Abstract
The treatment of hematological malignancies with umbilical cord blood transplantation (UCBT) is rapidly increasing for adult patients. Disadvantages of UCBT include insufficient cell numbers ...for adult patient reconstitution, a lack of antigen experienced cells, and deficits in T cell signal transduction mechanisms. Consequently, UCBT is frequently associated with impaired immune function and high infection-related mortality. To counter these difficulties, transplantation with two UCB units has been employed to improve immune reconstitution in adult patients. We evaluated both the quantitative and functional reconstitution of cellular immunity in a group of adult patients undergoing UCBT. Thirty-two patients with a median age of 50 years with hematopoietic malignancies were treated with reduced intensity conditioning (Flu/Mel/rATG) followed by infusion with two sequential UCB grafts and GvHD prophylaxis with tacrolimus and sirolimus. The grafts were at least a 4/6 match with each other and the recipient. Here we report the results of 27 patients who have completed at least one year of follow up. Assessments were done prior to transplant and at various time intervals until 12 months post UCBT. Neutrophil and platelet engraftment occurred at a median of 21 days and 42 days, respectively. CD3+ populations remained severely depressed until 8 wks post-transplant when they gradually began to re-emerge. However the CD4+ and CD8+ populations demonstrated distinctly different reconstitution kinetics. At 6 months the median value of absolute numbers of CD4+ lymphocytes was 35% of pre-transplant levels increasing to 42% at 1 yr post-transplant, a median value far below the normal range for adults. In contrast, at 6 months post-transplant CD8+ lymphocytes remained severely depressed to 12% of pre-transplant levels, but dramatically increased and reached normal levels by 1 yr after UCBT. Interestingly, both the CD14+ monocyte and the CD16+CD56+ NK cell populations expanded dramatically at 4 wks post-transplant and reached pre-transplant levels and were within the normal range by 6 months. CD20+ B cell repopulation began at 8 wks post-transplant, displayed a striking expansion leading to a 17-fold increase in the median value for B cell numbers over pre-transplant values at 1 year and resulting in a median value of absolute numbers near the top of the normal range. To evaluate functional T cell immune reconstitution in vivo, we performed IFN-γ ELISpot analysis on CMV stimulated PBLs and compared the results to a PCR-based assay for CMV viremia. Additionally, we assessed the reconstitution of thymopoiesis with the T cell receptor excision circle (TREC) assay and real-time PCR. 16/27 patients and 26/52 UCB products were CMV seropositive prior to transplant. In the post-UCB period, development of CMV-specific effectors as determined by ELISpot did not always correlate with clearance of CMV viremia. Specifically, prior to 8 weeks post-UCBT, 8 out of 12 (67%) patients with CMV-positive ELISpot displayed CMV viremia, between 8 weeks and 100 days post-UCBT 4 out of 11 (36%) patients with CMV-positive ELISpot displayed CMV viremia and after 100 days post-UCBT only 3 out of 10 patients (30%) who developed CMV-positive ELISpot remained positive for CMV viremia. Identification of functional CMV effectors was only associated with the numbers of CD8+CD45RA+ cells (p=0.01) and the development of high TREC concentrations (p=0.01) that were detected after 6 months of UCBT, and was independent of GvHD or mixed chimerism. Taken together these results indicate that reconstitution of T cell immunity after UCBT is characterized by delayed recovery of CD4+ and CD8+ T cells and correlates with reconstitution of thymopoiesis and increase of naïve CD8+CD45RA+ T cells that can develop into efficient pathogen-specific effectors.
Umbilical cord blood transplantation (UCBT) is associated with impaired immune reconstitution and high infection-related mortality. Proposed potential mechanisms involve lack of antigen-experienced ...cells in UCB and impaired generation of antigen specific effectors due to Th2/Tc2 skewing, imprinted by placental factors. Furthermore, transplantation of two UCB units and mixed chimerism, albeit transient, may affect immune reconstitution. We evaluated the capacity of the recovering immune system to establish antigen-specific responses by quantitative and functional assessment of cellular immunity, in adult patients undergoing UCBT. Thirty patients with a median age of 51 years with hematopoietic malignancies underwent reduced intensity conditioning (Flu/Mel/rATG) followed by two sequential UCB infusions. GvHD prophylaxis consisted of tacrolimus and sirolimus. Here, we report our preliminary results of 15 patients who completed one year of follow up. Assessments were conducted prior to and at various time intervals until 12mo post-UCBT. CD4+ and CD8+ T cells decreased until 8wks post-UCBT and gradually increased thereafter. However, CD4+ and more strikingly CD8+ cells remained below normal by 12mo (median values 0.2x103/ul and 0.06x103/ul respectively). Most of the recovery was in the CD4+CD45RA+ population, which showed a slight initial drop but subsequently increased to normal. In contrast, CD4+CD45RO+ cells after the initial drop reached a low-level plateau by 6mo and never achieved normal levels. B lymphocytes remained low until 8wk but rapidly increased thereafter, exceeding normal levels by 12mo. CD16+CD56+ NK cells and CD14+ monocytes gradually increased to reach normal values by 6 and 12mo respectively. We evaluated functional immune reconstitution in vivo by assessing CMV-specific effectors by IFN-g ELISpot and additionally, in HLA-A*0201 positive patients, by HLA-A*0201/CMV-pp65 pentamer (NLVPMVATV) and flow cytometry. 14/15 patients and 2/30 UCB products were CMV seropositive prior to transplant. At a median time of 4 weeks, CMV antigenemia was detected in 6/15 UCB recipients and 5 of them developed functional CMV-specific effectors as determined by ELISpot. Functional CMV effectors were also detected in all 8/15 patients who did not develop detectable CMV antigenemia. The one seronegative patient developed neither CMV antigenemia nor CMV effectors. In HLA-A*0201 patients CMV-specific effectors could also be detected by pentamers. Unexpectedly, identification of functional effectors by ELISpot was only associated with the numbers of naive cells (CD4+CD45RA+ (p=0.03), CD8+CD45RA+ (p=0.02)) and CD16+CD56+ NK cells (p=0.01) and was independent of GvHD or mixed chimerism. Taken together these results indicate that immune reconstitution after UCBT is characterized by delayed recovery of CD4+ and more notably CD8+ cells and predominance of naive T lymphocytes. However, reconstituted naive T cells are capable of developing into functional effectors after in vivo exposure to antigen. This raises the possibility that vaccination, in vivo expansion or adoptive transfer of ex-vivo expanded antigen-specific effectors may provide successful strategies to overcome infection-related mortality in UCB recipients.
Background: Cytogenetic risk categories based on conventional chromosome studies are widely used in clinical practice to make treatment decisions for AML. We evaluated the efficacy of interphase FISH ...to detect chromosome anomalies in the workup of young (<60 years) patients with AML.
Methods: Study subjects were enrolled in E1900, a front-line Eastern Cooperative Oncology Group (ECOG) clinical trial for AML. This is an on-going Phase III clinical trial with Daunorubicin dose-intensification ± Gemtuzumab-Ozogamicin consolidation therapy prior to stem cell transplant. This trial opened December 2002; as of February 2005, 223 patients were enrolled. The protocol was designed to collect bone marrow for both cytogenetic and FISH studies at study entry (diagnosis). Cytogenetic studies were done by local laboratories with results reviewed centrally by the ECOG Cytogenetics Committee. Each case was classified as acceptable or unacceptable based on predefined ECOG cytogenetic criteria. FISH for each patient was performed in the ECOG FISH laboratory at Mayo Clinic and utilized eight probe sets to detect t(8;21), t(9;22), t(11;var), t(15;17), inv(16), +8, −5/5q, and −7/7q (Vysis, Downer Grove, IL).
Results: 64 (29%) of 223 specimens had incomplete cytogenetic and/or FISH results. We analyzed the remaining 159 (71%) specimens with complete cytogenetic and FISH results. Results for each specimen were classified by probe set into one of the following categories:
Normal cytogenetics and normal FISH;Abnormal cytogenetics and abnormal FISH for the anomaly the probe was designed to detect;Abnormal cytogenetics and abnormal FISH for an anomaly the probe was not primarily designed to detect;Normal cytogenetics and abnormal FISH;Abnormal cytogenetics and normal FISH; orAbnormal cytogenetics and abnormal FISH that further defined the karyotype.
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The concordance rate between cytogenetic and FISH results ranged from 97 to 100% for all probe sets and kappa analysis for concordance had a p value of <0.0001. Of the total 159 cases, discrepancies between FISH and cytogenetic results occurred in only 4 cases; two with normal cytogenetics and abnormal FISH and two with abnormal cytogenetics and normal FISH results.
Conclusions: The high level of agreement between cytogenetics and FISH demonstrates the accuracy of a panel of 8 FISH probe sets for the detection of significant abnormalities in AML. The data from this investigation support the use of FISH as an adjunct in cases of failed cytogenetic analyses to increase the yield of useful cytogenetic results in large cooperative trials. Furthermore, because of the strong correlation between cytogenetics and FISH, our results demonstrate the potential of FISH as a follow-up study of minimal residual disease in ECOG trials.
Increased methylation of the tumor suppressor and all-trans retinoic acid (ATRA) target gene RARβ2 has been reported in a variety of neoplasias. In APL, this has been related to the recruitment of ...DNA methyltransferases (DNmts) by PML-RARα to the RA response element-containing RARβ2 gene promoter (Di Croce, et al, Science 295, 1079, 2002). In this study, we applied a highly quantitative method, pyrosequencing of bisulfite-converted DNA, to precisely measure the level of methylation (% of total dinucleotides;dnts) at each of 14 CpG dnts in the RARβ2 promoter from APL cases with de novo vs 1st relapse vs ≥2nd relapse disease. Additionally, we assessed whether patients with PML-RARα mutations at relapse, which increase binding to the co-repressor complex and might secondarily increase DNmt recruitment to this complex, are associated with increased methylation of this ATRA target gene. Non-parametric 2-sided Wilcoxon tests were utilized to evaluate potential differences in median values. Surprisingly, the methylation value for de novo APL cells (N = 10) was only slightly greater than that for normal peripheral blood mononuclear cells (N=4) (medians of sums of all 14 dnts were 95.9 and 68.9, respectively; p = 0.14). However, in a matched set of pretreatment and 1st relapse samples from 7 patients treated on intergroup protocol INT0129/E2491, the methylation level was significantly higher in the relapse samples at 6/14 dnts (p <0.05 each) and in the sums of all dnts (p = 0.03). Compared to these 7 1st relapse cases, the median values for each of the 14 dnts and the sums of all dnts were higher for 6 cases with ≥2 relapses (median of sums 149.8 vs 385.6, respectively) but this difference was not significant (p = 0.22), primarily related to 1 double-relapse case with exceptionally low values (excluding this case, p = 0.05). Similarly, compared to 8 relapse cases without PML-RARα mutations, 5 relapse cases with mutations had higher median values of 13/14 dnts and of the sums of all dnts (160.5 vs 402.0) but this was not statistically significant (p = 0.27). These results indicate that higher levels of RARβ2 promoter methylation are present in relapse compared to de novo APL cells and suggest that increasing methylation is frequently associated with disease progression and with mutations in PML-RARα.
Regulatory T cells (Treg) play a key role in controlling immune responses following allogeneic hematopoietic stem cell transplantation (HSCT). In murine models, infusion of purified CD4+CD25+ Treg at ...the time of transplant is sufficient to prevent acute GVHD. In humans, development of acute as well as chronic GVHD has been associated with reduced numbers of Treg following allogeneic HSCT, suggesting that defective reconstitution of this functional cell type can contribute to exacerbation of alloimmune responses. Based on these results, adoptive cellular therapy using purified and in vitro expanded populations of Treg has been proposed as a therapeutic strategy to correct chronic GVHD. Treg are mainly characterized by the constitutive expression of the IL-2 receptor ? chain, CD25 and proliferate in response to IL-2 in vitro. In vivo, the effects of IL-2 on Treg populations are unknown. To examine this question we quantified changes in Treg in 9 patients with CML who previously received low dose IL-2 following allogeneic HSCT. Patients enrolled in this protocol received a daily intravenous infusion of 2 X 105 U IL-2/m2 for 3 months, starting 3 months after CD6 depleted allogeneic bone marrow transplantation (BMT). No patient developed GVHD following IL-2 administration and overall toxicity was minimal. The predominant immunologic effect of IL-2 reported in the initial study was a marked increase in NK cell populations characterized as CD3-CD16+CD56+ as well as total CD56+ cells. In this retrospective analysis we investigated populations of CD4+CD25+ T cells before and 1 to 2 months after the beginning IL-2 treatment. Using RNA extracted from patient PBMC we also assessed the level of expression of the specific transcription factor FOXP3 by quantitative PCR as an alternate marker of Treg in vivo. As initially reported, all 9 patients showed a marked increase in CD3-CD56+ cells 1 to 2 months post IL-2 administration. In contrast, the percent of CD3+ T cells were either unchanged or slightly decreased. The percent of CD4+CD25+ cells within the CD3+ T cell population increased during IL-2 treatment (median: 5.8 pre IL-2 vs 7.6 post IL-2, p-value=0.02). Likewise, FOXP3 expression in the CD3+ population showed 5 to 19 fold increase in 8 of 9 patients during this period (median: 3817 AU pre IL-2 vs. 18924 AU post IL-2, p-value=0.055). These results indicate that administration of low dose IL-2 can augment Treg cells in vivo as reflected by increased ratio of CD4+CD25+/CD3+ T cells as well as higher levels of FOXP3 expression. These studies suggest that prolonged treatment with low dose IL-2 can effectively expand CD4+CD25+ Treg in vivo. This represents a novel strategy for expanding regulatory T cells in vivo and may be useful alone or in conjunction with adoptive cellular therapy with Treg.
Highlights • Intensive topical therapy with dexamethasone .5 mg/5 mL solution is effective for managing patients with new-onset symptomatic oral chronic graft-versus-host disease • The dexamethasone ...solution results support the most recent National Institutes of Health consensus ancillary care guidelines for management of oral chronic graft-versus-host disease • Topical tacrolimus .5 mg/5 mL solution is less effective than dexamethasone solution for managing patients with new-onset symptomatic oral chronic graft-versus-host disease; however, its relative efficacy and safety at higher concentrations are unknown
Abstract We evaluated the efficacy of FISH to detect chromosome anomalies in the evaluation of young (<60 years) patients with AML. Patients were enrolled in E1900, an ECOG clinical trial for AML. ...The protocol was designed to collect bone marrow or blood for both cytogenetic and FISH studies at study entry (diagnosis). FISH for each patient was performed and utilized eight probe sets to detect t(8;21), t(9;22), t(11;var), t(15;17), inv(16), +8, −5/5q, and −7/7q. We analyzed 237 specimens with complete cytogenetic and FISH results. Results for each specimen were classified by probe set into one of six categories. The concordance rate between cytogenetic and FISH results ranged from 98 to 100% for all probe sets and kappa analysis for concordance had a p -value of <0.0001. The high level of agreement between cytogenetic and FISH results demonstrate the accuracy of a panel of eight FISH probe sets for the detection of significant abnormalities in AML. Data from this investigation support the use of FISH as an adjunct method to increase the yield of useful cytogenetic results in large cooperative trials and demonstrate the potential of FISH as a follow-up study of minimal residual disease in ECOG trials.
The etiology and pathogenesis of type 2 diabetes mellitus
(T2DM) are not completely understood although it
is often associated with other conditions such as obesity,
hypertension, and dyslipidemia. ...Lipoprotein lipase
(LPL) is a key enzyme in human lipid metabolism
that facilitates the removal of triglyceride-rich lipoproteins
from the bloodstream. LPL hydrolyzes the core of triglyceride-rich lipoproteins (chylomicrons
and very low density lipoprotein) into free fatty acids
and monoacylglycerol. To gain insight into the possible
role of LPL in T2DM, nine single nucleotide polymorphisms
(SNPs) of LPL were analyzed for the association
with T2DM using 944 unrelated Koreans, including
474 T2DM subjects and 470 normal healthy
controls. Of the nine LPL SNPs we analyzed, a significant
association with multiple tests by the false discovery
rate (FDR) was observed between T2DM and
SNP rs343 (+13836C＞A in intron 3). SNP rs343 was also
marginally associated with some of T2DM-related
phenotypes including total cholesterol, high density
lipoprotein cholesterol (HDLc), and log transformed
glycosylated hemoglobin in 470 normal controls, although
no significant association was detected by
multiple tests. In total, our results suggest that the control
of lipid level by LPL in the bloodstream might be
an important factor in T2DM pathogenesis in the
Korean population. KCI Citation Count: 16
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