High efficacy and safety of low-dose CD19-directed CAR-T cell therapy in 51 refractory or relapsed B acute lymphoblastic leukemia patients.

Refractory or relapsed B lymphoblastic leukemia (B-ALL) patients have a dismal outcome with current therapy. We treated 42 primary refractory/hematological relapsed (R/R) and 9 refractory minimal residual disease by flow cytometry (FCM-MRD+) B-ALL patients with optimized second generation CD19-directed CAR-T cells. The CAR-T-cell infusion dosages were initially ranged from 0.05 to 14 × 105/kg and were eventually settled at 1 × 105/kg for the most recent 20 cases. 36/40 (90%) evaluated R/R patients achieved complete remission (CR) or CR with incomplete count recovery (CRi), and 9/9 (100%) FCM-MRD+ patients achieved MRD-. All of the most recent 20 patients achieved CR/CRi. Most cases only experienced mild to moderate CRS. 8/51 cases had seizures that were relieved by early intervention. Twenty three of twenty seven CR/CRi patients bridged to allogeneic hematopoietic stem cell transplantation (allo-HCT) remained in MRD- with a median follow-up time of 206 (45-427) days, whereas 9 of 18 CR/CRi patients without allo-HCT relapsed. Our results indicate that a low CAR-T-cell dosage of 1 × 105/kg, is effective and safe for treating refractory or relapsed B-ALL, and subsequent allo-HCT could further reduce the relapse rate.

[Comparison of three different types of donor hematopoietic stem cell transplantation for intermediate and high-risk myelodysplastic syndrome].

Objective: To investigate three different types of donor hematopoietic stem cell transplantation (HSCT) for intermediate and high-risk myelodysplastic syndrome (MDS) . Methods: Between August 2001 and May 2015, 167 consecutive patients with MDS in intermediate and high-risk who underwent allogeneic HSCT were analyzed retrospectively. Results: With the median follow up of 60 (12-177) months, The total 5-year DFS was 67.8% (95%CI 60.0%-75.6%) . Among three different types of donor, 5-year DFS rates were 68.0% (95%CI 54.1%-81.9%) in MSD-HSCT vs 77.4% (95%CI 62.1%-92.7%) in MUD-HSCT vs 64.0% (95% CI 52.4%-75.6%) in Haplo-HSCT (P=0.632) , respectively. Univariate analysis showed that median disease course before HSCT was the influencing factor of DFS (P=0.018) . Five-year relapse and TRM had no correlation with the above-mentioned factor. Conclusions: Haplo-HSCT for intermediate and high-risk MDS achieved similar effect produced by MUD or MSD, Haplo-HSCT could be used as an important alternative donor. allo-HSCT must be performed on intermediate and high-risk MDS patients as early as possible after diagnosis.

[Effect of minimal residual disease monitoring by multiparameter flow cytometry pre-conditioning on prognosis of acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation].

Objective: To investigate the effect of minimal residual disease (MRD) monitoring by multiparameter flow cytometry (MFC) pre-conditioning on prognosis of acute myeloid leukemia in first complete remission (CR(1)-AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) , and to explore the value of MRD monitoring by MFC in the prognosis evaluation on allo-HSCT in CR(1)-AML. Methods: Between April 2012 and March 2015, consecutive 186 patients with CR(1)-AML who underwent allo-HSCT were analyzed retrospectively. MRD in BM before conditioning was detected by eight-color MFC. Any level of residual disease was considered to be MRD positive. Results: ①Of 186 patients, MRD was negative in 151 patients, positive in 35 patients (<1% in 25 patients and 1% to 3% in 10 patients) . ② With the median follow up of 18 (5-41) months, two-year DFS was 80.0% (95%CI 68.5%-92.3%) . Univariate analysis showed that MRD positive patients had lower DFS[62.9% (95%CI 50.6%-75.2%) vs 88.9% (95%CI 76.6%-100.0%) , P<0.001], higher relapse[11.4% (95%CI 4.1%-29.0%) vs 3.3% (95% CI 0.6%-20.9%) , P=0.003] and higher NRM [25.7% (95% CI 8.1%-43.3%) vs 7.9% (95% CI 1.3%-26.5%) , P=0.001] after HSCT compared with that of MRD negative patients. Secondary AML showed lower DFS than primary AML [60.0% (95% CI 42.4%-76.6%) vs 86.0% (95% CI 68.4%-100.0%) , P=0.004]. ③Multivariate analysis indicated that MRD positive pre-HSCT was the independent risk factor on DFS [HR=4.565 (95%CI 2.918-9.482) , P<0.001], relapse [HR=5.854 (95%CI 1.538-22.288) , P=0.010] and NRM [HR=3.379 (95%CI 1.361-8.391) , P=0.009] after allo-HSCT in CR(1)-AML. Conclusion: MRD positive pre-conditioning was the only negative impact factor for patients with CR(1)-AML after allo-HSCT. MRD by MFC can be used to assess the prognosis of CR(1)-AML after allo-HSCT.

[Effect of NCCN (2015) risk stratification on prognosis of patients with acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation].

Objective: To analyze the effect of NCCN (2015) risk stratification on prognosis of patients with acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods: Retrospective analysis of 258 patients with AML in CR (186 cases in CR(1), 72 cases in CR(2)) who underwent allogeneic HSCT in our hospital between April 2012 and March 2015 according to NCCN (2015) risk stratification. Of them, 63 cases were classified as low risk, 112 cases intermediate risk and 83 cases high risk. Results: ①With the median follow up of 18 (5-41) months, two-year disease free surviva (DFS) in 258 patients was 78.0% (95% CI 60.4%-96.6%) . Two-year DFS in AML after transplantation was 78.6% (95% CI 61.0%-96.2%) in low risk, 76.0% (95% CI 84.0%-93.6%) in intermediate risk and 80.3% (95% CI 62.7%-97.9%) (P=0.886) in high risk groups respectively. ②Univariate analysis showed that DFS has no significant difference in patient age, the median disease course before HSCT, the WBC number at the beginning of the disease, blood routine and chromosomes examination before transplantation, extramedullary disease before transplantation, disease status before transplantation, conditioning regimen, donor type, donor and recipient sex, recipient blood type, transfused MNC number, transfused CD34(+) cell number and transfused CD3(+) cell number. DFS was significant lower in primary AML than that in secondary AML (P=0.006) and also lower in MRD positive than that in MRD negative (P=0.003) . The accumulative relapse was significant higher in CR(2) compared to that in CR(1) (P=0.046) . Accumulative non-relapse mortality (NRM) was significanlyt higher in secondary AML compared to that in primary AML (P=0.004) and also higher in MRD positive compared to that in MRD negative (P=0.010) . ③Multivariate analysis showed that MRD positive was the only significant factor in DFS and NRM. Conclusion: Allo-HSCT treatment of AML CR patients could achieve a high efficacy, which is similar between CR(1) and CR(2) patients. There is no significant correlation between NCCN (2015) risk stratification and the prognosis of AML patients with allo-HSCT treatment. Pre-conditioning MRD status monitored by multiparameter flow cytometry was the only impact factor on DFS and NRM in allo-HSCT for CR-AML patients.

Risk factors for recurrence of invasive fungal infection during secondary antifungal prophylaxis in allogeneic hematopoietic stem cell transplant recipients.

BACKGROUND: Invasive fungal infections (IFIs) are a major cause of mortality among allogeneic hematopoietic stem cell transplantation (allo-HSCT) patients. Thanks to the widespread use of secondary antifungal prophylaxis (SAP), a history of IFI is not an absolute contraindication to allo-HSCT. However, IFI recurrence remains a risk factor for transplant-related mortality. METHODS: To evaluate the risk factors for IFI recurrence in allo-HSCT patients receiving SAP, we performed a retrospective analysis of 90 individuals treated at our hospital. SAP antifungal agents included fluconazole (n = 28), voriconazole (n = 25), itraconazole (n = 23), caspofungin (n = 7), and micafungin (n = 7). RESULTS: By day +100, recurrent IFI had occurred in 23 (25.5%) patients. Our multivariate analysis identified 4 factors significantly associated with a risk of IFI recurrence within 100 days of allo-HSCT: duration of neutropenia >18 days, presence of severe acute graft-versus-host disease (aGVHD), <70-day interval between previous infection and transplantation, and use of a narrow-spectrum SAP agent (P = 0.008, 0.010, 0.041, and 0.001, respectively). Of the 87 patients who remained in the study for the duration of the follow-up period (median length: 551 days), 26 (29.9%) died; only 7 (8.0%) of these deaths resulted from a severe fungal infection. CONCLUSION: These results suggest that transplantation outcome can be improved by adequate antifungal treatment before transplantation, better prevention of, and therapy for, severe aGVHD, use of granulocyte colony-stimulating factor to reduce the duration of neutropenia, and use of broad-spectrum prophylaxis agents.

Hematopoietic SCT activity in Asia: a report from the Asia-Pacific Blood and Marrow Transplantation Group.

The hematopoietic SCT (HSCT) activity in nine Asian countries/regions was surveyed to overview the current situation. Data of 58 113 HSCTs (allogeneic: 63% vs autologous: 37%) performed between 1986 and 2006 by 432 transplant teams were collected. The number of HSCTs has been increasing in the past two decades in most countries/regions. The increase in allogeneic HSCTs is greater than in autologous HSCTs. The proportion of unrelated donors among allogeneic HSCTs in 2006 varied widely from <1% (Iran and Vietnam) to 62% (Japan). The use of each stem cell source, that is, BM, PBSC, cord blood and others (including co-infusion of BM and PBSC), also varied widely (36, 58, 0.1 and 6% in HSCT from related donors, respectively, and 53, 11, 35 and 1% in HSCT from unrelated donors, respectively). HSCTs have been continuously increasing for all indications except for chronic myelogenous leukemia and solid tumors. Hemoglobinopathy is a common indication among non-malignant diseases in many Asian countries/regions except for China, Japan and Korea. This survey clearly shows the recent progress of HSCTs in Asia and also some differences in donor and stem cell selection and disease application among countries/regions.

Blood and marrow transplantation in mainland China.

As of 1981, allogeneic bone marrow transplantation (allo-BMT) was applied in an acute leukaemia patient with success. Since then, the number of BMT has been increasing gradually, especially since the 1990s. Approximately 2000 BMTs per year have been performed in recent years in more than 100 BMT units in mainland China. A recent survey of 12 major BMT units indicates that the predominant types of transplantation performed are identical sibling (38.6%), related mismatched/haploidentical (19.4%), unrelated (17.2%), and autologous (24.5%). The indications of major disease entities are acute myeloid leukaemia (32.8%), acute lymphoblastic leukaemia (20%), chronic myeloid leukaemia (CML) [18.9%], and lymphoid malignancy (13.5%). The number of transplants from unrelated donor or related mismatched/haploidentical donor has been increasing significantly in recent 6 years. Granulocyte colony-stimulating factor-mobilised bone marrow plus peripheral blood are routinely used as a source of stem cells for haploidentical BMT. Umbilical cord blood is used less often. Although the total number of patients who received allo-BMT continues to increase, the increase in BMT for CML has been flattened since 2004. By the end of 2008, more than 960 000 volunteer's human leukocyte antigen (HLA) data are available in Chinese Marrow Donor Program (CMDP), and more than 1100 stem cell donations have been performed from it. Stem cells for unrelated BMT in mainland China are mainly from Taiwan Tzu Chi Stem Cell Center and CMDP. Related HLA-mismatched/haploidentical BMT has reached fairly good outcomes in terms of severe acute graft-versus-host disease (GVHD), chronic GVHD, relapse, treatment-related mortality, disease-free survival, and overall survival, which are comparable with HLA-identical-sibling BMT in the author's BMT units. Syngeneic BMT started successfully in 1964 and has still very good outcomes in more than 23 BMT units from the statistics of Chinese Society of Blood and Marrow Transplantation.

Unmanipulated haploidentical blood and marrow transplantation: where we are.

Human leukocyte antigen (HLA)-mismatched/haploidentical blood and marrow transplants (haplo-BMT) from family donors have been intensively studied because of the decreasing family size in mainland China, and also because the Chinese Marrow Donor Program is still not big enough. The protocol for unmanipulated haplo-BMT has been designated as 'GIAC' by Dr DP Lu--'G' represents granulocyte colony-stimulating factor mobilisation; 'I' stands for immunosuppression during pre-conditioning being prolonged and intensified; 'A' stands for the use of antithymocyte globulin; 'C' means combined use of bone marrow and peripheral blood as the graft. Haplo-BMT with GIAC regimen has been shown to be feasible for many applications as reported in 2004. Under this protocol, haplo-BMT has achieved comparable outcomes in terms of severe acute graft-versus-host disease (GVHD), chronic GVHD, relapse, treatment-related mortality (TRM), disease-free survival (DFS), and overall survival with HLA-identical sibling transplantation. The probabilities of DFS at 2 years in haplo-BMT setting were 70.7%, 49.6%, 22.2% in standard-risk, high-risk, advanced disease groups, respectively. As the third party cells, cord blood co-infusion could significantly reduce the incidence and severity of acute GVHD, and also 100-day TRM. The majority of refractory cytomegalovirus, Epstein-Barr virus and aspergillus infections can be controlled by adoptive cellular therapy. Many patients who early relapsed after BMT and failed, or are ineligible for standard therapy, have been salvaged with dendritic cell-primed cytokine-induced killer cells. With these new strategies, the lower TRM and improved DFS have been attained. Therefore, it is better to consider haplo-BMT for the patients with otherwise incurable haematological malignancies at earlier stage, when matched sibling or unrelated donors are not available.

Blood and marrow transplantation in the People's Republic of China.

Syngeneic BMT was first performed successfully in China in 1964. In 1981, allogeneic BMT was applied in an acute leukemia patient with success. Since then, the number of BMTs has been increasing gradually, especially since the 1990s. More than 2000 stem cell transplants per year have been performed in recent years in more than 50 BMT units in mainland China. A survey of 16 BMT units from 1986 to 2005 indicates that the predominant types of transplantation performed are identical sibling (36%), related mismatched/haploidentical (11.2%), unrelated (7.5%) and autologous (44.5%) and that the distribution of disease entities and prevalent diseases being transplanted are AML (31%), ALL (16.1%), CML (19.1%) and lymphoid malignancy (22.2%). The number of transplants from unrelated donor or related mismatched/haploidentical donor has increased significantly in the past 5 years. BM and G-CSF-mobilized peripheral blood are used about equally often as a source of hematopoietic stem cells, or they are used in combination. Umbilical cord blood is used least often. Leukemias for allogeneic and lymphoid malignancies for autologous BMT continue to increase, but the increase in BMT for CML has been slow since 2004. By the end of 2007, HLA data were available on more than 700,000 individuals in the Chinese Marrow Donor Program, and 800 stem cell donations have been carried out from these. Related HLA-mismatched/haploidentical BMT has achieved comparable outcomes in terms of severe acute GVHD, chronic GVHD, relapse, treatment-related mortality, disease-free survival (DFS) and overall survival (OS) with HLA-identical sibling transplantation in the author's two BMT units. Cord blood co-infusion as the third-party cells could significantly reduce the incidence and severity of acute GVHD, steroid-refractory acute GVHD and extensive chronic GVHD without an increase in leukemia relapse and could improve DFS and OS.

Correlations of human herpesvirus 6B and CMV infection with acute GVHD in recipients of allogeneic haematopoietic stem cell transplantation.

Human herpesvirus 6 (HHV-6) and CMV reactivation were monitored in a cohort of 72 consecutive haematopoietic stem cell transplant (HSCT) patients using RQ-PCR and antigenaemia assay, respectively. The association between acute GVHD (aGVHD) and HHV-6B/CMV was evaluated. We found that on day 100 the cumulative incidence of grades I-IV aGVHD, grades II-IV aGVHD and grades III-IV aGVHD was 55.6, 27.8 and 13.9%, respectively. Multivariate analysis indicated that HHV-6B reactivation was closely correlated with a higher probability of grade II-IV aGVHD by day 30 (Hazard ratio (HR), 8.9; 95% confidence interval (CI), 2.6-31.0; P=0.0006), by day 50 (HR, 6.1; 95% CI, 2.1-17.8; P=0.0010) and by day 100 (HR, 4.8; 95% CI, 1.7-13.6; P=0.0028). However, CMV reactivation did not significantly affect the development of aGVHD by day 50 (HR, 0.8; 95% CI, 0.1-6.7; P=0.8236) and by day 100 (HR, 0.5; 95% CI, 0.1-4.4; P=0.5330) after HSCT. In conclusion, this study demonstrated that active HHV-6B infection, but not CMV, is significantly associated with an increased risk of aGVHD development after HSCT.

CD34+CD38+CD19+ as well as CD34+CD38-CD19+ cells are leukemia-initiating cells with self-renewal capacity in human B-precursor ALL.

The presence of rare malignant stem cells supplying a hierarchy of malignant cells has recently been reported. In human acute myelogenous leukemia (AML), the leukemia stem cells (LSCs) have been phenotypically restricted within the CD34+CD38- fraction. To understand the origin of malignant cells in primary human B-precursor acute lymphocytic leukemia (B-ALL), we established a novel in vivo xenotransplantation model. Purified CD34+CD38+CD19+, CD34+CD38-CD19+ and CD34+CD38-CD19- bone marrow (BM) or peripheral blood (PB) cells from three pediatric B-ALL patients were intravenously injected into sublethally irradiated newborn NOD/SCID/IL2rgamma(null) mice. We found that both CD34+CD38+CD19+ and CD34+CD38-CD19+ cells initiate B-ALL in primary recipients, whereas the recipients of CD34+CD38-CD10-CD19- cells showed normal human hematopoietic repopulation. The extent of leukemic infiltration into the spleen, liver and kidney was similar between the recipients transplanted with CD34+CD38+CD19+ cells and those transplanted with CD34+CD38-CD19+ cells. In each of the three cases studied, transplantation of CD34+CD38+CD19+ cells resulted in the development of B-ALL in secondary recipients, demonstrating self-renewal capacity. The identification of CD34+CD38+CD19+ self-renewing B-ALL cells proposes a hierarchy of leukemia-initiating cells (LICs) distinct from that of AML. Recapitulation of patient B-ALL in NOD/SCID/IL2rgamma(null) recipients provides a powerful tool for directly studying leukemogenesis and for developing therapeutic strategies.

Haploidentical hematopoietic stem cell transplantation without in vitro T-cell depletion for the treatment of hematological malignancies.

Many patients who require allogeneic hematopoietic stem cell transplantation (allo-HSCT) lack a human leukocyte antigen (HLA)-matched donor. Here, we report a protocol for haploidentical allo-HSCT that combines granulocyte-colony stimulating factor primed bone marrow (G-BM) and peripheral blood stem cells (PBSC) without in vitro T-cell depletion (TCD). In this study, 171 patients, including 86 in high-risk group, underwent transplantation from haploidentical family donors. All patients achieved sustained, full donor chimerism. One hundred and eleven patients were alive in remission at a median of 682 (253-1502) days. The cumulative incidence of grade III-IV acute graft-versus-host disease (GVHD) was 23% and that of extensive chronic GVHD, 47%; these were not influenced by HLA disparity. Patients younger than 15 years had less grade III-IV acute GVHD than older patients (P=0.044). The 2-year probability of relapse was 12% for standard-risk disease and 39% for high-risk disease. The 2-year probability of leukemia-free survival (LFS) was 68% for standard-risk patients and 42% for high-risk patients (P=0.0009). Grade III-IV acute GVHD was associated with better LFS (P=0.0017). The results require confirmation and show that G-BM combined with PBSC from haploidentical family donors, without in vitro TCD, may be used as a good source of stem cells for allo-HSCT.

Identification of a novel HLA-DRB1 allele (HLA-DRB1*111902*).

In this paper, we report a new HLA-DRB1 allele identified in a male acute myeloid leukaemia Chinese patient. This sample was initially typed as DRB1*11XX using commercial polymerase chain reaction-sequence-specific primers kit. When it was typed using a chip-based sequence-specific oligonucleotide technique, a novel hybridization pattern that does not match any known alleles was observed. Through sequencing, we have identified this allele as a new HLA-DRB1 allele, which was later named HLA-DRB1*111902 by the WHO Nomenclature Committee. The sequence of this new allele differs from DRB1*111901 by one nucleotide (from G to C) at 203nt of exon 2 but does not cause any amino acid substitution.

Managing acute withdrawal syndrome on patients with heroin and morphine addiction by acupuncture therapy.

Though there are articles and case reports about using acupuncture to detoxify and to break the narcotic addiction, few articles describe in the West about using acupuncture therapy to treat the emergence of acute withdrawal symptom due to heroin, opium, or morphine. Most often the method of treatment are using the methadone or benzodiazepine and phenoziazine drugs this article describes many years of clinical experience with non-drug approach to treat the acute withdrawal symptoms with acupuncture therapy. Unlike the drug approach, which usually has side effects, there is no adverse effect with acupuncture therapy.

Expression of cytokeratins (CKs) 8, 13 and 18 and their mRNA in epithelial linings of radicular cysts: implication for the same CK profiles as nasal columnar epithelium in squamous epithelial lining.

OBJECTIVE: Our objective was to specify cytokeratin (CK) patterns in lining epithelia of radicular cysts which are sometime lined with ciliated columnar epithelia as seen in the nasal epithelia. MATERIALS AND METHODS: We examined the CK expression in 52 radicular cysts obtained from 32 maxillary and 20 mandibular lesions and investigated CK-mRNA expression using in situ hybridization in 24 maxillary and 13 mandibular cysts and reverse transcription-polymerase chain reaction (RT-PCR) in 24 maxillary cysts. RESULTS: Of the maxillary cysts, 20, 29 and 19 squamous epithelial linings were positive for CK8, CK13 and CK18, respectively; of the mandibular cysts, 10, 20 and 11 linings were positive for these CKs, respectively. The expression patterns of CK18(+)-CK13(-), CK18(+)-CK13(+) and CK18(-)-CK13(+) were observed in 3, 16 and 13 linings of the maxillary cysts and 0, 11 and 9 linings of the mandibular cysts, respectively. In situ hybridization revealed the expression of CK18-mRNA in 9 and 4 linings of 24 maxillary and 13 mandibular cysts examined, respectively. With RT-PCR, we explored that both CK18- and CK13-mRNA were expressed not only in the normal nasal and gingival epithelia but also in the examined maxillary cyst linings although their expression levels differed correlating with the difference in CK staining. CONCLUSION: It is concluded that CK13- and CK18-mRNA are constitutively expressed in columnar and squamous epithelial cells, respectively, and that the variant CK expression patterns with CK18-mRNA expression in maxillary radicular cysts are indicative of the possibility of phenotypic transformation in the cyst linings.

Serum TNFalpha levels in patients with acute graft-versus-host disease after bone marrow transplantation.

Tumor necrosis factor alpha (TNFalpha) has been implicated in the pathogenesis of graft-versus-host disease (GVHD). In this study, serum levels of TNFalpha were assessed by ELISA in 243 sera samples from 40 patients who had undergone allogeneic bone marrow transplantation (BMT). Serum TNFalpha levels were measured before BMT and at different time points after BMT. The results were correlated with acute GVHD (aGVHD), infection and conditioning regimen. Serum TNFalpha levels were significantly higher in patients with grades II-IV aGVHD than in those with grade 0 or I aGVHD, but there was no clear correlation between serum TNFalpha and severity of aGVHD. Serum TNFalpha levels in infected patients were not statistically different from those in patients without infection. The conditioning regimen did not cause a significant rise in TNFalpha levels. These results indicate that TNFalpha may be useful for the diagnosis of aGVHD and for differentiating between aGVHD and other BMT-related complications, such as infection.

Asian Pacific cooperative study of allogeneic bone marrow transplantation.

The first cooperative study of the Asian Pacific bone marrow transplantation group included 75 patients with early leukemia who received human leukocyte antigen-matched sibling bone marrow transplants and were randomized into granulocyte colony-stimulating factor and control groups. The selected patients were registered from 10 centers in six countries and areas within Asia (Beijing, Taipei, Hong Kong, Japan, Korea, and Malaysia). The incidence of grades II-IV acute graft-vs.-host disease was 22.2%, and the 2-year survival rate was 62.7%. The period of protective isolation (27.1-66.7 days), period of hospitalization (38.6-130.5 days), and medical costs for 4 months (US $10,300-US $80,803) varied considerably. Good cooperation, i.e., low rate of protocol violation or rapid and precise presentation of case reports, was obtained.