Myeloablative Hematopoietic Cell Transplantation Research Articles

Puberty, Gonadal Function and Pregnancy in Female Patients Following Hematopoietic Cell Transplantation (HCT).

With increasing success of myeloablative HCT for patients with hematologic disorders, development of pre-pubertal children through puberty, return of gonadal function and the ability to become pregnant are of concern. To determine the impact of the preparative regimen on these issues, long-term follow-up records of more than 2,000 patients have been evaluated. Preparative regimens include cyclophosphamide (CY) 200 mg/kg, busulfan (BU) 16 mg/kg plus 120 or 200 mg/kg CY, or CY 120 mg/kg and 12–15.75 Gy total body irradiation (TBI). Among girls who were pre-pubertal at HCT and are now >12 years of age, 24/26 (92%) CY recipients, 11/23 (52%) BUCY recipients, and 46/96 (51%) CY + TBI recipients have developed normally through puberty. Among 148 post-pubertal women transplanted with CY only, 75 (50%) recovered gonadal function at a median of 6 months after HCT and 45 (30%) have become pregnant. This is in contrast to 2 (1%) of the 207 women who received BUCY who have shown gonadal function recovery and 1 has become pregnant. Among the fractionated TBI recipients, 39 (8%) of 460 women who received CY + 12 Gy TBI recovered ovarian function and 17 (3%) have become pregnant whereas among the 366 women who received 14.0–15.75 Gy TBI, 14 (4%) have recovered ovarian function and 4 (1%) have become pregnant. All women who recovered ovarian function after BUCY or TBI regimens were <25 years of age at HCT. The primary pregnancy complication was preterm labor and delivery, but all babies born were normal. For those women who do not recovery ovarian function, in vitro fertilization may be an option, and 14 women in this category have had successful pregnancies with 21 babies born using donated oocytes or pre-HCT stored gametes. These data demonstrate that pre-pubertal girls have more than 50% rate of developing normally through puberty and among women <25 years of age at HCT, recovery of ovarian function varies between 1%-50% depending on preparative regimen and some have become pregnant. For those who do not recover ovarian function, in vitro fertilization may be an option.

Impact of Total Body Irradiation Technique on Interstitial Pneumonitis and Clinical Outcomes of 623 Patients Undergoing Hematopoietic Cell Transplantation for ALL: 25 Years at the University of Minnesota

Impact of Total Body Irradiation Technique on Interstitial Pneumonitis and Clinical Outcomes of 623 Patients Undergoing Hematopoietic Cell Transplantation for ALL: 25 Years at the University of Minnesota

Prognostic utility of the acute kidney injury network (AKIN) criteria for acute kidney injury in myeloablative haematopoietic cell transplantation

Prognostic utility of the acute kidney injury network (AKIN) criteria for acute kidney injury in myeloablative haematopoietic cell transplantation

Pharmacogenomic associations in ABCB1 and CYP3A5 with acute kidney injury and chronic kidney disease after myeloablative hematopoietic cell transplantation

Renal disease is a major complication in patients following myeloablative allogeneic hematopoietic cell transplantation (HCT). Post-HCT patients receive immunosuppressive regimens containing calcineurin inhibitor (CNIs), cyclosporine or tacrolimus, for graft-versus-host disease prophylaxis. In this retrospective trial, we investigated pharmacogenomic associations in the multidrug resistance (ABCB1) and cytochrome P450 3A5 (CYP3A5) genes and acute kidney injury (AKI) and chronic kidney disease (CKD) in a cohort of 121 patients. ABCB1 and CYP3A5 are responsible for the renal disposition of CNIs, which are known to be nephrotoxic. AKI was defined as doubling of baseline serum creatinine during the first 100 days post-HCT, and CKD as at least one glomerular filtration rate <60 ml/min/m2 between 6 and 18 months post-HCT. Patients were genotyped for CYP3A5*1>*3 and ABCB1 single nucleotide polymorphisms (SNPs) (1199G>A, 1236C>T, 2677G>T/A and 3435C>T). Odds ratios were calculated using logistic regression. Haplotype estimation and univariate association analyses were performed because of strong ABCB1 linkage disequilibrium (LD). AKI occurred in 48 of 121 patients (39.7%) and CKD in 16 of 66 patients (24.2%). No pharmacogenomic associations were found between ABCB1 and CYP3A5 SNPs and the incidences of AKI or CKD. The degree of LD(r2) between ABCB1 SNPs was estimated as follows: 2677G>T/3435C>T (0.44), 1236C>T/3435C>T (0.42) and 1236C>T/2677G>T (0.72). ABCB1 1199G>A showed no LD to other SNPs (<0.05). No associations were found between the most common ABCB1 haplotypes and AKI or CKD. Since no significant pharmacogenomic associations were observed, tailoring CNIs dosing based on these genotypes is unlikely to lower significantly the risk of renal injury following myeloablative HCT.

Assessment of the hematopoietic cell transplantation comorbidity index in non-Hodgkin lymphoma patients receiving reduced intensity allogeneic hematopoietic cell transplantation

7096 Background: Several patient-related factors influence outcomes after allogeneic hematopoietic cell transplantation (HCT). The Hematopoietic Cell Transplantation Comorbidity Index (HCT-CI), a weighted index of 17 pre-transplant comorbidities, has been validated in non- myeloablative HCT studies, but it has not been tested in non-Hodgkin's lymphoma (NHL) patients receiving reduced-intensity conditioning (RIC) HCT nor has it been compared with a number of other pre-transplant factors. Methods: We performed an analysis of 63 NHL patients treated with RIC HCT to assess the HCT-CI's impact on overall survival (OS), treatment-related mortality (TRM) and disease-related mortality (DRM). Individual factors, including performance status, single comorbidities, CD34 dose, prior chemotherapy number, pre-transplant induction chemotherapy response, and NHL histology, were also analyzed relative to their impact on OS. Prior to transplant, all patients received an identical induction regimen (EPOCH-fludarabine). All patients received identical RIC (Cy/Flu) and were transplanted from HLA-matched siblings. Results: In univariate analysis, each of the above factors, except for histology and CD34 dose, exhibited a trend toward an association with OS (adjusted p&lt;0.10). The HCT-CI (0–2 comorbidities vs. 3+ comorbidities) demonstrated an association with TRM at 100 days (4.5% vs. 26.3%) and 1 year (13.6% vs. 36.8%) post-transplant, but not DRM. The factor most strongly associated with OS was response to induction chemotherapy (CR/PR/SD vs. PD; p=0.0025). In a Cox model for OS, induction chemotherapy response remained the sole important factor, while the HCT-CI and other pre-transplant factors added little predictive value to the induction chemotherapy response. The HCT-CI was more strongly associated with OS and TRM in a subset analysis restricted to patients in CR/PR following induction chemotherapy. Conclusions: These findings suggest that the HCT-CI may be useful for estimating probabilities of TRM and OS in NHL patients being considered for RIC HCT. The data further suggest that chemotherapy response at transplant may have significant impact on transplant outcomes. No significant financial relationships to disclose.

Unrelated donor hematopoietic cell transplantation (HCT) as treatment for acute myeloid leukemia (AML) in first remission

7000 Background: Unrelated donor HCT has been used with increasing frequency for the treatment of AML patients in first remission. Methods: Between 1990 and 2005, 105 adult patients with AML [66 (62%) primary and 39 (38%) secondary] received T- cell-replete HCT from unrelated donors in Seattle. Results: Median age was 44 (range, 18–74) years. Seventy-nine patients received ablative conditioning for HCT (57% conditioned with cyclophosphamide plus total body irradiation (TBI), 43% with chemotherapy alone). Non- myeloablative doses of fludarabine (FLU; 25 mg/m2 daily for 3 days) and TBI (200 cGy) were delivered to 26 patients. Ninety-six percent of patients who underwent reduced-intensity conditioning received unrelated peripheral blood stem cells (PBSC) while 59% of myeloablative HCT patients received PBSC as their source of stem cells. For all patients, the leukemia-free survival (LFS) and non-relapse mortality estimated at 2 years were 53%, and 26%, respectively, as 27 patients died of transplant-related causes (pulmonary-6, cardiac-1, graft versus host disease (GVHD)-6, infection-5, and unknown-9). Twenty-two patients (21%) relapsed 2 to 83 months (median 5) post-transplant, and 56 patients are surviving disease-free 1 to 168 months (median 27) post-transplant. For 90 patients (86%) with intermediate-risk cytogenetics, 51 (57%) are surviving disease-free, with 16 (18%) relapsing. Fourteen patients (13%) were considered high risk based on unfavorable cytogenetics; 4 of these 14 (29%) are surviving disease-free and 6 (43%) have relapsed. The probability of grade 3/4 acute GVHD was 19% while the probability of clinical extensive chronic GVHD was 54% one year after transplant. Unfavorable cytogenetics (HR=2.16, p=.08), secondary leukemia (HR=1.71, p=.10), higher co-morbidity score (p&lt;.0001), and HLA mismatching (HR=2.35, p=.02) were associated with an increased hazard of mortality. After controlling for these factors, non-ablative conditioning was not statistically worse than ablative conditioning (HR=1.21, p=.61). Conclusions: These data suggest that HCT using unrelated allografts has the potential to provide significant LFS for patients undergoing HCT for AML in first remission when an HLA family match is not available. No significant financial relationships to disclose.

Comparable Antileukemia/Lymphoma Effects in Nonremission Patients Undergoing Allogeneic Hematopoietic Cell Transplantation with a Conventional Cytoreductive or Reduced-Intensity Regimen

To evaluate the potential of allogeneic hematopoietic cell transplantation (HCT) with a reduced-intensity conditioning regimen (RIST) for the treatment of patients with hematologic malignancies not in remission, we retrospectively reviewed the medical records of 132 patients (89 leukemia or myelodysplastic syndrome, 40 malignant lymphoma, and 3 others) who received conventional myeloablative HCT (CST, n = 52) or RIST (n = 80). The median age of the RIST group was significantly higher than that of the CST group (53 years versus 40 years, P < .01). The RIST group also included a higher proportion of patients with an HCT-specific comorbidity index (HCT-CI) of 1 or more than the CST group (65% versus 37%, P = .03). The probabilities of achieving complete remission and the incidences of grades II-IV and III-IV acute graft-versus-host disease (aGVHD) in the CST and RIST groups were, respectively, 77% and 64%, 50% and 50%, and 23% and 28%, with no significant differences. Similarly, there was no difference in the 2-year probabilities of nonrelapse mortality (NRM, 36% and 38%), progressive disease or relapse (PD 51% and 49%), overall survival (OS, 31% and 38%), and progression-free survival (PFS, 28% and 29%). Multivariate analyses revealed that a higher HCT-CI score and transplant from donors other than HLA-matched relatives were associated with increased risks of NRM and poor OS, and patients who received chemotherapy within 2 months before HCT were associated with increased risks of PD, poor OS, and PFS after transplantation. After adjusting for these variables, the risks of NRM, PD, OS, and PFS in the RIST group were not significantly different from those in the CST group. In conclusion, these results suggest that the antileukemia/lymphoma effect associated with RIST is comparable to that associated with CST. RIST appears to be feasible for the treatment of hematologic malignancies not in remission.

Matched‐related donor transplantation for sickle cell disease: report from the Center for International Blood and Transplant Research

We report outcomes after myeloablative haematopoietic cell transplantation (HCT) from human leucocyte antigen (HLA)-matched sibling donors in 67 patients with sickle cell disease transplanted between 1989 and 2002. The most common indications for transplantation were stroke and recurrent vaso-occlusive crisis in 38% and 37% of patients respectively. The median age at transplantation was 10 years and 67% of patients had received >10 red blood cell transfusions before HCT. Twenty-seven percent of patients had a poor performance score at transplantation. Ninety-four percent received busulfan and cyclophosphamide-containing conditioning regimens and bone marrow was the predominant source of donor cells. Most patients achieved haematopoietic recovery and no deaths occurred during the early post-transplant period. Rates of acute and chronic graft-versus-host disease were 10% and 22% respectively. Sixty-four of 67 patients are alive with 5-year probabilities of disease-free and overall survival of 85% and 97% respectively. Nine patients had graft failure with recovery of sickle erythropoiesis, eight of who had recurrent sickle-related events. This report confirms and extends earlier reports that HCT from HLA-matched related donors offers a very high survival rate, with few transplant-related complications and the elimination of sickle-related complications in the majority of patients who undergo this therapy.

Clinical significance of donor–recipient HLA matching on survival after myeloablative hematopoietic cell transplantation from unrelated donors

The application of unrelated donor hematopoietic cell transplantation can be expanded with the use of mismatched donors if human leukocyte antigen (HLA) disparity does not lead to increased morbidity and mortality. The rules that govern permissibility of HLA mismatches are not well defined. The International Histocompatibility Working Group in hematopoietic cell transplantation measured the risks associated with locus-specific disparity in 4796 patients transplanted for low, intermediate, or high-risk hematologic diseases. The permissibility of a given HLA mismatch is in part defined by the locus and by disease risk.

Hematopoietic stem cell transplantation: killer immunoglobulin‐like receptor component

Recognition of recipient human leukocyte antigen (HLA) class I ligand by donor natural killer cell killer immunoglobulin-like receptors (KIR) has been proposed as the basis for donor allograft reactivity against malignancy leading to reduction in posttransplant relapse and higher survival for acute myelogenous leukemia. Analysis of KIR ligand effects in 1770 patients undergoing myeloablative T-replete hematopoietic cell transplantation (HCT) from HLA-matched or mismatched unrelated donors showed that lack of KIR ligand in patients for inhibitory KIR was associated with lower hazards of relapse in leukemia patients with in HLA-mismatched transplants [hazard ratio (HR): 0.061; 95% confidence interval (CI): 0.43-0.85; P-value = 0.004]. Absence of HLA-C group 2 or HLA-Bw4 KIR ligands were each associated with lower hazards of relapse (HR: 0.47; 95% CI: 0.28-0.79; P-value = 0.004; HR: 0.56; 95% CI: 0.33-0.97; P-value = 0.04, respectively). Based on these analyses, recipient homozygosity for HLA-B or -C epitopes that define KIR ligands is likely to be a predictive factor for leukemia relapse following myeloablative HCT from unrelated donors. KIR genotyping for unrelated donors and recipients will clarify the role of these receptors in transplant outcome.

Nephrotic syndrome associated with thrombotic microangiopathy following allogeneic stem cell transplantation for myelodysplastic syndrome – response to Nakamura et al

We thank Dr Nakamura et al for their comment on our report describing nephrotic syndrome following cyclophosphamide/ fludarabine-based non-myeloablative allogeneic haematopoietic cell transplantation (HCT) (Srinivasan et al, 2005). Nakamura et al describe a patient who developed cyclosporine-associated thrombotic microangiopathy (TMA) and nephrotic syndrome following reduced-intensity HCT. They further suggest that TMA might have contributed to nephrotic syndrome in our patient series. Clinically significant thrombotic thrombocytopenic purpura (TTP)/haemolytic uremic syndrome (HUS), often associated with the use of calcineurin inhibitors, is a well recognised complication of allogeneic HCT (Holler et al, 1989; Rabinowe et al, 1991). Furthermore, proteinuria – including frank nephrotic syndrome – is a well described and not uncommon manifestation of TMA (Magee et al, 2000; Galesic et al, 2006). Therefore, this syndrome should be entertained in the differential diagnosis of nephrotic syndrome in all patients who present with clinical or laboratory features suggestive of TMA (such as thrombocytopenia, haemolysis, neurologic symptoms or renal failure). Six of the seven patients in our series had no clinical evidence of TMA; a renal biopsy was obtained in three of these patients and confirmed a diagnosis of membranous glomerulonephritis without evidence of microangiopathy. One patient (Patient 7) died from a cerebral haemorrhage related to TTP; however, proteinuria preceded the onset of clinical features suggestive of TTP by several months and examination of renal tissue obtained at autopsy revealed incontrovertible evidence of membranous glomerulonephritis in addition to microangiopathic changes. These data suggest that factors other than TMA were responsible for the nephrotic syndrome observed in the cohort of patients described in our report (Srinivasan et al, 2005). Since our original report, we have observed four additional cases of nephrotic syndrome following HCT at our centre. Two of these occurred in patients who received nonmyeloablative conditioning with cyclophosphamide/fludarabine while the other two cases occurred following a T-cell depleted HCT with myeloablative conditioning using cyclophosphamide and total body irradiation. Renal biopsy performed in all four cases showed a variety of renal histopathologic features including membranous glomerulonephritis (one patient), focal segmental glomerulosclerosis (one patient), TMA (one patient) and features suggestive of TMA and a vasculitic process (one patient). It is clear from our experience as well as from previously published reports in patients undergoing myeloablative HCT that nephrotic syndrome in the post-transplant period can result from diverse aetiologies that may require equally diverse management strategies. Consequently, it is our current practice to obtain a histologic diagnosis in every patient who presents with nephrotic syndrome to help guide appropriate management.

Marrow Fibrosis as a Risk Factor for Posttransplantation Outcome in Patients with Advanced Myelodysplastic Syndrome or Acute Myeloid Leukemia with Multilineage Dysplasia

Marrow fibrosis is considered a poor prognostic factor in patients with myelodysplastic syndrome (MDS). The affect of fibrosis on outcomes after hematopoietic cell transplantation (HCT) in patients with MDS has not been examined. We performed a retrospective analysis in 471 patients with MDS or acute myeloid leukemia with multilineage dysplasia arising from MDS, 113 with and 358 without marrow fibrosis, who received myeloablative allogeneic HCT. Post-HCT follow-up was 0.3–10 years (median, 3.6 years) for patients with, and 0.6–12 years (median, 5 years) for patients without fibrosis. Engraftment was significantly delayed in patients with fibrosis (hazard ratio [HR] = 0.4; P < .001). Overall, there were no significant differences in overall survival (OS), relapse-free survival (RFS), and nonrelapse mortality (NRM) between patients with and without fibrosis. However, among patients with advanced disease (int-2 or high-risk disease by the International Prognostic Scoring System), OS ( P = .03), RFS ( P = .04), and NRM ( P = .04) were inferior when marrow fibrosis was present. Given that marrow fibrosis is a poor prognostic factor for patients with MDS, and that it does not appear to affect outcome of transplantation in patients with earlier-stage disease but has a negative impact on outcome for patients with advanced disease, patients with earlier-stage MDS and marrow fibrosis might be considered for HCT earlier than their disease stage would normally dictate.

Lack of Utility of Chimerism Studies at Day 80 after Myeloablative Hematopoietic Cell Transplantation for Acute Lymphocytic Leukemia.

Between January 1998 and December 2004, 89 adults (≥18 years old) received allogeneic myeloablative hematopoietic stem cell transplants for treatment of acute lymphocytic leukemia (ALL) and had chimerism studies performed at a median of 77 days (range = 65–113 days) after transplant to assess the degree of donor engraftment. Median patient age was 33.5 yrs (range = 18.1–55.9 yrs). Seventy-eight patients had B-cell ALL, 8 had T-cell ALL, and 3 were not phenotyped. Conventional cytogenetic analyses were available for 70 patients at diagnosis: 19 had normal karyotypes, 24 were Philadelphia chromosome positive (Ph+), and 27 were Ph−, but had other clonal abnormalities. Four of the 19 patients without conventional cytogenetic studies had fluorescent in-situ hybridization (FISH) or polymerase chain reaction (PCR) studies positive for the bcr/abl gene rearrangement. Thirty-nine patients were transplanted in first remission, 29 in ≥2nd remission, and 21 in relapse. Eighty-four of the 89 patients received preparative regimens of cyclophosphamide (120 mg/kg) plus 12 or 13.2 Gy TBI. Thirty-two donors were HLA-identical family members, 2 were HLA-mismatched relatives, 36 were HLA-identical unrelated donors (URDs), and 19 were HLA-mismatched URDs. Thirty-four patients received stem cells from a bone marrow (BM) harvest, 54 received peripheral blood (PB) stem cells, and one received a cord blood product. The primary GVHD prophylaxis regimen was cyclosporine and methotrexate (n=80). Seventy-seven patients developed grades II-IV acute GVHD at a median of 19 days after transplant. Fifty-seven patients developed extensive chronic GVHD at a median of 140 days after transplant. Only one patient failed to engraft and died early; no one experienced graft rejection. Chimerism studies were done using either unfractionated BM or fractionated (CD3+ and CD33+ cells) PB. Prior to June 2002, a panel of 10 Variable Number Tandem Repeat (VNTR) loci were amplified and analyzed for same sex donor/recipient pairs. Subsequently, PCR amplification and analyses of Short Tandem Repeat (STR) loci were compared for same sex donor/recipient pairs. FISH studies using X and Y chromosome-specific probes were used for sex-mismatched donor/recipient pairs. Forty-seven patients had chimerism studies performed on unfractionated BM specimens and 58 had studies of fractionated PB samples (20 patients had both BM and PB studies). Only 2 patients who had unfractionated BM chimerism analyses had <95% donor engraftment. Only 1 patient who had a fractionated PB chimerism study had <95% donor engraftment in the CD33+ fraction. Eighteen patients had <95% donor engraftment (range = 45–94%) in the CD3+ fraction. Lower percentages of CD3+ engraftment did not correlate with survival, relapse, or incidence and severity of acute or chronic GVHD. The laboratory fees for one chimerism study range from $750–$1,000. Given the high percentage of complete donor engraftment on day 80 after myeloablative transplant in this patient population, the lack of correlation of incomplete CD3+ donor engraftment with transplant outcome, and the cost of chimerism analysis, these studies are not indicated in patients with ALL undergoing myeloablative transplants in the absence of clinical evidence of graft failure or graft rejection.

Identifying Biomarkers for Acute GVHD.

Acute graft versus host disease (aGVHD) is a complication of allogeneic hematopoietic cell transplantation (HCT). Current prevention and treatment approaches are effective in many but not all cases. Acute GVHD after a myeloablative HCT has a median onset of ~21 days, with a few cases occurring as late as 3 months post-HCT. We hypothesized that global gene expression profiling of RNA from white blood cells (WBC) could identify biomarkers associated with onset of aGVHD and potentially provide insight into the mechanisms responsible for causing clinically significant aGVHD. Patients were enrolled prospectively and blood samples collected weekly until onset of aGVHD or day 90. RNA samples adequate for hybridization were collected between day 19 and 24 from 23 patients who developed aGVHD (GVHD+) and compared with RNA from 13 patients who remained free of aGVHD through day 90 (GVHD−). All blood samples from GVHD+ patients were obtained prior to initiation of steroid therapy.Biotin-labeled cRNA was hybridized on Affymetrix HG-U133A. Realizing quantitative complexities underlying gene expression assessment and different assumptions required by different algorithms, we used 5 different summary algorithms (Mas5, PLIER, RMA, gcRMA, Dchip) to compute gene expression levels. Subsequently, we carried out analyses and identified genes that were consistently identified in more than one analyses; replications imply robustness of discoveries, even though discovery efficiency is compromised to some degree. A total of 141 genes showing differential expression were discovered.Thirteen functional classes were identified for 101 genes. The remaining 40 genes had unknown function.Sixteen transcription factors were identified, 12 of them showing increased expression such as the early response genes EGR1, EGR2, FOS and JUN. Among 12 immune response genes identified increased expression was observed for CD83 and IL-18, associated with antigen presentation and adaptive immunity; and NRS2, MMP9 and CD157, associated with innate immune response. Decreased expression was observed for CD52 and PTPN11.Among 7cell cycle progression and proliferation genes, we observed increased expression for CSF2, VEGF, PTN and TGFa. These data are consistent with the hypothesis that the development of acute GVHD is associated with acute inflammation. Ongoing validation studies using Taqman have confirmed up regulation of CD83 and EGR2 and down regulation of CD52 in GVHD+ patients.To further understand the functional implications of the 141 genes identified, probe sets were also input into Ingenuity Pathway Analysis software to identify possible signaling and regulatory pathways. The Ingenuity defined pathways most densely populated by the 141 genes included pathways controlling cytokine production and inflammation. Network analysis showed upregulation of transcriptional pathways responsible for cell proliferation, growth and activation: IL18, CSF2, TGFa and the transcription factors EGR1, EGR2, FOS and JUN were linked to networks that appear to have important role in regulation of expression of VEGF suggesting that it may be involved in sustaining inflammatory response in GVHD.This study demonstrates that whole genome transcriptional analysis of WBC RNA can be informative for detecting biomarkers associated with aGVHD. Furthermore these findings suggest that these biomarkers can be used to better define cellular pathways associated with clinical onset and/or severity of acute GVHD and thereby provide potential targets for therapy.

Pre-Transplant 5-Azacitidine (Vidaza®) May Improve Outcome of Allogeneic Hematopoietic Cell Transplantation (HCT) in Patients with Myelodysplastic Syndrome (MDS).

Most patients with MDS are older than 60 yrs and, because of age, many have previously been excluded from HCT due to the high risk of transplant related mortality. Reduced intensity conditioning has decreased regimen related mortality in HCT and has increased the age of patients for whom this treatment is offered. There remains substantial risk of relapse after HCT especially in patients with > Int-1 MDS who benefit the most from immediate HCT. One strategy to suppress leukemia burden and potentially decrease the risk of relapse is pre-transplant treatment with 5-azacitidine (Vidaza®). This DNA hypomethylating drug is FDA approved for the treatment of MDS. We analyzed the outcome for 34 MDS patients who received a myeloablative HCT from an HLA compatible sibling or unrelated donor from July 2004 through June of 2006, to investigate the potential impact of pre-transplant 5-azacitidine on response and post-transplant relapse. Fourteen of thirty-four patients received a median of 2 (1–7) cycles of 5-azacitidine prior to HCT. Median age was 59 (49 – 69) yrs. Diagnosis and IPSS category included MDS (8) [Int-1 (1) and Int-2 (7)], MDS-AML (5) and CMML (1). Two patients had induction chemotherapy with residual MDS prior to 5-azacitidine. Subsequent to 5-azacitidine: ten responded [CR (2), PR (1) improvement (7)] three progressed and one was not evaluable. The IPSS category after 5-azacitidine treatment and prior to HCT was Int-2(1), Int-1 (4), and Low (5; 3 in CR) in addition to AML (2) and CMML (1). One was unable to assess. The median follow-up in the survivors is 191 (47 – 524) days. Of the fourteen 5-azacitidine treated patients, none have relapsed post-HCT including patients who progressed on 5-azacitidine. Four have died from non-Hodgkins lymphoma (1), DAH (1), adenovirus pneumonia (1) and chronic GVHD (1). Ten are alive in remission, two for greater than one year. Twenty patients did not receive 5-azacitidine. Diagnosis and IPSS category included MDS (9) [Int-1 (3) and Int-2 (6)], MDS-AML (9) and CMML (1) and one not determined due to lack of cytogenetics. Their median age was 53 (33 – 68) yrs. Treatment included supportive care, induction chemotherapy (9) and SCIO-469 (1). IPSS distribution prior to HCT was Int-2 (7), Int-1 (5) Low (5; 4 in CR); in addition to AML (1) and CMML (1). One was unable to assess. The median follow-up in the survivors is 420 (21 – 742) days. Ten of twenty are alive without relapse and eight have relapsed. Five have died, with relapse (3), fungal pneumonia (1) or multi-organ failure (1). The 1 year K-M estimates of overall and progression free survivals are 64% (SEM 15%) and 64% (SEM 15%), respectively for 5-azacitidine group and 70% (SEM 11%) and 51% (SEM 13%) for non-5-azacytidine group. We conclude from this preliminary analysis that pre-HCT conditioning treatment with 5-azacitidine may reduce the risk for MDS relapse after allogeneic transplant in higher risk patients. In addition to its direct anti-tumor effect, 5-azacitidine may sensitize neoplastic cells to the effects of high dose chemotherapy or promote the expression of antigens critical to effective graft-vs-tumor response. This treatment strategy will be evaluated in a prospective trial to investigate the role of pre-transplant 5-azacitidine on transplant outcomes in patients with higher risk MDS.

Global Assessment of B Cell Alloimmunity Using 5000 Human Protein Microarrays.

Allogeneic Hematopoietic Cell Transplantation (HCT) can cure hematologic malignancies through beneficial graft-v-leukemia (GVL) allo-immune responses, but its full potential is limited by graft-versus-host disease (GVHD). Recent studies demonstrate allogeneic antibodies develop against mHA encoded on the Y-chromosome, called H-Y antigens develop after sex-mismatch HCT in association with chronic GVHD and persistent disease remission. We hypothesize novel mHA can be serologically identified as targets of allogeneic antibody responses that develop after transplant and were absent pretransplant. Over 70,000 nonsynonymous single nucleotide polymorphisms (nsSNPs) encode polymorphic amino acid residues in human proteins that are potential mHA, and their analysis requires a novel high-throughput approach. ProtoArray™ (Invitrogen) displays five thousand full-length human proteins expressed as N-terminal GST-fusion proteins in a baculovirus system that are affinity purified under native conditions maintaining their cellular enzymatic activities/native conformations. These human antigens are printed in duplicate on nitrocellulose-coated glass slides. In order to determine if targets of allogeneic antibodies can be detected using microarray technology we tested three cGVHD patients for de novo Ab development after HCT using ProtoArrays™.Plasma from three male patients with AML was collected 1) prior to myeloablative HCT 2) from each HLA-identical sibling donor, and 3) one year after HCT. All three had developed extensive chronic GVHD. Plasma was diluted 1:150 and incubated on two microarrays providing replicate results. After washing, each microarray was incubated with anti-human IgG conjugated to Alexa Fluor 647 dye and detected. Negative controls include buffer, BSA, and GST, and their maximum fluorescent signals with these samples were 5,631 units with mean SD of 546. In contrast, influenza A protein is a positive control with fluorescent signal ranging 30,000–45,000 units. Correlation coefficients (R2 values) between duplicate slides ranged from 0.85 to 0.91.Data analysis was performed using Invitrogen's Prospector Analyzer Software. Fluorescent intensity is measured for each “spot” and individual antigen results are reported as both flourescent signal intensity and a Z-score which is a measure of the intensity of a given signal relative to all of the other human protein targets reported in units of standard deviation. In order to identify human protein targets of allogeneic antibodies, each target antigen's pretransplant Z-score was subtracted from his respective one year post-transplant Z-score yielding the “ΔZ” for that antigen. Using a conservative ΔZ of 0.1, these three patients developed new antibody responses for 67, 66, and 74 human proteins. Ninety-two percent of these proteins have known nsSNPs. No single protein was recognized by “new” antibodies in all three proteins, however polymorphic proteins Growth Arrest Specific-7 (GAS7), laminin A/C, and ribosomal protein S19 (RPS19) were recognized by two of the three patients after HCT. Results from plasma samples collected 3 and 6 months after HCT demonstrate the progression of alloimmune immune response with few new antibody responses at 3 months.Conclusion: Protein microarrays are an innovative, powerful tool for high-throughput global assessment of B cell alloimmunity after HCT. Microarray technology provides sufficient reproducibility for candidate mHA discovery. These novel mHA require validation by large clinically characterized patient samples.

Factors Associated With Outcomes in Allogeneic Hematopoietic Cell Transplantation With Nonmyeloablative Conditioning After Failed Myeloablative Hematopoietic Cell Transplantation

Several studies have investigated the feasibility of allogeneic hematopoietic cell transplantations (HCTs) after reduced-intensity conditioning in patients who experienced relapse after myeloablative HCT. Although most studies showed relatively low nonrelapse mortality (NRM) rates and encouraging short-term results, it has yet to be defined which patients would benefit most from these approaches. We analyzed data from 147 patients with hematologic malignancies who experienced treatment failure with conventional autologous (n = 135), allogeneic (n = 10), or syngeneic (n = 2) HCT and were treated with HLA-matched related (n = 62) or unrelated (n = 85) grafts after conditioning with 2 Gy of total-body irradiation with or without fludarabine. Three-year probabilities of NRM, relapse, and overall survival were 32%, 48%, and 27%, respectively, for related recipients, and 28%, 44%, and 44%, respectively, for unrelated recipients. The best outcomes were observed in patients with non-Hodgkin's lymphoma, whereas patients with multiple myeloma and Hodgkin's disease had worse outcomes as a result of high incidences of relapse and progression. Being in partial remission (PR) or complete remission (CR) at HCT (P = .002) and developing chronic graft-versus-host disease (GVHD; P = .03) resulted in lower risks of relapse and progression. Factors associated with better overall survival were PR or CR (P = .01) and lack of comorbidity (P = .03) at HCT and absence of acute GVHD after HCT (P = .06). Encouraging outcomes were seen with allogeneic HCT after nonmyeloablative conditioning in selected patients who had experienced relapse after a high-dose HCT, particularly in patients with non-Hodgkin's lymphoma. Results with unrelated grafts were comparable with results with related grafts.

KIR Ligands and Prediction of Relapse after Unrelated Donor Hematopoietic Cell Transplantation for Hematologic Malignancy

Recurrent malignancy remains a significant complication after allogeneic hematopoietic cell transplantation (HCT). Efforts to decrease relapse have included donor lymphocyte infusion to stimulate donor anti-recipient T-cell allorecognition of major and minor histocompatibility differences. Recently, alloreactive effects of donor natural killer cell-mediated inhibitory killer immunoglobulin-like receptor (KIR) recognition of recipient HLA-C and -B ligands have been described. We examined KIR ligand effects on risk of relapse in 1770 patients undergoing myeloablative T-replete HCT from HLA-matched or -mismatched unrelated donors for the treatment of myeloid and lymphoid leukemias. KIR ligands defined by HLA-B and -C genotypes were used to determine donor-recipient ligand incompatibility or recipient lack of KIR ligand. Among HLA-mismatched transplantations, recipient homozygosity for HLA-B or -C KIR epitopes predicted lack of KIR ligand and was associated with a decreased hazard of relapse (hazard ratio, 0.61; 95% confidence interval, .043-0.85; P = .004). Absence of HLA-C group 2 or HLA-Bw4 KIR ligands was associated with lower hazards of relapse (hazard ratio, 0.47; 95% confidence interval, 0.28-0.79, P = .004; hazard ratio, 0.56; 95% confidence interval, 0.33-0.97; P = .04, respectively). The decrease in hazard of relapse in patients with acute myelogenous leukemia was similar to that in patients with chronic myelogenous leukemia and acute lymphoblastic leukemia ( P = .95). Recipient homozygosity for HLA-B or -C epitopes that define KIR ligands is likely to be a predictive factor for leukemia relapse after myeloablative HCT from HLA-mismatched unrelated donors. This effect was not observed in HLA-identical unrelated transplants.

Chronic Kidney Disease in Long-Term Survivors of Hematopoietic Cell Transplantation

High-dose myeloablative hematopoietic cell transplantation is becoming an increasingly common treatment modality for a variety of diseases. Patient survival may be limited by substantial treatment-related toxicities, including chronic kidney disease (CKD). Although the majority of CKD after transplantation is idiopathic, thrombotic microangiopathic syndromes and nephrotic syndrome have been described. Epidemiology, pathogenesis, and potential treatment options for the various clinical syndromes that are associated with CKD in hematopoietic cell transplantation patients is reviewed. As the indications for and the numbers of transplants that are performed worldwide increases, so will the burden of CKD. The nephrologists and oncologists will have to work together to identify patients who are at risk for CKD early to prevent its development and progression to end-stage kidney disease.

Folic acid supplementation during methotrexate immunosuppression is not associated with early toxicity, risk of acute graft-versus-host disease or relapse following hematopoietic transplantation

Methotrexate (MTX) is used as an immunosuppressive agent for acute graft-versus-host disease (GVHD) prophylaxis following hematopoietic cell transplantation (HCT). Concerns that folate intake may impair MTX effectiveness or selectively rescue leukemic cells have led to variations in clinical practice regarding supplemental folic acid during MTX administration. A retrospective, observational study was undertaken to determine the association between folic acid intake (days 0-18 post transplant) and MTX toxicity and efficacy following HCT. The study population consisted of 311 adult patients who received a myeloablative HCT for chronic myelogenous leukemia, all four scheduled doses of MTX, and did not require leucovorin rescue. Multiple linear regression models were used to assess the relationships between folic acid intake (days 0-18 post-HCT) and oral mucositis index (OMI) scores, time to engraftment and risk of detectable acute GVHD. No statistically significant differences in mean OMI scores, time to engraftment, risk of acute GVHD, days to acute GVHD, risk of relapse or survival were observed when comparing patients taking, on average, <400 (14%), 400 (58%) or >400 microg (28%) folic acid per day. Our results suggest that concurrent folic acid supplementation does not change MTX effectiveness or toxicity in this patient population.