Chimeric antigen receptors T cells for treatment of 48 relapsed or refractory acute lymphoblastic leukemia children: long term follow-up outcomes / 中华血液学杂志

Objective: To evaluate the safety and efficacy of chimeric antigen receptors T cells (CAR-T) in childhood acute B lymphoblastic leukemia (B-ALL) to probe the prognosis-related factors. Methods: Forty-eight children, 29 boys and 19 girls, aged 3-17years old (median age was 8 years old) , with recurrent or refractory CD19 positive B-ALL, were treated by the CD19 specific CAR-T cells. A total of 48 cases received 61 infusions. Flow cytometry or real-time quantitative polymerase chain reaction method were used to monitor micro residual disease (MRD) . The follow-up period was from 16 to 1 259 days with the median follow-up of 406 days. SPSS software was used to statistical analysis. Results: No adverse reaction was observed during 61 infusions. The most common adverse reaction after CAR-T cell infusions was cytokine-release syndrome (CRS) . Only 2 cases experienced level 3 CRS performance, including continuous high fever, convulsions, delirium, serous cavity effusion, and decreasing of blood pressure. Tocilizumab was given to release CRS performance. No treatment-related death occurred. Thirty-seven patients showed response during 7 to 28 days after infusions. The early response rate was 77.1%, with MRD before infusion less than 5% group higher than the MRD more than 5% group (87.1% vs 58.8%, χ2=4.968, P=0.036) . For the 37 patients who showed response to CAR-T cell infusions, univariate analysis identified that age, disease status at the time of treatment, MRD before infusion affected 2-year OS rate (P<0.05) . Multivariate prognostic analysis for EFS disclosed that the MRD before infusion more than 5% (RR=3.433, 95% CI 1.333-8.844, P=0.011) and not bridge to HSCT (RR=4.996, 95% CI 1.852-13.474, P=0.001) were the independent risk factors. Conclusion: The fourth generation CAR-T cells directed against CD19 could effectively and safely treat relapsed and refractory B-ALL, which implicated that CAR-T therapy as a novel therapeutic approach could be useful for patients with relapsed or refractory B-ALL who have failed all other treatment options. Reducing MRD as far as possible by effective pretreatment chemotherapy was in favor of increasing the response rate. Bridging HSCT after CAR-T cell treatment might be a better therapeutic strategy for the patient with refractory or molecular relapsed B-ALL.

HSC transplantation-associated intestinal thrombotic microangiopathy: clinical pathological features, diagnosis criteria and treatment / 中国实验血液学杂志

Thrombotic microangiopathy (TMA) is a lethal transplantation-associated complication which exactly likes acute intestinal graft-versus-host disease (GVHD) in the clinical manifestation. 373 consecutive patients with hematological diseases received family HLA matched or mismatched HCT from May, 2002 to July, 2004. To analyse the clinical and pathological characteristics of TMA, 30 patients who suffered from severe diarrhea and received colonoscopic examination and gut biopsy were retrospectively analyzed. The results indicated that 7 patients originally diagnosed as gut GVHD showed the pathological evidence of enteric TMA. The incidence of TMA was 7 out of 30 specimen (23.3%). Pathological evidence of enteric TMA shown microvascular disorder characterized by thrombus in the capillary without infiltration of lymphocytes and perivascular hemorrhages in the mucosa, swelling and focal denudation of epithelial cells. All patients with TMA were associated with cytomegalovirus (CMV) antigenemia/disease. Among these patients, 4 cases, who only showed TMA without the evidence of gut GVHD pathologically, displayed treatment-resistant bloody diarrhea, renal failure, veno-occlusive disease, hemorrhagic cystitis, hemolytic anemia as well as thrombocytopenia. But the other 3 cases, with co-existence of both TMA and GVHD pathological characteristics had better treatment response. Survival analysis indicated that 3 patients with TMA-GVHD survived for 461 to 536 days but three out of four TMA patients died from VOD with liver failure as well as multiple organ failure during 101 to 254 days after HCT. In conclusion, to better diagnose those patients with severe and refractory diarrhea following HCT, pathological examination may indicate crux evidence to identify intestinal TMA from gut GVHD. Furthermore, this primary report has first evidenced that TMA and TMA-GVHD are two pathologically well-recognized subtypes with the difference between the pathological characteristics, treatment response and clinical outcomes.

Prevalence of human herpesvirus-6 in allogeneic hematopoietic stem cell transplant recipients in correlation with cytomegalovirus infection / 中国实验血液学杂志

In order to study the prevalence of human herpesvirus 6 (HHV-6) in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients in China and to analyze the relationship between HHV-6 and cytomegalovirus (CMV) infection in post-HSCT patients, nested polymerase chain reaction (PCR) was used to monitor HHV-6 DNAemia in 72 consecutive allo-HSCT recipients. 680 EDTA anticoagulated peripheral blood specimens were gathered before HSCT or weekly until 12 weeks after allo-HSCT. The variants of HHV-6 were identified by Hind III restriction analysis. CMV-pp65 antigenemia was detected by immunofluorescence stain. The results showed that HHV-6 DNAemia was detected at least once in 62.5% (45/72) of the patients on the median day 14 (range, 7 - 63 days) after allo-HSCT, and HHV-6B was the predominant variant. CMV antigenemia was detected at least once in 65.3% (47/72) of the patients on the median day 43 (range, 14 - 105 days) after allo-HSCT. Co-infection of HHV-6 and CMV (HHV-6+/CMV+) occurred in 52.8% (38/72) recipients. The onset of HHV-6 DNAemia was earlier than that of CMV antigenemia (P < 0.0001). Patients with HHV-6 DNAemia positive were more likely to have concurrent CMV antigenemia than HHV-6 DNAemia negative patients (84.4% vs 33.3%, P = 0.0001) after allo-HSCT. Among the herpesvirus related disease, the relatively high incidence of hemorrhage cystitis (HC) occurred in 23.6% (17/72) of post-HSCT patients. 88.2% (15/17) of HC developed in HHV-6 positive patients, and 82.3% (14/17) occurred in CMV+/HHV-6+ patients. It is concluded that infection of HHV-6, co-infection of HHV-6 and CMV, commonly occurred in post-HSCT patients in China, HHV-6 infection closely related to CMV antigenemia.

Correlation between human herpesvirus 6 activation and acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the potential relationship between HHV-6 activation and acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic cell transplantation (HSCT).</p><p><b>METHODS</b>Peripheral blood samples were collected before and weekly after HSCT from 72 consecutive recipients. HHV-6 DNAemia was monitored by nested polymerase chain reaction (PCR). The genotypes of HHV-6 were identified by Hind III restriction assay.</p><p><b>RESULTS</b>Of the 72 patients, HHV-6 DNAemia were detected in 45 (62.5%) on a median of day 14 (range, 7 - 63 days) after HSCT. Grade I - IV aGHVD occurred in 40 (55.6%) on a median of day 26 (range, 9 -73 days). The median onset time of HHV-6 DNAemia was significantly earlier than that of aGHVD (P = 0.018). Compared with that in HHV-6 DNAemia negative [HHV-6(-)] patients, the cumulative incidence of grade I - IV aGHVD was higher (68.9% vs. 33.3% , P = 0.003) in HHV-6 (+) patients. Cumulative incidence of grade II - IV aGVHD in HHV-6 (+) cohort was also higher than that in HHV-6 (-) cohort (35.6% vs 14.8% , P = 0.027). Cumulative incidence of grade I - IV aGVHD was higher in patients with both HHV-6 and CMV positive (CMV+/HHV-6+) than in those with either CMV (CMV+/HHV-6-) or HHV-6 positive (CMV+/HHV-6+) and neither of them positive (CMV-/HHV-6-) [78.9% (30/38), 55. 6% (5/9) , 14. 3% (1/7) and 22. 2% (4/18), respectively, P = 0. 0001]. Cumulative incidence of grade II - IV aGVHD in CMV+/HHV-6+ group was also higher than that in CMV+/HHV-6-, CMV-/HHV-6+ and CMV-/HHV-6- groups [42.1% (16/38), 22.2% (2/9), 0% (0/7) and 11.1% (2/18), P = 0. 008].</p><p><b>CONCLUSIONS</b>Patients with HHV-6 activation or HHV-6/CMV co-infection maybe involved in the occurrence of aGVHD after HSCT.</p>

Analysis of risk factors for the development of acute graft-versus-host disease in 151 cases of allo-hematopoietic stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the incidence and risk factors of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT).</p><p><b>METHODS</b>The clinical data of 151 cases of allo-HSCT in 150 patients from Nov 2001 to Jan 2004 was analyzed.</p><p><b>RESULTS</b>aGVHD was developed in 60 cases (40.2%), including 43 cases with grade I - II and 17 with grade III - IV aGVHD, the mean time of aGVHD development was 21 days (range 1 - 85 days) after allo-HSCT, 35 out of 55 cases achieved complete response (CR) (63.6%). The early survival rate for grade I - II aGVHD was more than 90%, while that for grade III - IV aGVHD was 46%. Nineteen factors possibly correlated with the development of aGVHD were analyzed. The univariate analysis showed that recipient age, donor's sex, recipient's sex, sex and ABO blood group disparity between donor and recipient, diagnosis, the status of disease, the stage of disease, stem cell source, conditioning regimen (TBI/without TBI), CD34(+) cell number, CD3(+) cell number, early engraftment and neutropenic infection were not closely associated with the occurrence of aGVHD (P > 0.05). On the Cox regression model, 2 independent factors for grade I - IV aGVHD were identified:HLA mismatch (RR = 1.681, P < 0.05) and positive surface antigen (HBsAg) (RR = 1.907, P < 0.05). In addition, the univariate analysis showed aGVHD was strongly associated with CMV infection (P < 0.01).</p><p><b>CONCLUSION</b>aGVHD is a common complication after HSCT, HLA mismatch and HBsAg positivity are independent risk factors for aGVHD.</p>

KIR and allogeneic hematopoietic cell transplantation - review / 中国实验血液学杂志

The profound graft-versus-leukemia (GVL) effect presented after allogeneic hematopoietic cell transplantation (allo-HSCT) has been evidenced. In contrast to T cell mediated GVL, natural killer (NK) cells recognize target cells and introduce GVL effect by using an integration of activating and inhibitory receptors. This review has summarized current literatures from 2001 - 2003 on human killer cell immunoglobulin receptors (KIR) and other NK cell receptors involved in recognition of tumor targets and the polymorphism of KIR genes of donor/recipient pairs of related and unrelated Allo-HSCT. KIR epitope mismatch may facilitate engraftment and reduce leukemia relapse post transplant by mediating lysis of recipient's cells and introducing GVL effect under the condition of KIR epitope mismatch. Clinical roles of KIR in Allo-HSCT and immunotherapy are discussed. Technologic approach in allogeneic reactive NK cells introduction, identification and selection in vitro, development of inhibitory receptor blockade as well as up-regulation of activating NK cells may significantly enhance GVL immune response. Further investigation on the regulation of KIR inhibitory receptors enables to design novel strategy in cancer immunotherapy over the forthcoming decade.

Mismatched hematopoietic stem cell transplantation using GIAC protocol: report of 100 cases / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the clinical application of human leukocyte antigen (HLA) mismatched hematopoietic stem cell transplantation (HSCT) for malignant hematological diseases using a new GIAC protocol.</p><p><b>METHODS</b>One hundred patients with malignant hematological disease received G-CSF mobilization, intensive immunosuppression, ATG and combination of bone marrow and peripheral blood stem cell transplantation at least 1 locus mis-matched hematopoietic stem cell transplant performed with GIAC protocol. The conditioning regimen was intensified and prolonged with combined use of CsA, MMF and ATG for GVHD prophylaxis.</p><p><b>RESULTS</b>All patients achieved sustained, full donor-type engraftment. The cumulative incidence of grade II approximately IV aGVHD was 48.39%, and grade III approximately IV aGVHD was 12.90%. Thirty-eight patients had cGVHDs which were of extensive type in 11 patients. Twelve patients relapsed, 11 of them were high-risk patients, and 3 returned to CR after donor lymphocyte infusion. Twenty-two patients died, owing to recurrent diseases in 6 and transplant-related complications in 16 cases. Seventy-two patients were alive and disease free, with 1 year disease-free survival probabilities for standard and high risk patients of (83.52 +/- 7.41)% and (47.63 +/- 8.49)%, respectively.</p><p><b>CONCLUSION</b>The GIAC protocol for at least 1 locus mismatched hematopoietic stem cell transplantation is relatively safe and efficient for patients with hematological malignancies.</p>

Identification of acute leukemia-specific genes from leukemia recipient/sibling donor pairs by distinguishing study with oligonucleotide microarrays / 中国实验血液学杂志

To explore differentially expressed genes in leukemia gene expression profile and identify main related genes in acute leukemia, gene expression profiles were analyzed in bone marrow/leucopheresis peripheral blood stem cells samples from 9 acute leukemia patients and their sibling donors with the use of oligonucleotide microarrays. 163 reported leukemia-related genes were involved in the study. The oligonucleotide primers were designed, synthesized and spotted on the chemical-material-coated-glass plates in array. The total RNAs were isolated from nine patients' bone marrow or leucopheresis peripheral blood cells and from nine their sibling donors peripheral blood stem cells treated by G-CSF, then collected by CS-3000 cell selection machine, and were reversely transcribed to cDNAs with the incorporations of fluorescent dUTP. The mixed probes were then hybridized to the oligonucleotide microarray. The results showed that in four patient/donor pairs with B-ALL, 5 up-regulated (RIZ, STK-1, T-cell leukemia/lymphoma 1A, Cbp/p300, Op18) and 1 down-regulated genes (hematopoietic proteoglycan core protein) were identified; In five patient/donor pairs with AML-M(4) and AML-M(5), 6 up-regulated (STAT5B, ligand p62 for the Lck SH2, CST3, LTC4S, myeloid leukemia factor 2 and epb72) and 1 down-regulated genes (CCR5) were identified. In conclusion, on the basis of distinguishing study of specific genetic related recipient/sibling donor pairs, screening leukemia-related genes with oligonucleotide microarrays, a set of 13 up-regulated or down-regulated genes among 163 leukemia-related genes has been identified. The result has further confirmed that above genes play critical role in the molecular mechanism of acute leukemia.

Role of matrix metalloproteinases in the pathogenesis and therapy of leukemia / 中国实验血液学杂志

The invasion and metastasis of malignant tumor cells are important factors causing the death of cancer patients. The proteolytic activity of proteinases to most of the extracellular matrix macromolecules is closely correlated with the invasion and metastasis of malignant tumor cells. Matrix metalloproteinases (MMP) are key proteinases involved in these processes. MMP is a type of Zn(2+)-depended proteinases. MMP2 and MMP9 are the unique types of proteinase that hydrolyze the bone structure of excellulary matrix (type IV collagen). So they are particularly correlated with leukemia cells infiltration and metastasis. This review aims to introduce the function of MMP and the regulation of matrix gene expression, as well as their roles in leukemia cell invasion and metastasis. A new strategy that MMP may be a therapeutic target in the treatment of leukemia is particularly introduced.

Aberrant expression and deletion of FHIT gene in leukemias / 中国实验血液学杂志

FHIT (fragile histidine triad) gene at chromosome 3p14.2 usually expresses at a very low level in human tissue and cells. A high frequency of abnormalities in FHIT gene has been demonstrated in various cancers. FHIT is proposed as a putative tumor-suppressor gene. To evaluate the expression of the FHIT gene in various leukemias, bone marrow or peripheral blood samples from 98 leukemia patients were tested by RT-PCR: 38 from patients with AML-[M(2)(9), M(3)(12), M(4)(8), M(5)(9)], 16 with ALL, and 34 with CML-[CP(20), AP(4), BC(10)] of various FAB types, as well as 10 patients with other hematological malignancies. To detect a deletion in sequencing the FHIT gene, the representative aberrant PCR products were cloned and then sequenced. The results showed that 22/38 (58%) patients with AML, 9/16 (56%) patients with ALL and 19/34 (56%) patients with CML were detectable of aberrant FHIT mRNA transcripts or deletion of FHIT. In 6 (16%) AML patients, 3 (19%) ALL patients, and 5 (15%) CML patients, the wild-type product was absent. Some patient's samples - 6 (42%) AML, 6 (38%) ALL, and 14 (41%) CML revealed aberrant FHIT transcripts in addition to a normal-sized band. Samples from healthy donors (PB, n = 12; BM, n = 5) did not indicate any abnormal expression. Eleven isolated fragments from various patterns of FHIT gene expression were investigated using cDNA sequencing. Sequence analysis revealed deletion of exon 4-8, exon 5-8, and exon 5-6 in various leukemias, as well as the deletion of the full FHIT gene sequence. The fused transcripts included: exon 3 and exon 9, exon 3 and exon 7, exon 4 and exon 9, exon 5 and exon 7. Sequence analysis of aberrant fragments present in samples from an AML and a CML patients was detected for point mutations and insert mutations located in exons 2, 8 and 10, plus a variety of aberrant transcripts. Deletion or aberrant FHIT mRNA transcripts in 50/98 (51%) leukemia patients were found. All samples with aberrant FHIT lacked gene product. A Kaplan-Meier plot of survival in patients with AML in relation to FHIT expression revealed that aberrance or loss of FHIT gene significantly correlated with a low clinical remission rate and poor overall survival.

Selected CD34+ cell autologous transplantation for advanced malignant tumors / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To determine the clinical results of selected CD34(+) cell autologous transplantation in advanced malignant tumors.</p><p><b>METHODS</b>After pretreatment, fifteen patients aged 12 - 70 (49.5) years with various Stage III or IV malignant tumors were given the sorted CD34(+) cells collected by magnetic-activated cell sorting (Clini MACS, Milteny Biotech, Germany).</p><p><b>RESULTS</b>Peripheral blood progenitor cells (PBPC) from the patients were mobilized by chemotherapy and G-CSF 5 micro g/kg per day. CD34(+) cells gave 2.0 - 5 log depletion after cell sorting, with a median yield of CD34(+) selected cells of 2.4 (0.15 - 12.03) x 10(6)/kg. It gave a median recovery of 64 (52 - 81.4)% and median purity of 98.2 (83.2 - 99.7)%. The median time of neutrophil recovery > 1.0 x 10(9)/L and platelet recovery > 20 x 10(9)/L post-transplantation were 14 (8 - 26) days and 13 (11 - 35) days, respectively. On follow-up of 2 - 33 (11) months, the event-free survival rate was 53.3% (8/15) and the overall survival rate was 66.7% (10/15).</p><p><b>CONCLUSION</b>Transplantation of autologous selected PBPC CD34(+) cells gives prompt and stable engraftment. Selected CD34(+) cell transplantation, being a safe approach, may improve the clinical outcome even in patients with advanced malignant tumors.</p>

Cytomegalovirus infection and disease in allogeneic hematopoietic stem cells transplantation / 军事医学科学院院刊

Objective: To investigate the incidence of CMV infection(CMV-I) and CMV related diseases (CMV-D) after allogeneic hematopoietic stem cells transplantation in 70 consecutive allogeneic hematopoietic stem cells transplantation(allo-HSCT) patients and to search for the optimal prophylactic strategy.Methods: Blood samples were monitored using the CMV pp65 antigenemia assay.Of the 70 patients observed,30 patients with chronic myeloid leukemia［CML:CP(27),AP(2),BC(1)］,12 with acute myeloblastic leukemia(AML),10 with acute lymphoblastic leukemia(ALL)and other cases were NHL(3), AA(5), MDS(7), SCLC with pancytopenia (1),CLL(1), and MF (1). Sixty six patients received HLA - identical siblings transplantation and four received tranplants from their HLA- haploidentical donors. Seventy cases included allo-PBPCT (64 cases) , allo-BMT (4 cases) and allo-PB+BMT (2). Before transplantation, all patients and donors received CMV serological examination except 4 pairs of donors/recepients. All 66 patients (3 cases were CMV IgM positive) and 64/66 donors were CMV IgG positive. Results：After transplantation, 64/70 patients developed CMV viremia during monitoring period. Forty three of 70 patients developed CMV-D.Thirty five of them suffered from CMV-associated interstitial pneumonia(CMV-IP). The high peak levels of CMV antigenemia were associated with development of CMV disease　. Close correlation was found between acute graft vs host disease(GVHD) and CMV disease. The patients were followed up for 2 to 24 months. The patients who received preemptive therapy(group A)had significantly better outcome than CMV disease group(group B, P=0.0001). Conclusions: The results suggest that CMV antigenemia has high predictive value for subsequent CMV disease and CMV pp65 antigenemia -guided early therapy has particular advantage for avoiding morbidity and mortality caused by CMV disease.

Detection of Cytomegalovirus Antigenemia Guides Prophylaxis of Cytomegalovirus Infection after Allogeneic Hematopoietic Stem Cell Transplantation / 中国实验血液学杂志

Cytomegalovirus infection (CMV-I) and CMV related diseases (CMV-D) occurred after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) seem to be with high morbidity and mortality. This study is a retrospective analysis of the incidence of CMV infection and diseases in Allo-HSCT patients known to be CMV seropositive before transplantation. To review the efficacy of CMV pp65 antigen-guided ganciclovir prophylaxis in preventing CMV infection and to search the optimal determination methods, 45 consecutive Allo-HSCT patients have been observed. Using the CMV pp65 antigenemia assay and serological analysis monitored blood samples from 23 patients with chronic myeloid leukemia (CML), 7 acute myeloblastic leukemia (AML), 6 acute lymphoblastic leukemia (ALL); other: 4 myelodysplastic syndrome (MDS), 3 non-Hodgkin's lymphoma (NHL) and 2 aplastic anemia. Forty-three patients received HLA-identical siblings transplantation and 2 from their HLA-haploidentical donors. Forty-five cases included Allo-PBPCT (38 cases), Allo-BMT (2 cases) and Allo-PBPCT + BMT 5 cases. Before transplantation, all donors/recipients have taken CMV serological detection. All donor/recipients were CMV IgG positive and one donor and one recipient with CMV IgM positive, respectively. After transplantation, all patients developed CMV antigenemia during monitoring period. Twenty-five patients developed CMV related interstitial pneumonia (CMV-IP). Patients have been followed from 6 to 28 months (median of 18 months) after transplantation. The patients who received preemptive therapy had a significantly better outcome than patients who did not received preemptive therapy. CMV related mortality was 1/29 cases in preemptive group vs. 12/16 cases in non-preemptive group. The results suggest that prompt and early institution of effective therapy with ganciclovir upon detection of CMV pp65 antigenemia, provides optimal protection against progress of CMV disease for patients undergoing Allo-HSCT.