ara-c, 100mg/m2/day for 7 days) in combination with HHT (2mg/m2/day for 7 days; HA group; n=19), or DNR (40mg/m2/d for 3 days; DA group; n=16), or idarubicin (8mg/m2/d for 3 days; IDA group; n=18). In the HA group, 42.1% (8/19) of patients achieved complete remission (CR), 26.3% (5/19) of patients had partial remission (PR). In the DA group, the CR and PR rates were each 18.8% (3/16). In the IDA group, 55.5% (10/18) of patients achieved CR, 5.9% (1/18) patients had PR. The CR and OR rates were not significantly different between the three groups. However, whereas in the IDA and DA groups the early death rate within one month after chemotherapy was 33.3% (6/18) and 23.5% (4/16) respectively, there was no early death in the HA group. The estimated OS (overall survival) times were 23.2±7.9 months, 7.6 ±2.1 months, 14.0±3.4 months in HA, DA, and IDA groups (HA versus DA, P = 0.048; HA versus IDA, P = 0.678). The estimated mean disease-free survival (DFS) time of those patients who achieved CR in the HA group (44.3±17.3) were also significantly higher than those in the DA group (7.8±2.7; P = 0.047), and comparable with those in the IDA group (18.0±4.2; P = 0.598). In summary, the response to HA induction therapy was at least equal to that of DA and IDA induction, with relatively mild extramedullary toxicity and lower myocardial toxicity. So HHT is a particularly suitable candidate for the treatment of elderly AML patients.

Objective To explore the significance of reduced-intensity conditioning allogeneic peripheral blood hematopoietic stem cell transplantation (PBSCT) in the treatment of chronic myeloid leukemia. Patients and Methods Twenty-six consecutive patients received reduced-intensity conditioning (Flu 30mg/m2.d−1×5d+BU 4mg.kg−1.d−1×3d or CTX 100mg.kg−1+TBI≤6.0Gy) PBSCT(RIC group). Among them, 21 were male and 5 female with median age of 36 (23~49). Twenty three of them were in chronic phase and 3 in progressive phases. The median time from diagnosis to transplantation was 12 (3~84) months. Twenty-four consecutive patients received standard conditioning (CTX 120mg.kg−1+ TBI≥7.0Gy) prior to PBSCT (STAND group) were used as historic controls. In this group of patients, 22 were in chronic phase and 2 in progressive phases. Twenty patiens were male and 4 female with median age of 35 (18~49). The median time from diagnosis to transplantation was 13 (3~48) months. All patients received PBSC from HLA matched related donors. Mycophenolate mofetil (MMF),CsA and MTX were given for prophylaxis of acute graft-versus-host-disease (aGVHD). Results All patients were successfully engrafted and achieved a complete cytogenetic remission. One patient developed graft rejection 6 months post-transplantation in RIC group. The median time when granulocyte exceeded 0.5 ×109/L and platelets exceeded 20 ×109/L was 15(12–23) days and 19(12–32) days in STAND group and 13(11–18) and 17(11–30) days in RIC group, respectively. The cumulative incidence of aGVHD was 45.83% (11/24) in STAND group and 23.08% (6/26) in RIC group(P>0.05). The incidence of Grade III–IV aGVHD in STAND group and RIC group was 16.67% and 0%, respectively(P0.05). The incidence of extensive cGVHD in STAND group and RIC group was 35% and 0%, respectively(P0.05). 4 patients experienced a cytogenetic relapse, which was successfully treated with donor PBSC infusions. The Cumulative incidence of TRM was 41.67% (10/24) for the STAND group and 19.23% (5/26) for the RIC group, respectively (P>0.05). GVHD copplicated with interstitial pneumatitis or severe infection were the main causes of death. The estimated 3-year probabilities of disease-free-survival (DFS) was 62% in STAND group and 76% in RIC group, respectively (P>0.05). Conclusion Our results indicate that reduced-intensity conditioning allogeneic peripheral blood stem cells transplantation is a safe, less toxic and curative approach for patients with chronic myeloid leukemia.