The level of Tim-3+CD8+ T cells can serve as a potential marker for evaluating the severity of acute graft-versus-host disease after haplo-PBSCT

Early and accurate diagnosis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation is crucial for the prognosis of patients. This study identified a potential biomarker for the severity of aGVHD after human leukocyte antigen (HLA)-haploidentical peripheral blood hematopoietic stem cell transplantation (haplo-PBSCT). We included 20 healthy subjects and 57 patients who underwent haplo-PBSCT. Of these patients, 22 developed aGVHD after haplo-PBSCT. The results showed that patients with aGVHD had significantly increased levels of Tim-3+/Perforin+/Granzyme B+CD8+ T cells, but significantly decreased Galectin-9. The differences in Galectin-9 and Tim-3+/Granzyme B+CD8+ T cells between grade I-II aGVHD and III-IV aGVHD were also significant. In vitro, the apoptosis of CD8+ T cells from aGVHD patients was significantly increased after Tim-3/Galectin-9 pathway activation, which decreased Granzyme B secretion. As revealed by univariate analysis, the level of Tim-3+CD8+ T cells was a risk factor for severe aGVHD. ROC analysis demonstrated that high levels of Tim-3+CD8+ T cells had a significant diagnostic value for severe aGVHD, with an area under the curve of 0.854 and cut-off value of 14.155%. In conclusion, the binding of Tim-3 with exogenous Galectin-9 can promote apoptosis of CD8+ T cells and affect the secretion of Granzyme B. Tim-3+CD8+ T cells have the potential to serve as immunological markers for assessing the severity of aGVHD after haplo-PBSCT and identifying patients at a higher risk for severe aGVHD.

Immature Reticulocyte Fraction in Guiding Stem Cell Harvest in Autologous Peripheral Blood Stem Cell Transplant

Peripheral blood (PB) CD34+ cells enumeration is currently the most reliable method to guide the timing of stem cell harvest. However, its usage is restricted by being technically challenging, costly, and time-consuming. Immature reticulocyte fraction (IRF) determination, which is simpler and cheaper and has a faster turn-around time, has been proposed for a similar purpose. The purpose of this study is to evaluate the value of IRF in guiding stem cell harvest and examine the correlation between IRF and PB CD34+ cells count. Daily pre-harvest tests, i.e. PB CD34+ cells and IRF from 21 patients scheduled for autologous PBSC transplant were assessed. Stem cells harvests were commenced when the PB CD34+ cell count were more than 10 cell/ul. A total of 205 pre-harvest tests were analysed. Following stem cell mobilisations, both the IRF and PB CD 34+ cell counts rose with a variable pattern. In this study, we observed that the IRF peaks preceded the PB CD34+ count by 2 days. On the day of stem cell harvest, all the peak IRF values were >0.3. The PB CD34+ cell counts correlated with the harvested stem cell yield, whereby r2 = 0.77, p 0.3 may be used as a cut-off value for the initiation of PB CD34+ quantifi cation prior to stem cell harvest.

HLA Chimerism in allogenic haplo-identical peripheral blood stem cell transplant.

HLA antigens were used as markers to establish the presence of chimerism (i.e. simultaneous presence of two lymphocyte populations from recipient as well as donor) in a patient with chronic granulomatous disease treated with one haplotype matched stem cell transplant. Neutrophil engraftment occurred on Day 6 post peripheral blood stem cell transplant (PBSCT). Platelet counts were maintained above 20x10[9]/L. Six months after the allogenic PBSCT, lymphocyte population was chimeric and cells of both donor (father) and host HLA type were present. The patient revealed a shift in his HLA antigen profile and there was evidence of donor cell engraftment. The HLA phenotype A26,CwXX,B8,DRB1*03//A32,Cw4,B35,DRB1*16// represented his true phenotype whereas A11,Cw7,B62,DRB1*14 represented donor (father) origin.. HLA system as a genetic marker is a useful additional approach to determine engraftment following an allogenic haplo-identical stem cell transplantation.

The Effect of Preemptive Therapy with Intravenous Ganciclovir Followed by Oral Ganciclovir against Cytomegalovirus Infection after Allogeneic Peripheral Blood Stem Cell Transplantation / 대한혈액학회지

BACKGROUND: Recently, in vitro studies demonstrated faster immune reconstitution after allogeneic peripheral blood stem cell transplantation (PBSCT) compared to bone marrow transplantation (BMT). In consequence, it can be expected that better immune reconstitution against cytomegalovirus (CMV) will lead to a reduced CMV-related morbidity and mortality after allogeneic PBSCT. METHODS: Forty seven patients who received allogeneic PBSCT were enrolled. CMV was routinely sought by at least weekly screening for CMV-related matrix protein pp65 antigenemia after engraftment (WBC >1,500/nL) was achieved. CMV antigenemia was treated with ganciclovir 5mg/kg twice daily i.v. as preemptive therapy for at least 10 days. After then, ganciclovir i.v. was switched to oral ganciclovir for maintenance therapy. RESULTS: CMV antigenemia was detected 8 (17%) out of 47 patients and CMV disease developed in only 1 case (2.1%). The medianperiod of time until the detection of CMV antigenemia was 51.5 days (range, 35~230). In 7 out of 8 cases, CMV antigenemia disappeared with ganciclovir treatment in 7 days. One patient with CMV disease (CMV interstitial pneumonitis) showed persistent CMV antigenemia for 3 months and expired due to restrictive lung disease. CONCLUSION: The incidence of CMV antigenemia and resistance to ganciclovir treatment was lower than the incidence of those reported in allogeneic BMT trials. These findings suggest that faster immune reconstitution against CMV after allogeneic PBSCT might have a stronger role in the prevention of emergence of CMV antigenemia and ganciclovir treatment than after allogeneic BMT.

Autologous Peripheral Blood Stem Cell Transplantation Using G-CSF Combined Conditioning in AML Patients

BACKGROUND: The possibility that G-CSF recruits leukemic cells from the G0 to S phase, which may lead to a greater susceptibility to cytotoxic drugs, such as ara-C, has been presented in Harada's study. METHODS: In this study, we referred to the protocol of Harada et al 1 to try G-CSF combined marrow-ablative chemotherapy and autologous PBSCT, for the treatment of AML patients in CR1 status. Between January 1997 and March 1998, six AML patients (3: children, 3: adults) in CR1 status were autografted and followed up to 3 years. RESULTS: The major regimen related toxicity was composed of mucositis and diarrhea without death. The time of ANC recovery to 500/L and 1,000/L was 11~48 and 16~81 days, respectively. The mean time of platelet recovery to 20,000/L and 50,000/L was 21~233 and 35~370 days, respectively. The platelet recovery time to 50,000/L was markedly prolonged for more than 100 days in four patients (66.7%). Moreover, four patients (66.7%) experienced a relapse of leukemia after transplantation, with a mean interval of 147.5 days after PBSCT. Two patients were in CR status for 53 and 51 months after PBSCT, respectively. CONCLUSION: The G-CSF combined marrow-ablative chemotherapy and autologous PBSCT resulted in a markedly delayed platelet recovery and no advantages for decreasing the relapse rate of AML. But, further studies will be warranted.

Allogeneic Peripheral Blood Stem Cell Transplantation in Hematological Malignancies: Stem Cell Mobilization with GM-CSF alone or a combination of GM-CSF and G-CSF from Normal Healthy Donors / 대한혈액학회지

BACKGROUND: Granulocyte-colony stimulating factor (G-CSF) has been used in normal heathy donors to mobilize hematopoietic progenitors. Recently, it was reported that an addition of granulocyte-macrophage-CSF (GM-CSF) mobilized more primitive CD34+ subsets than did G- CSF alone. We investigated the result of the allogeneic peripheral blood stem cell transplantation (PBSCT) with stem cells mobilized with GM-CSF alone or a combination of GM-CSF and G-CSF from normal healthy donors in hematological malignancies. METHODS: Twenty-nine patients with hematologic malignancies had allogeneic PBSCT from normal sibling donors. Nine healthy donors were mobilized with GM-CSF (Leucogen (R)) alone and 20 with a combination of GM-CSF and G-CSF (Leucostim (R)). After 5~8 days of cytokine treatment, PBSCs were collected by large volume leukapheresis and analyzed. RESULTS: Stem cells were collected from the HLA matched normal healthy sibling donors. The mean harvested cell content was 8.74+/-3.22X10(8) MNCs/kg, 15.65+/-16.02X10(6) CD34+ cells/kg of the patients. There were significant differences in the harvested MNC count between mobilization group with GM-CSF alone and group with a bination of GM-CSF and G-CSF. Observed side effects of cytokine mobilization were myalgia (76%), headache (41%), febrile sense (24%) and skin rash (10%). These complications disappeared within 48 hours after discontinuation of cytokines. The median interval to achieve a WBC count>500/uL was 15.00+/-4.23 days, and 14.00+/-33.01 days to a platelet count>20,000/uL. The actual incidence of acute GVHD was 36.4%, 22.7%, and 4.5% for skin, GIT, and liver, respectively. Immunosuppressant responsive chronic GVHD developed in 63.1% (12/19) of assessable patients including 6 cases who had donor lymphocyte infusions. CONCLUSION: In this study, GM-CSF based cytokine mobilization was able to collect sufficient numbers of stem cells and allow rapid engraftment in the allogeneic PBSCT. Mobilization protocol with a combination of GM-CSF and G-CSF seemed to be superior to GM-CSF alone. Acute GVHD in patients with allogeneic PBSCT didn't appear to be more severe than in patients undergoing allogeneic BMT.

Effects of Low Dose Interleukin-2 Therapy after High Dose Chemotherapy and Autologous PBSCT in Childhood AML and Stage 4 Neuroblastoma / 대한소아혈액종양학회지

PURPOSE: Interleukin-2 (IL-2) exerts anti-cancer effect by increasing NK cell activity when the tumor burden is low. Earlier study conducted with high dose intravenous IL-2 exhibited significant toxicities such as capillary leak syndrome, fever, rash, etc. This study was designed to study the effect of low dose IL-2 in children after autologous PBSCT when the cancer is at minimal level. METHODS: A total of 12 patients (6 AML, 6 NBL) were enrolled in this study from May 1997 to Oct 1999. The age of the patients was between 0.9~15 yr (Median age: 4.35 yr). The AML patients were treated with AML-BFM-87 (5 cases) or CCG-2891 (1 case) protocol, and all the patients underwent autologous PBSCT at CR1. The NBL patients were treated with CCG-3891 (4 cases) or '6 in 1' (2 cases) protocol, and they had operation for residual tumor before PBSCT. The conditioning regimen for AML patients was busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) (4 cases) or BCVAC (2 cases), while NBL patients were conditioned with carboplatin (1200 mg/m2), etoposide (800 mg/m2) and melphalan (180 mg/m2). Infused stem cell dose was MNC: (4.5+/-1.7) 108/kg, CD34 : (8.6+/-3.2) 106/kg. IL-2 (Proleukin , Chiron) was started subcutaneously after neutrophil engraftment (ANC<500/mm3) with the dose of 3~5 MU/m2 for the first 2 days, 1MU/m2 for the subsequent 12 days, then followed by 14 days of rest. IL-2 was restarted with the same regimen for more than 6 cycles as outpatient. The CBC, total eosinophil count (TEC) and T lymphocyte subsets were checked before and after IL-2 therapy. RESULTS: The mean neutrophil engraftment was achieved on 12.0+/-3.4 days, and mean platelet recovery to more than 50,000/mm3 was achieved on 23.7+/-10.3 days. Common toxicities associated with IL-2 were fever and mild tenderness on injection site, but there was no need to discontinue IL-2. A total of 75 cycles of IL-2 therapy was given. During follow-up for 8~30 months (median 21 months), only 1 relapse occurred until now (neuroblastoma stage IV). All parameters of T lymphocyte subsets increased after IL-2 therapy. TEC increased in mean value after IL-2 and it was statistically significant (P<0.05). The absolute count of CD4 and CD8 was significantly increased (CD4 : 410 to 640, P<0.005, CD8 : 720 to 980, P<0.05). CD4/CD8 ratio remained reversed (<1) throughout the course of IL-2 in most patients. The total NK cell count was increased from 510 to 820 (P<0.005). CONCLUSION: Low dose IL-2 therapy was well tolerated as OPD basis and there was a significant change in T lymphocyte subsets, especially in NK cell count. Even though the follow up duration was short, the high relapse free survival indicates the beneficial effect of low dose IL-2. In the setting of low tumor burden, such as after autologous PBSCT, low dose subcutaneous IL-2 seems to provide effective anti-cancer effect.

Peripheral Blood Stem Cell Harvest by Transient Radial Arterial Catheterization in Pediatric Patients / 대한소아혈액종양학회지

BACKGROUND: Peripheral blood stem cell transplantation in children has some difficulties compared with adult cases. Despite the children's total blood volume is small, they have a relatively higher volume of blood per weight than adults. Also, it is hard for the children to maintain 2 central vein during leukapheresis to shorten the time consumed. We compared the artery-harvest and central vein-harvested group for possible risk and stem cell yield in pediatric patients with malignancies. METHODS: From August, 1995 to January, 1997, 21 leukapheresis was performed by 7 patients. The patients who could have 2 central veins for leukapheresis were included in vein-harvested group and the patients who could have only 1 central vein were included in artery-harvested group. The peripheral vein was not used for leukapheresis. COBE Spectra(COBEBCT, Lakewood, CO) was used and priming was done by packed RBCs in patients weighing less than 20kg. Stem cell yield was assessed by MNC, CD34+, CFU-GM, respectively. During leukapheresis, the patients were closely monitored for change in vital sign, evidence for thrombosis, bleeding, hypocalcemia, etc. RESULTS: There was no serious complication in each group of patients. After the leukapheresis, WBC and platelet count decreased but Hb level was increased due to reinfusion of primed packed RBCs. Average flow rate was higher in vein-harvested group but there was no difference in time consumed and results in stem cell assay. CONCLUSION: Peripheral stem cell harvest in children by radial artery can be performed safely and easily without an increased risk or complication. In younger children, it is possible to achieve even more higher stem cell yield. If the patient is unable to maintain 2 central vein for leukapheresis, transient radial arterial catheterization is a safe and convenient method.