Myeloid Recovery Research Articles

Impact of hematopoietic chimerism at day +14 on engraftment after unrelated donor umbilical cord blood transplantation for hematologic malignancies

Cord blood transplant is a feasible treatment alternative for adult patients with hematologic malignancies lacking a suitable HLA-matched donor. However, the kinetics of myeloid recovery is slow, and primary graft failure cannot be detected easily early after transplantation. We investigated the impact of hematopoietic chimerism status from unselected marrow cells 14 days after transplantation on predicting engraftment after a cord blood transplant. Seventy-one adult patients with hematologic malignancies undergoing single-unit unrelated donor cord blood transplantation after a myeloablative conditioning regimen were included in the study. All patients received conditioning regimens based on busulfan, thiotepa and antithymocyte globulin. Chimerism status was assessed analyzing short tandem repeat polymorphisms. The cumulative incidence of myeloid engraftment at 1 month was significantly lower in patients with mixed chimerism than in those with complete donor chimerism (55% vs. 94%; p<0.0001). For patients achieving myeloid recovery, the median time of engraftment was 16 days when donor chimerism at day + 14 was higher than 90%, compared with 24 days when donor chimerism was below this level (p<0.001). A donor chimerism level of 65% was found to be the best cut-off point for predicting primary graft failure, with a sensitivity of 97% and a specificity of 80%. The incidence of primary graft failure was 67% for patients with less than 65% donor chimerism at day +14 as compared to only 2% for those with more than 65% donor chimerism (p<0.001). Patients with mixed chimerism also had a lower cumulative incidence of platelet engraftment than those with complete chimerism (62% vs. 89%; p=0.01). Donor-recipient chimerism status at day +14 predicts engraftment after a single-unit cord blood transplant in adults.

Pegfilgrastim for PBSC mobilization and autologous haematopoietic SCT

To date, G-CSF is the most favoured cytokine administered for PBSC mobilization because of its great efficacy and lack of serious toxicity. Recently, a pegylated filgrastim (pegfilgrastim) has been introduced. Attachment of the polyethylene glycol (PEG) moiety reduces renal excretion and masks proteolytic cleavage sites resulting in elevated G-CSF serum levels for up to 14 days after a single injection. As single-dose pegfilgrastim had similar effects in the prophylaxis of chemotherapy-induced neutropenia as the daily administration of the unconjugated drug, its capability for the mobilization of haematopoietic stem and progenitor cells has been assessed and presented to be at least equal to that of conventional G-CSF. Administration of pegfilgrastim following high-dose therapy and autologous blood SCT (BSCT) shortened the time to myeloid recovery as seen in conventional G-CSF. Plasma G-CSF levels were about 1 log higher with pegfilgrastim, but in the setting of autologous BSCT this did not translate into a faster haematopoietic recovery. Only few data exist on the biological effects of pegfilgrastim. Still, these data suggest that pegfilgrastim-stimulation results in different functional properties of haematopoietic stem and progenitor cells compared with G-CSF.

Non-T-Cell-Depleted HLA-Haploidentical Hematopoietic Stem Cell Transplantation for Children with Hematological Malignancies: Single-Center Pilot Study

Allogeneic hematopoietic stem cell transplantation is the only curative approach for a number of patients with hematological malignancies. For patients who do not have an HLA-identical related or unrelated donor, a related haploidentical transplantation is an alternative option. Recent advances in effective T-cell depletion of stem cells have significantly decreased graft-versus-host disease (GVHD) and early transplantationrelated mortality (TRM). However, the problems related to delayed immune reconstitution causing infectious complications and relapse remain, limiting the efficacy of haploidentical transplantation. Here, we report the results of ten children with hematological malignancies who received non-T-cell-depleted haploidentical hematopoietic stem cell transplantation. Malignancies included acute lymphoblastic leukemia (ALL, n = 6), acute myeloid leukemia (n = 2), chronic myeloid leukemia (n = 1) and Epstein Barr virus (EBV)-associated peripheral T cell lymphoma (n = 1). Median patient age was 5 years, and all patients presented with clinical and biologic features that indicated a very high risk of relapse with conventional chemotherapy. At the time of transplantation, seven patients were in remission, and two of these were urgent cases with primary graft failure of the first cord blood transplantation. The remaining three patients were not in remission. HLA disparity was two loci mismatches in one case and three loci mismatches in nine cases. The conditioning regimen consisted of the myeloablative method in five cases and reduced intensity conditioning in five cases. GVHD prophylaxis was conducted with tacrolimus and short-term methotrexate (sMTX) in the initial two cases and the remaining eight cases were given a combination of tacrolimus, sMTX and prednisolone (PSL). The stem cell source for the initial two cases was peripheral blood stem cells mobilized with G-CSF, and that for the remaining eight cases was bone marrow. Nine (90%) of the ten patients engrafted showed myeloid and platelet recovery on days +15 and +31 (median), respectively. All of these patients achieved full donor chimerism by day +30, and it persisted, except in one patient who relapsed. Acute GVHD of Grades II–IV and III–IV occurred in six (66.7%) and two (22.2%) patients, respectively, all of which responded to temporary augmentation of PSL. Chronic GVHD occurred in five (62.5%) patients, three of whom had extensive GVHD. Although one of these developed steroid refractory chronic GVHD, it was not difficult to control the symptoms of GVHD of the remaining patients. Infectious complications, including cytomegalovirus (CMV) antigenemia (n = 2), interstitial pneumonia associated with CMV (n = 1), temporary elevation of EBVDNA (n = 1), zoster (n = 1), invasive aspergillus infection (n = 1) and sepsis (n = 1), were observed. TRM occurred in two patients, and one patient with ALL relapsed on day +472 after transplantation. The Karnofsky performance scales of six of the eight survivors were 100%, and more than half of the evaluable patients were expected to discontinue immunosuppressive therapy within 2 years after transplantation. Because only one patient had relapsed by the time of the last follow-up, we believe that our GVHD prophylactic regimen suppresses the incidence of both acute and chronic GVHD to an acceptable level, while preserving the graft-versus-leukemia effect. Median follow-up of the eight patients who survived event-free was 34 months (range, 3 to 95 months). The 5-year probability of overall survival and event-free survival (EFS) were 77.6% and 66.6%, respectively. In the analysis of disease status at transplantation, encouraging results were obtained in patients who received the transplantation while in remission, with the rate of engraftment and 5-year probability of EFS at 100% and 80%, respectively. Although the number of patients in this series was small, the results described here indicate the feasibility of non-T-cell-depleted haploidentical stem cell transplantation for children with hematological malignancies who do not have an HLA-identical donor.

Rapid Myeloid Recovery as a Possible Mechanism of Whole-Body Radioadaptive Response

We investigated the mechanism underlying the radioadaptive response that rescues mice from hematopoietic failure. C57BL/6 mice were irradiated with low-dose acute X rays (0.5 Gy) for priming 2 weeks prior to a high-dose (6 Gy) challenge irradiation. Bone marrow cells, erythrocytes and platelets in low-dose-preirradiated mice showed earlier recovery after the challenge irradiation than those in mice subjected only to the challenge irradiation. This suggests that hematopoiesis is enhanced after a challenge irradiation in preirradiated mice. The rapid recovery of bone marrow cells after the challenge irradiation was consistent with the proliferation of hematopoietic progenitors expressing the cell surface markers Lin-, Sca-1- and c-Kit+ in low-dose-preirradiated mice. A subpopulation of myeloid (Mac-1+/Gr-1+) cells, which were descendants of Lin-, Sca-1- and c-Kit+ cells, rapidly recovered in the bone marrow of low-dose-preirradiated mice, whereas the number of B-lymphoid (CD19+/B220+) cells did not show a statistically significant increase. Plasma cytokine profiles were analyzed using antibody arrays, and results indicated that the concentrations of several growth factors for myelopoiesis after the challenge irradiation were considerably increased by low-dose preirradiation. The rapid recovery of erythrocytes and platelets but not leukocytes was observed in the peripheral blood of preirradiated mice, suggesting that low-dose preirradiation triggered the differentiation to myelopoiesis. Thus the adaptive response induced by low-dose preirradiation in terms of the recovery kinetics of the number of hematopoietic cells may be due to the rapid recovery of the number of myeloid cells after high-dose irradiation.

Prospective single-arm study of pegfilgrastim activity and safety in children with poor-risk malignant tumours receiving chemotherapy

The objective of this study was to assess the efficacy of an injection of 100 microg/kg of pegfilgrastim in haematopoietic recovery and mobilization in children following 32 courses of chemotherapy. End points were duration of neutropaenia, myeloid recovery and PBMC collection. Neutropaenia lasted a mean of 4.7 days (+/-2.13 days). Myeloid recovery occurred at a median of 10 days (inter quartile range (IQR) 8-11). Febrile neutropaenia complicated 13 courses (40.6%). Mobilization was observed in 20 out of 26 assessable courses (76.9%). The rise in CD34+ cells occurred at a median of 6 days (IQR 4-7) after PEG and remained >20 per microl for 6 days (IQR 4-8), with a median value of 80 per microl (IQR 48-170.5). The median CD34+ cell peak was 165 per microl (IQR 82.5-331), 9 days (range 6-14) after PEG. PBMC were collected on average at day +5 (+4 to +9) after PEG. In 93.3% of collections, at least 3 x 10(6) per kg CD34+ cells were collected through a single apheresis. Myeloid recovery occurred in all cases within 15 days, without concomitant thrombocytopaenia. The incidence of primary febrile episodes is in line with data in the literature and with our own historical experience. A long-lasting period of circulating CD34+ cells allowed for more accurate scheduling of apheresis.

Early identification and management of graft failure after unrelated cord blood transplantation

Delayed hematologic recovery is common after unrelated donor umbilical cord blood transplants (UCBT). Clinically it is important to quickly differentiate slow engraftment from graft failure (GF). We report the engraftment data on 110 pediatric UCBT recipients. By day 28, 71 patients achieved an ANC >0.5 x 10(9) per liter, and 6 others died early without recovery. Of the remaining 33 patients who were still neutropenic, 20 eventually attained donor myeloid recovery, 3 died of transplant-related complications or recurrent leukemia and 10 survived without donor-derived hematopoiesis. These patients received a second UCBT 33-95 days after the first transplant, after additional immunosuppression. One patient died early, the remaining nine patients were engrafted; eight demonstrated complete, and one mixed, donor chimerism (with subsequent graft loss). Acute GVHD developed in three, and chronic GVHD in six of the eight engrafted patients. Two patients developed EBV-lymphoproliferative disorder. Infections, especially viral, were common and protracted. Six of 10 patients are alive, 165-1375 (median 1147) days after second UCBT. Chimerism studies correlated with subsequent engraftment course. Any result showing <5% donor cells was associated with irreversible graft loss. In conclusion, early second UCBT after primary GF is a feasible treatment option. Chronic GVHD and viral reactivation are common post transplant.

Leukocyte transfusions revisited: Stem cell graft stimulation in graft failure

The aim was to evaluate the role of leukocyte transfusions (LTX) in relation to graft function during prolonged neutropenia after stem cell transplantation (SCT). In 1989-2005, 22 SCT patients have received LTX in our unit. In all patients severe infection with profound neutropenia (<0.2 x 10(9)/L) preceded LTX. 13/22 had multi-organ failure or thrombotic microangiopathy. Irradiated leukocytes from pooled random donor products at 1.0 x 10(10) leukocytes/m(2)/day for a minimum of 3 days were used. Myeloid recovery and clinical benefit were analyzed. Engraftment defined as an absolute neutrophil count (ANC) of >0.5 x 10(9)/L on three consecutive days was observed in 15/22 (68%) patients at median of 6 (range 2-25) days after LTX, and sustained engraftment (ANC of >0.5 x 10(9)/L for 4 weeks) in 11/22 (50%) patients. Clinical benefit was observed in 6/22 (27%) patients with a prolonged antecedent neutropenia, who still demonstrated rapid sustained engraftment after LTX. Ongoing thrombotic microangiopathy was associated with persistent graft failure. LTX from random donors was associated with a rapid recovery of myeloid function in severely ill SCT patients. We speculate that allostimulation by LTX may have a role.

Efficacy of delayed administration of post-chemotherapy granulocyte colony-stimulating factor: evidence from murine studies of bone marrow cell kinetics

The optimal schedule of post-chemotherapy granulocyte colony-stimulating factor (G-CSF) administration has not been determined. G-CSF is customarily started 24 hours after chemotherapy; however, clinical data demonstrated that delaying G-CSF until 5 days after completion of chemotherapy has not resulted in a longer duration of neutropenia. Here, we examined the optimal timing of post-chemotherapy G-CSF administration in a mouse model, to show that delayed administration does not postpone the appearance of mature granulocytes in the peripheral blood. We also investigated the mechanism of decreased efficacy of the early G-CSF application after chemotherapy by characterizing the changes in bone marrow cellular composition. To our knowledge, we demonstrate for the first time, that early after chemotherapy, the bone marrow is predominantly composed of mature residual granulocytes and very few progenitors and precursors, on which G-CSF would act to generate granulocytes. The point when immature progenitors reappear does not occur in murine bone marrow until 48 hours after a single dose of cyclophosphamide. Our results indicate that the bone marrow cellular composition early after discontinuation of chemotherapy is not optimal for G-CSF action on acceleration of myeloid recovery. Given the high cost of G-CSF prophylaxis, its delayed administration may potentially result in substantial economic benefits.

Kinetics of peg-filgrastim after high-dose chemotherapy and autologous peripheral blood stem cell transplantation

Peg-filgrastim is a form of G-CSF with a sustained duration of action due to self-limited clearance. We administered 6 mg peg-filgrastim to 18 autograft recipients on day +1 after transplantation for hematologic malignancies. Plasma samples were collected at baseline and during transplantation. Hematopoietic recovery and clinical outcomes were compared to the historical data of 54 patients not receiving G-CSF. Patients receiving peg-filgrastim achieved a serum level of 115 000 pg/ml on day +2, 24 h after drug administration. Drug level maintained a plateau until day +8 and, after day +10, declined concomitantly with myeloid recovery. Patients experienced prompt neutrophil recovery: days +9 and +10 to 500 and 1000 neutrophils per microliter, and 4 days with an absolute neutrophil count <100 cells per microliter. Duration of antibiotic therapy was significantly shortened, but we did not observe significant differences in other end points. In conclusion, peg-filgrastim was well tolerated and efficacious, and hastened myeloid recovery.

Impact of Post-Remission Therapy on Survival of Older Patients with AML Treated with Intensive Chemotherapy: Results of the Multicenter ALFA-9803 Trial.

Background: More than 50% of newly-diagnosed patients with AML are older than 60 years of age. In this age group, the benefits of intensive chemotherapy are debated, due to excessive toxicity and short duration of response. Negative impacts of unfavorable cytogenetics and associated comorbidities are assumed to explain these results. Prolonged survival may, however, be observed in some patients even there is no well-established post-remission therapy at the present time.Methods: In the randomized ALFA-9803 trial in AML patients aged 65 years or more, we randomly compared idarubicin or daunorubicin throughout the study (R1) and two different post-remission strategies (R2): one single intensive consolidation course similar to induction versus six ambulatory cycles with one dose of idarubicin/daunorubicin (day 1) and 2x60 mg/m2/d cytarabine SC (day 1 to 5) delivered in out-patients on a monthly basis. Primary endpoint was 2-year overall survival (OS). From Nov 1999 to Apr 2006, 416 patients with de novo (#353) or post-MDS AML (#63) were included (median actuarial follow-up, 34 months). Median age was 72 years. High-risk cytogenetics was defined as complex (5 abns or more), monosomy 7, chromosome 5 and 7 abns, or 3q abn. Associated comorbidities were scored prospectively.Results: Complete remission (CR) rate was 57%, with 4% CRi and 10% induction deaths (ID), after a 4+7 induction with either 45 mg/m2/d daunorubicin or 9 mg/m2/d idarubicin (day 1 to 4) and cytarabine 200 mg/m2/d CIV (day 1 to 7), followed by lenograstim G-CSF until myeloid recovery. Salvage with intermediate-dose cytarabine and mitoxantrone was used in 54 patients. By multivariate analyses, only high comorbidity score (p=0.001) and advanced age (p=0.02) predicted ID. High-risk cytogenetics (p<0.001 and <0.001), high comorbidity score (p=0.001 and <0.001), high WBC (p=0.05 and <0.001), and advanced age (p=0.13 and =0.003) predicted lower CR rate and shorter OS, respectively. Median OS was only 4 months in a subset of about 20% of patients (high-risk cytogenetics and/or high comorbidity score and/or age 80+) who could thus be considered as “unfit” for intensive chemotherapy, even if meeting all usual selection criteria for an intensive approach. Median OS of the remaining 80% patients was 15 months. Long-term outcome, CR and ID rates did not differ according to R1. In the 164/236 CR patients randomized for R2 (69%), estimated 2-year OS from CR was 55% (95% CI, 43 to 66) in the ambulatory arm as compared to 36% (95% CI, 18 to 43) in the intensive arm (p=0.045). Remission duration was similar among both consolidation arms, while incidence of death in CR was lower in the ambulatory arm (1% versus 9% at 2 years).Conclusion: These results indicate that an ambulatory post-remission strategy has an anti-leukemic efficacy similar to intensive consolidation in older patients with AML and could become a reference arm to which alternative strategies incorporating new agents should be compared. In addition, such an “out-patient” strategy is easier to apply in most patients who have reached CR after intensive induction.

Umbilical Cord Blood (UCB) Graft T-Cells Correlate with Myeloid Recovery in Adults with Hematologic Malignancies Treated with Reduced Intensity Conditioning (RIC) and Single Unit Infusion.

Experience with unrelated allogeneic UCB stem cell transplantation in the non-myeloablative setting is limited because of concerns of graft failure due to limited cell content. Dual UCB graft infusion has been explored to overcome cell dose limitations, but influence on engraftment is unclear. This single institution phase I trial examined RIC followed by single UCB transplantation for patients unable to tolerate fully myeloablative regimens and lacking a matched adult donor. Conditioning consisted of TBI 200 cGy, fludarabine 175 mg/m², cyclophosphamide 2 gm/m², ATG 60 mg/kg. Trial design specified selection of UCB grafts to be matched at 4/6 HLA loci or better with one unit infused if cryopreserved nucleated cell dose exceeded 2.5 x 107/kg recipient weight. If no adequate single UCB unit to meet these criteria was available, study patients received 2 UCB units containing a combined cell dose exceeding 1.5 x 107/kg. Grafts were analyzed via flow cytometry post thaw for stem cell and lymphocyte expression of surface antigens, among them CD4, CD8, CD7, CD34, CD38, CD45RA, CD45RO. 23 patients have been enrolled, the majority diagnosed with AML (n=17). Median age was 47 (range 25–68) years. Single UCB units meeting stated criteria were identified for all but one study patient, who was excluded from graft analysis. The time to engraftment was measured from the date of transplantation to the date of 3 successive days ANC≥500/μL attained. In addition, we assessed time to peripheral blood donor lymphocyte chimerism ≥ 60%. Patients were censored at the date of last follow-up or the date of death. 94% (95%CI: 77-.99%) of patients achieved ANC≥500/μL by T+36 with median of 26.5 ( range 11–55) days. 68% (95%CI: 48–85%) of patients (n=15) achieved >60% donor derived chimerism by median of 22 (range 14–35) days. Median cryopreserved and infused total nucleated cell dose was 2.85 x 107 and 2.5 x 107 cells/kg, respectively. Median infused CD34 cell dose was 1.71 (range 0.21–5.39) x 105 cells/kg. By univariate analysis the dose of infused UCB graft T-cells including CD4 and CD8 T-cells co-expressing CD45RO, and CD34 stem cells co-expressing CD7 and CD38 correlated with neutrophil recovery, p=0.046, 0.008, and 0.033, respectively. Median overall survival at this early interim analysis has not been reached; with 86% and 65% survival at 3 and 24 months, respectively. Event free survival is 78% and 44.5% at 3 and 24 months, respectively. In summary, in this heavily pretreated group of advanced age patients, RIC and single unit unrelated allogeneic UCB stem cell transplantation is safe. Identification of a single UCB graft of HLA match 4/6 meeting cell dose requirement 2.5x107/kg is identified in the vast majority of full size adult patients. Engraftment and survival rates are higher than reported in single UCB transplants following administration of myeloablative regimens and similar to recent reports for RIC with double UCB graft infusion. The potential benefit of infusion of 1 vs. 2 UCB units after RIC in adult patients remains to be fully evaluated.

Nucleotide Receptor P2Y14 Modulates Hematopoietic Stem Cell Response to Tissue Injury Altering Stem Cell Preservation and Tissue Recovery.

P2 receptors are functionally diverse cell surface receptors that bind nucleotides adenine (ADP, ATP) and uridine (UDP, UTP). P2Y receptors are metabotropic G protein-coupled receptors that mediate vascular and immune responses to injury. We previously reported the differential expression cloning of the UTP-glycoconjugate receptor, P2Y14 from quiescent primary human bone marrow (BM) hematopoietic stem cells (HSCs). Using P2Y14−/− mice, we now report that the presence of P2Y14 protects HSCs from apoptosis in the face of cytotoxic chemical injury. P2Y14 null mice develop normally and showed no significant differences in peripheral blood cell counts, BM cellularity or the absolute number/proportion of lin−cKit+Sca1+ (LKS+) and CD34−/lowLKS+ (34-LKS+) cells compared to their wildtype littermates. Similarly, cell cycle status, in vitro colony-forming cell (CFC) capacity, in vivo homing and in vivo colony-forming unit-spleen (CFU-S) function were unaffected. Since the role of nucleotide receptors in injury response have been reported, we examined BM HSC content following IP injection of 200mg/kg cyclophosphamide (CTX) and found that the relative protection of LKS+ and 34-LKS+ cells from CTX-induced apoptosis was lost in P2Y14 null animals (WT LKS+: 12.7% AnnexinV+7AAD-, KO LKS+ 36.8% AnnexinV+7AAD−, n=5 each, p=0.004; WT 34-LKS+: 13.2% AnnexinV+7AAD−, KO LKS+ 38.7% AnnexinV+7AAD−, n=5 each, p=0.007). In addition, the kinetics of long-term myeloid recovery after a single injection of 5-Fluorouracil (5FU) IP 150mg/kg was significantly more accentuated in P2Y14 null animals, with significantly greater peripheral blood Gr-1+ cell count at days 21–56 post-injection (n=10 each, p=0.009). When sorted BM LKS+ cells were exposed in vitro to UDP-glucose, a putative P2Y14 ligand known to be released from cytoplasm during cellular injury, BrDU incorporation was significantly reduced (n=3 each, p<0.05), suggesting that P2Y14 activation with UDP-glucose reduces HSC cell cycle entry in response to injury. While these in vivo models examine HSC response to injury to both BM microenvironment and the HSCs themselves, when uninjured HSCs were reintroduced into injured microenvironment in the setting of hematopoietic reconstitution following lethal irradiation, P2Y14 null BM HSCs performed better in serial transplantation (n=10 each, p<0.01 for primary, secondary and tertiary transplantation), showing greater reconstitution and self-renewal capacity compared with WT littermates. From these findings, we propose that P2Y14 protects HSCs from chemical injury by acting as a sensor for metabolic “danger signal” in the form of released intracellular UDP-glucose during acute chemical injury in the BM and maintaining relative resistance of HSCs to toxin-induced apoptosis by restricting cell cycle entry. In the setting of injury exclusive to BM microenvironment (HSC transplantation), P2Y14 null HSCs, unable to detect UDP-glucose, respond to highly proliferative environment following lethal irradiation, resulting in greater reconstitution and self-renewal.

Posttransplantation Engraftment and Safety of Cord Blood Transplantation with Grafts Containing Relatively Low Cell Doses in Adults

The cell dose of a graft is a critical determinant of hematopoietic recovery and survival following unrelated cord blood transplantation. Most studies have found that the minimum acceptable nucleated cell dose should be between 1.5 X 10(7) and 2.0 X 10(7) nucleated cells per kilogram of body weight to reduce the time to myeloid recovery and increase the probability of engraftment. For some patients who have indications for hematopoietic cell transplants and for whom no other graft source except cord blood is available, it is difficult to decide whether they can receive cord blood grafts containing lower cell doses. In our study, patients who received cord blood grafts containing 1.0 X 10(7) to 2.0 X 10(7) cells/kg (n = 7) exhibited slower neutrophil and platelet recoveries compared with patients who received grafts containing total nucleated cell doses of 2.0 X 10(7) cells/kg and above (n = 93); however, 4 of those low-cell-dose recipients survived with a longer follow-up. Based on these preliminary results, cord blood grafts containing less than 2.0 X 10(7) cells/kg may be useful for cases where no grafts with higher cell doses or other stem cell sources are available.

A pilot phase II study of alternate day ganciclovir and foscarnet in preventing cytomegalovirus (CMV) infections in at‐risk pediatric and adolescent allogeneic stem cell transplant recipients

Prophylaxis with ganciclovir or foscarnet post allogeneic stem cell transplant (AlloSCT) reduces cytomegalovirus (CMV) disease. Combination ganciclovir/foscarnet is more effective than monotherapy in HIV patients with CMV retinitis. We hypothesized that alternate day ganciclovir and foscarnet for the prevention of CMV during the first 100 days after AlloSCT would be safe and effective. Fifty-three pediatric and adolescent AlloSCT recipients receiving 57 AlloSCTs where donors and/or recipients were CMV seropositive received ganciclovir (5 mg/kg/48 hr) alternating with foscarnet (90 mg/kg/48 hr) from myeloid recovery (>or=ANC 750/mm3) until Day +100. Patients were: M:F 31:22; age 6 years (0.8-18 years); donor sources: 25 related peripheral blood/bone marrow, 3 unrelated adult peripheral blood, 26 unrelated cord blood, and 3 related cord blood. GVHD prophylaxis included tacrolimus/mycophenolate mofetil (MMF). Median-nucleated and CD34 cell counts were 7.3x10(8)/kg and 5.07x10(6)/kg, respectively, for BM/PBSC; 4.07x10(7)/kg and 1.69x10(5)/kg, respectively, for CB. Despite a 36.5% probability of Grades II-IV acute GVHD, no patient developed systemic CMV disease. Five percent had Grade IV hematological toxicity that required discontinuation of ganciclovir. Twenty-five percent required discontinuation of foscarnet secondary to electrolyte abnormalities and/or renal dysfunction that were presumed to be multifactorial in origin. Probability of 1-year overall survival was 58.8%. Alternate day ganciclovir/foscarnet in AlloSCT recipients where recipient and/or donor is seropositive appears to be tolerable and 100% effective in preventing CMV systemic disease. A randomized study will be required to determine if this approach is superior to other CMV prophylactic designs.

Randomised comparison of two B‐cell purging protocols for patients with B‐cell non‐Hodgkin lymphoma: in vivo purging with rituximab versus ex vivo purging with CliniMACS CD34+ cell enrichment device

We investigated the feasibility, safety and efficacy of two B-cell purging methods in patients with CD20+ non-Hodgkin lymphoma (NHL) receiving autologous stsem cell transplantation. Myeloid and immune recoveries between the methods were compared. Twenty-seven patients were randomised to either in vivo purging with rituximab or ex vivo purging by CD34+ cell selection. Both purging methods were efficient at eliminating B-cells in infusates. When compared with in vivo purging, ex vivo purging was associated with CD34+ cell loss and delayed median neutrophil (10 d vs. 11 d) and platelet (12.5 d vs. 17 d) count recoveries. Lymphocyte recovery was similar in both groups, but immunoglobulin recovery was delayed after in vivo purging. Late-infectious complications were few in both arms. At a median follow-up of 27 months, 2-year probabilities of event-free survival (EFS) rates were 81% for in vivo purging and 76% for ex vivo purging (P = 0.66). When compared with 53 unpurged patients, all 27 purged patients had improved 3-year probabilities of overall survival (89% vs. 70%, P = 0.014) and a trend for improved EFS (78% vs. 57%, P = 0.075). In conclusion, although both purging methods were feasible and safe, rituximab purging was superior as it did not impair CD34+ cell mobilisation and was associated with faster myeloid recovery. Further studies are needed to determine whether rituximab purging is more effective than the use of unpurged autografts.

Does Early Myeloid Recovery Predict Superior Survival in Children and Young Adults Undergoing Hematopoietic Stem Cell Transplantation?.

Introduction: Slow rate of myeloid recovery at the end of induction chemotherapy reflected by a low absolute neutrophil count (ANC) is highly predictive of relapse in children with ALL (Laughton et al, 2005). Whether rate of myeloid recovery is predictive of outcome post-hematopoietic stem cell transplantation (HSCT) in children and young adults has not been studied.Objective: To explore the association between early ANC recovery and survival in children and young adults undergoing allogeneic and autologous HSCT for leukemia or lymphoma.Methods: Retrospective chart review was performed for all consecutive patients (age 6 months to 21 years) undergoing first HSCT for hematologic malignancies and lymphomas at Mayo Clinic, Division of Pediatric Hematology/Oncology from 1995 to 2004. Day 15 and day 28 ANC were calculated for all patients. The primary end point was 1-year relapse free (RFS) and overall survival (OS). To explore the association between ANC and primary outcome, patients were stratified in 2 groups: ≥ or < than median ANC and Kaplan Meier survival curves were generated for both the groups and compared with the log-rank test. Matched related donor (MRD), matched unrelated donor (MUD) and haploidentical (as one group) and autologous transplantations were analyzed separately.Results: There were a total of 50 patients. 28 received MRD, 2 MUD, 10 haploidentical and 10 autologous. The underlying malignancies were (AML n =25, Biphenotypic Leukemia n=1, ALL n =7, NHL n=3, HD n=7, JMML n=5, CML n=2, CLL n=1). No patient was lost to follow up during that time and data was available for all except (day 28 ANC) for one patient. The 1-year RFS and OS rate was the same; 81% for the MRD, 50% for MUD and haploidentical and 70% for autologous transplant populations, respectively. In patients receiving MRD transplants, higher ANC at either day 15 or day 28 post transplantation was associated with better, albeit statistically non-significant, RFS/OS rates at 1 year (86% for ≥ median (0.68) vs. 76% for < median p=0.44 and 92% for ≥ median (2.2) vs. 72% for < median p= 0.18 respectively). Opposite results were observed for MUD and Haploidentical transplantation group (33% for ANC ≥ median (0.47) at day 15 vs. 66% for < median p=0.28, 43% for ANC ≥ median (1.9) at day 28 vs. 60% for < median p=0.48). For autologous transplant group, higher ANC at day 15 was associated with better, albeit statistically non-significant, RFS/OS rates at 1 year (80% for ANC ≥ median (0.42) vs. 60% for < median p=0.60) while opposite results were observed on day 28 (60% for ANC ≥ median (0.46) vs. 75% for < median p=0.54).Conclusions: Our data generated conflicting findings of the association between ANC and RFS/OS in different patient populations undergoing HSCT. However, our results are limited by the small sample size. Further collaborative studies are needed to explore this association.

Patterns of Early Infectious Complications in Adult Unrelated Cord Blood Transplantation (UCBT). A Single Center Experience.

The major cause of non-relapse transplant related mortality (TRM) in UCBT continues to be infectious, with blood stream infections (BSI) constituting the majority of these events. However, CMV antigenemia and BK virus associated late onset hemorrhagic cystitis (LOHC) are increasingly important causes of morbidity and mortality. We review our single institution experience. We identified 13 patients with hematological malignancies in our database that had undergone UCBT in the period June 2002 - January 2005 and analyzed these data to document the incidence and patterns of infectious complications. The median age was 48 (range 19–62) years. All patients received infectious prophylaxis with Ciprofloxacin, Acyclovir, Voriconazole, Bactrim/nebulized Pentamidine. Eleven patients underwent conditioning with standardized Thiotepa/Busulfan/Cytoxan and GVHD prophylaxis with Tacrolimus/Mycphenalate Mofetil/ATG and the remaining 2 patients received TBI based conditioning. The median total nucleated cell (TNC) was 1.70E+07 and the median CD34 (+) cell count was 8.0E+04. The HLA matches were 4/6 in 9, 5/6 in 3 and 6/6 in 1 patient. All patients engrafted with a median time to neutrophil engraftment (TNE) of 25 (range 16–34) days. Twelve of thirteen patients achieved full donor chimerism at day 30 and 10 of 12 patients with full chimerism at day 100. Five patients developed grade II-IV acute GVHD requiring additional steroid therapy and 1 patient died of steroid refractory GVHD at day 50. Four patients had positive blood cultures at transplant. All patients developed documented bacterial infections: Gram positive in 12 (92%) (VRE in 23%, Steotrophomonas maltophilia in 31%); Gram negative in 8 (61%) (E. cloacae in 15%, Acinobacter spp. in 2 cases). Ten patients were CMV seropositive and 9 of these developed antigenemia requiring Gancyclovir/Foscarnet therapy. Nine patients had documented BK viruria with consequent late onset hemorrhagic cystitis (LOHC) and 5 patients developed BK viremia. There was 1 case of fungal BSI, while 8 patients had documented fungal tongue cultures. Four patients had positive bronchial washings (3 bacterial, 1 C. glabrata). Twelve (92%) patients were alive at day 100. Of note 62% of BSI and all cases of LOHC and CMV antigenemia developed after neutrophil engraftment. In our series of patients, viral infections (CMV, BK virus) were the most significant causes of morbidity. The high rate of BSI, especially after myeloid recovery, argues for a major influence of impaired immune recovery.

VEGF Levels Aid Prediction of Chemotherapy Induced Myeloid Toxicity and Presence of Extranodal Disease in Diffuse Large B-Cell Lymphoma (DLBCL).

Myeloid toxicity and their complications after chemotherapy for diffuse large B-cell lymphoma (DLBCL) are frequent and can be dose limiting. Most predictive models have focused on neutropenia and neutropenic fever. In our study we studied the role of conventional clinical factors & baseline vascular endothelial growth factor (VEGF) on all 3 lineage cytopenia i.e. thrombocytopenia, anemia and neutropenia.Methods: 106 newly diagnosed DLBCL patients between 2001 to 2005 had evaluation of their baseline white blood count (WBC), absolute neutrophil count (ANC), platelet count (PLT), hemoglobin (HB), serum albumin (ALB), creatinine (CRE), age, stage, presence of B-symptoms, high-LDH, performance status, bone marrow involvement (BM+). VEGF levels were performed in peripheral blood of 38 cases using Human VEFG Immunoassay Kit (Biosource International). All patients received standard CHOP chemotherapy +/− immunotherapy with rituximab. The clinical and laboratory factors were correlated with day-21 HB, PLT, ANC & cycle-1 neutropenic fever (NEU-FVR). Statistical analysis was done using SPSS v 11.5 software.Results: 106 patients had age distribution from 18–78-y (median 47.9y). 38 were >60y & 27>65y. Male: Female ratio was 67:39. Stage distribution was I=15, II=50, III=29& IV=12. 57 presented with B-symptoms. Only 11 had b-WBC (baseline) <4.0k/μl. 59 had b-HB <12g% and 17 <10g%. 13 had b-PLT <150K/μl. 25 developed neutropenic fever after first cycle of chemotherapy. Number of patients with D-21 HB<10g%, ANC<1.5 K/μl & PLT <100K/μl were respectively 30, 31 & 21. By linear regression analysis the significant predictors of D-21 Hb <10g% were BM+, baseline anemia & thrombocytopenia (p<0.05) and age >65y, ALB<35g/L & presence of B-symptoms (p<0.01). By backward stepwise methods of linear regression D-21 HB was predicted by (p<0.01) baseline anemia, thrombocytopenia, B-symptoms, low albumin & BM+. Similarly d-21 ANC was predicted by (p<0.001) advanced age, low albumin, basal anemia & thrombocytopenia. Thrombocytopenia and high LDH predict D-21 PLT<100K (p<0.001). By logistic regression neutropenic fever (NEU-FVR) after first cycle were predicted by low albumin, leukopenia (WBC<4.0K/μl) and age > 65 (all p <0.05). Baseline VEGF levels varied from 5–643pg/ml. Levels over 100pg/ml predicted baseline HB <12g%, extranodal disease, d-21 HB<10g% and d-21 ANC <1.5K/μl as shown in Table-1. A simple model based on age, baseline counts, albumin and VEGF correctly predicted D-21 counts & neutropenic fever with an accuracy of 96%.Conclusions: In our series clinical variables such as advanced age, baseline cytopenia and low albumin were important clinical variables for predicting myeloid toxicity and neutropenic fever (Table 2). Serum VEGF levels as a single parameter shows promise in predicting myeloid recovery before next chemotherapy. Its incorporation along with clinical variables presents an attractive predictive model for myeloid toxicity.Table 1:Elevated VEGF compared to important clinical parametersParameterVEGF <100pg/mlVEGF >100pg/mlp-valueBaseline HB <12g%15/2711/110.008Extranodal disease4/2711/11<0.001Day-21 Hb <10g%6/278/11<0.01Day-21 ANC <1.5K7/277/11<0.05Day-21 PLT <100K5/273/11NSCycle-1 NEU-FVR7/274/11NSChi square test performed in all. NS-Not significantTable 2:Significant predictors of myeloid toxicityAgeLow albuminBasal HBBasal PLTBasal WBCHi-LDHVEGFD-21 HB++++−−+D-21-PLT−−−+−+−D-21 ANC++++−−+NEU FVR++−−+−−

TGF-β1, in Synergy with M-CSF Induce Generation of a Novel Type of Human Cord Blood Dendritic Cell That Is Both Anti-Inflammatory and Capable of Ex-Vivo Expansion of Myeloid Progenitor Cells.

Tolerogenic dendritic cells (DCs) could be a promising cellular therapeutic tool in human transplantation due to their potential for silencing unwanted immune reactions. However, it is not known whether tolerogenic DCs are capable of regulating human hematopoiesis. We recently reported that M-CSF, in combination with IL-4, induced cord blood monocyte differentiation into a population of dendritic cells (DC) with tolerogenic potential (M-DC) (Li et.al. J. Immol. 174: 4706, 2005.). TGF-β1 has potent immune regulatory functions on DCs. In this context, we investigated effects of TGF-β1 on cord blood M-DC differentiation by characterizing phenotype, cytokine production and allogeneic mixed lymphocyte reaction (MLR) of the resulting DC population (TGF/M-DC). We also evaluated effects of the resultant cells on expansion and growth factor withdrawal-induced apoptosis of human CD34+ progenitors isolated from cord blood. The effects were compared to those of TGF-β1, GM-CSF and IL-4-induced DCs (TGF/DC). Addition of TGF-β1 to highly purified cord blood monocytes cultured with M-CSF and IL-4 changed M-DC morphology to that of a homogeneous DC morphology (TGF/M-DC). Similar to M-DC, TGF/M-DC negatively stained for monocyte/macrophage markers CD14 and CD16, and positively stained for CD86 and HLA-DR, molecules involved in antigen presentation. It appeared that TGF/M-DC were bona fide DC with typical DC morphology and phenotype. In comparison with TGF-DC, TGF/M-DC produced high amounts of IL-10, but displayed reduced capacity in induction of allogeneic MLR, features categorizing them more appropriately into an anti-inflammatory DC subset. We generated conditioned medium (CM) from both types of DC by stimulation of these DC with LPS and studied effects of their CM on ex-vivo expansion of CD34+ progenitors isolated from cord blood. In the absence of DC CM, the combination of SCF, Flt3 ligand and TPO (SFT, a cytokine combination widely used for cell expansion) induced a 10.5 ± 1.8 fold expansion of myeloid progenitors (CFU-GM) in 1 week culture. TGF/M-DC CM (25% v/v), but not TGF DC CM, in synergy with SFT further expanded myeloid progenitors (CFU-GM) 19.4 ± 2.7 fold beyond that seen with SFT alone. The effects of TGF/M-DC CM was accompanied with a significant increase of CD34+ cell numbers after 1 week (TGF/M-DC CM in combination with SFT, TGF-DC CM in combination with SFT, and SFT alone expanded CD34+ cells by 8.4 ± 2.8 fold, 3.8 ± 2.8 fold and 5.9 ± 1.1 fold respectively). TGF/M-DC CM and TGF/DC CM prevented growth factor withdrawal-induced apoptosis of CD34+ cells, as analyzed by annexin-V staining. Therefore, TGF/M-DC might be capable of in vivo silencing of unwanted immune responses while promoting myeloid recovery. These features of TGF/M-DC make them an interesting cellular therapeutic candidate to potentially facilitate hematopoietic recovery as well as down-modulate unwanted immune responses after hematopoietic stem cell transplantation.

Protection against LethalAspergillus fumigatusInfection in Mice by Allogeneic Myeloid Progenitors Is Not Major Histocompatibility Complex Restricted

Invasive fungal infections are a leading cause of morbidity and mortality after myelotoxic chemotherapy or radiation exposure. The resulting depletion of myeloid precursors under these conditions appears to be the factor that limits approaches to accelerate immune reconstitution. In a murine model of myeloablation after radiation exposure, we demonstrated that highly purified common myeloid and granulocyte-monocyte progenitors (CMPs/GMPs) accelerated myeloid recovery and, thus, enhanced innate immunity as measured by survival after a lethal challenge with Aspergillus fumigatus. Of greatest significance was the demonstration that the protection afforded by CMPs/GMPs was not major histocompatibility complex restricted. Furthermore, the effect of CMP/GMP cellular therapy was additive with that of liposomal amphotericin B treatment. These observations greatly expand the potential donor pool and, thus, the clinical utility of CMP/GMP cellular therapy in patients with myeloid depletion.