Nonmyeloablative Allogeneic Hematopoietic Cell Transplantation Research Articles

Anterior Uveitis: A Manifestation of Graft-versus-Host Disease

To describe the occurrence of anterior uveitis along with systemic manifestations of chronic graft-versus-host disease (GVHD) after nonmyeloablative allogeneic hematopoietic stem cell transplantation (HSCT) in 3 patients with hematologic malignancies. Retrospective small case series. Three patients who underwent HSCT and 4 age- and gender-matched controls for cytokine analysis in ocular fluid. Interventional study of patients who underwent HSCT. Screening for evidence of infectious causes and immunological analysis of ocular fluid samples. Clinical features of uveitis and results of aqueous analysis. Anterior uveitis developed during an exacerbation of chronic GVHD in 3 patients after allogeneic HSCT for hematologic malignancies. Patients exhibited no abnormalities on extensive uveitis screening, and in addition, serologic and wide-ranging aqueous analysis showed no evidence of recent infections. We observed signs of inflammation in the ocular fluid in 2 of 3 patients by the determination of different cytokines. No other explanation for uveitis was found except the exacerbation of GVHD. Treatment, both systemic and topical, directed to chronic GVHD induced remission of uveitis in all patients along with amelioration of other signs of GVHD. Anterior uveitis occurred in the wake of the acute exacerbation of chronic GVHD after HSCT and may represent an ocular manifestation of chronic GVHD.

Ex Vivo Expanded T Regulatory (Treg) Cells Block Conversion of Mixed Chimeras To Complete Donor Chimerism.

A highly effective method to establish long-term, stable mixed hematopoietic chimerism was developed in the dog model. This involves nonmyeloablative allogeneic hematopoietic cell transplantation (HCT), consisting of 2 Gray (Gy) total body irradiation (TBI), dog leukocyte antigen (DLA)-identical marrow, and short-term post-grafting immunosuppression. We hypothesized that CD4+CD25+ Treg cells may be important regulators for the maintenance of cellular immune tolerance after allogeneic HCT. Previously, we showed in 8 mixed chimeras that naive donor lymphocyte infusion (DLI) did not change the level of donor chimerism. However, reconditioning mixed chimeras with 2 Gy TBI followed by DLI “breaks” tolerance and increases the level of donor chimerism. Seven mixed chimeras were reconditioned with 2 Gy TBI followed by DLI. Within 4 weeks after DLI, conversion to 100% donor chimerism was seen in 5 of 7 dogs and 2 dogs had a > 50% sustained increase in donor chimerism. Four recipients developed graft-versus host disease (GVHD). A control group of 3 mixed chimeras reconditioned with 2 Gy TBI without DLI had no change in donor chimerism. These results suggest that reconditioning with 2 Gy TBI followed by DLI can break the tolerance mechanism established in mixed chimeras. Next we asked if CD4+CD25+ Treg cells obtained from mixed chimeras before reconditioning could block the increase in donor chimerism following 2 Gy TBI and DLI. Peripheral blood mononuclear cells (PBMC) from 8 mixed chimeras were obtained by leukapheresis and cultured in bulk mixed leukocyte culture (MLC) with 3rd party DLA-mismatched, unrelated and irradiated CD34+ derived dendritic cells (10:1 responder: stimulator ratio) or PBMC (1:1). On day 4 of MLC, CD25+ cells were isolated by positive immunomagnetic selection. Next, artificial antigen presenting cells (aAPC, KT32) were added to expand the CD4+CD25+ Treg cells. The aAPC expressed Fcγ receptor CD32, canine CD86, and human IL-15, were loaded with the canine-specific mitogenic anti-CD3ε antibody 17.6F9 and irradiated prior to stimulation of CD4+CD25+ Treg. After 7 days, Treg were expanded a median of 23 (range, 8–36)-fold. Expanded CD4+CD25+ Treg were assessed for phenotype and in vitro function. The Treg cells were generated from 8 mixed chimeras and were infused back into the respective dogs (median dose 1× 107/kg) after reconditioning with 2 Gy TBI and immediately prior to DLI. In 6 of 8 dogs there was no change in the level of donor chimerism at 16–20 weeks follow-up; 2 dogs converted to complete donor chimerism within 6 weeks. Treatment with expanded Treg cells blocked conversion to complete donor chimerism after 2 Gy TBI +DLI in 6 of 8 dogs, compared with significant increases in donor chimerism for all 7 dogs after 2 Gy TBI +DLI not given Treg (p=.007). None of the 8 Treg recipient dogs developed GVHD, compared with 4 of 7 not given Treg, (p=0.02). A control group of 4 mixed chimeras were infused with expanded, non-Treg CD25− T cells. To this end, CD25+ T cells were immunomagneticaly depleted on day 4 of MLC. CD25− T cells were expanded with aAPC. Three of 4 dogs converted to the complete donor chimerism within 7 weeks after 2 Gy TBI, non-Treg and DLI. These results suggest that ex vivo expanded CD4+CD25+ Treg cells have in vivo function in a large animal model and can restore the tolerance mechanism in mixed chimeras that is broken by 2 Gy TBI and DLI.

The Joint Role of Comorbidity and Performance Status (PS) in Predicting Morbidity after Allogeneic Nonmyeloablative Hematopoietic Cell Transplantation (HCT).

An HCT-specific comorbidity index (HCT-CI) was developed to capture comorbidities among patients (pt) given HCT. Here, we asked whether HCT-CI could provide information independent from current measures, notably PS. PS before and toxicities after HCT were prospectively graded per Karnofsky and Common Toxicity Criteria, respectively, while comorbidities were retrospectively evaluated per HCT-CI. Spearman rank was used to evaluate correlations. This study included pts (n=107) diagnosed with hematologic malignancies and conditioned with nonmyeloablative regimen of 2 Gy total body irradiation alone (15%) or with 90 mg/kg of fludarabine (85%) before related (44%) or unrelated HCT (56%). There were no exclusion criteria for HCT-CI scores but pts with PS of <60% were excluded from HCT. Pretransplant parameters included age (median=56 years); prior regimens (median=4); interval between diagnosis and HCT (median=24.5 months); prior HCT (34%); low, standard, and high disease risks (10%, 63%, and 27%) as described (Kahl et al. Blood-2005).; PS scores (median=90% and range=60–100%); and HCT-CI scores (median=3 and range=0–7). After HCT, 44%, 9%, and 2% of pts developed grades II, III, and IV acute graft-versus-host disease (GVHD) and 24% and 15% developed overall grades III and IV toxicities of solid organs, respectively. There were no correlations between PS and age, prior regimens, interval before HCT, prior HCT, or disease risk. HCT-CI scores had weak correlations with prior regimens (r =0.16, p=0.09) and disease risk (p=0.03) and no correlations with age, interval before HCT, and prior HCT. Pts with high-risk disease and HCT-CI scores of 0–2 vs ≥3 had median prior regimens of 3 vs 5. There was inverse correlation between HCT-CI and PS scores (r = −0.32, p=<0.001). Both higher HCT-CI and PS scores predicted increased incidences of grades II-IV acute GVHD and grades III-IV HCT-related toxicities while only HCT-CI scores predicted increased risks of 1-year NRM and worse survival (Table 1). Table 2 illustrates a composite scale combining the HCT-CI and PS scores and its relationship with short-term morbidity. We conclude that the HCT-CI and PS are only weakly correlated and therefore both should be assessed at HCT. HCT-CI was a strong predictor of morbidity and mortality, while PS predicted morbidity only. The consolidation of both scales could provide a refined stratification of risk groups for HCT-related morbidity. Larger pt cohorts are required to better define such risk groups.Table 1:Impacts of HCT-CI and PS scores on 1-year HCT outcomesHCT-CI scoresPS scores0–2≥3p>80%≤80%p%%Grades II-IV GVHD43670.0148620.02Grades III-IV toxicities25530.00430520.02Grade IV toxicities8230.0312200.2NRM11300.0120220.5survival84500.000668640.2Table 2:Composite index of HCT-CI and PS scoresGroupsGrades II-IV GVHDGrades III-IV toxicitiesGrade IV toxicities%AHCT-CI=0–2 and PS=≥80%37206BHCT-CI=0–2 and PS=<80%563713CHCT-CI=≥3 and PS=≥80%644420DHCT-CI=≥3 and PS=<80%696125p0.020.0090.1

Comparison of T Cell (CD3+) Chimerism after Myeloablative (MY) and Nonmyeloablative (NM) Allogeneic Hematopoietic Stem Cell Transplantation (AHSCT).

Assessment of chimerism after AHSCT has been important to monitor donor hematopoietic engraftment and to assess for residual disease or relapse. Achievement of T cell (CD3+) complete donor chimerism (CDC), particularly after NM AHSCT, has been considered important to achieve a graft-vs.-malignancy effect. Formal comparisons of the onset and frequency of T cell CDC in pts treated with MY vs. NM conditioning regimens have not been well described. The current analysis compared rates of achieving T cell CDC for 116 pts transplanted from 1/10/00–5/19/06 who were categorized into the following 4 groups based upon type of transplant conditioning: 1) 200 cGy total body irradiation (TBI) + fludarabine 30 mg/m2/d × 3 days (NM200; n=47); 2) 400 cGy TBI + fludarabine (NM400; n=23); 3) MY AHSCT with busulfan/cyclophosphamide without TBI (MY-noTBI; n=31); 4) MY AHSCT with TBI (MY-TBI; n=15). All NM AHSCT pts received peripheral blood stem cells and all MY AHSCT patients received bone marrow as their stem cell source. The total nucleated cell (TNC) and CD34+ cell doses were higher in the NM AHSCT patients. 36 (31%) pts had matched unrelated donors, all with at least an 8/8 match (HLA-A, -B, -Cw, -DR) by HLA class I and II DNA-based typing [10 (21%) NM200, 7 (30%) NM400, 9 (29%) MY-noTBI, 10 (67%) MY-TBI; p=0.011]. Graft-vs-host disease prophylaxis consisted of mycophenolate mofetil and cyclosporine or tacrolimus for most pts. Short tandem repeat analysis for T cell (CD3+) chimerism was performed on peripheral blood post transplant and CDC was defined as achievement of >95% DNA of donor origin isolated from CD3+ T cells. Post-transplant T cell chimerism was found in both the MY and NM AHSCT groups. The number of pts who achieved CDC and the median time to its occurrence for each group was as follows: NM200 - 34 (72%) at 4 mos; NM400 - 18 (78%) at 2.7 mos; MY-noTBI - 20 (65%) at 3.3 mos; and MY-TBI - 13 (87%) at 1.3 mos.The Kaplan-Meier curves for achievement of CDC are shown above (p=0.23). The group of pts who received MY-TBI developed T cell CDC more rapidly than the NM200 pts (p=0.05). No significant differences were observed between the NM400, MY-noTBI and MY-TBI groups with regards to achieving CDC. NM conditioning with either 200 cGy or 400 cGy TBI did not show a significant difference in rate of achieving T cell CDC as compared to MY conditioning with busulfan/cyclophosphamide without TBI. This may be related in part to the higher TNC and CD34+ cell doses in the NM AHSCT pts. However, the increased TBI doses utilized for MY conditioning may more effectively suppress anti-donor immune effector cells from the recipient, which resulted in the increased CDC compared to the NM200 group. In conclusion, post-transplant monitoring for T cell CDC is important in both MY and NM AHSCT to allow for immune manipulation to maintain a state of donor-host tolerance in order to prevent graft rejection. [Display omitted]

Non-Myeloablative Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Acute Myelogenous Leukemia.

Despite increases in CR rates, most patients with AML will relapse following successful induction treatment. Allogeneic stem cell transplantation (SCT) remains the most effective post-induction relapse preventing treatment. Similarly, for patients with relapsed/chemorefractory disease, SCT represents the only possible curative treatment modality. However, because of the significant morbidity and mortality of myeloablative conditioning regimens, SCT has been mainly restricted to young patients without co-morbidities. Because of these limitations, currently most transplant centers limit the application of SCT to patients younger than 50–55 years old.This study included 73 patients (M=37, F=46, median age 39-years, range (17–65)) with AML treated with a single nonmyeloablative (NST) regimen in the BMT department of Hadassah University Hospital. The majority of patients had advanced AML pre-transplant (CR1 n=27; CR2 n=14; CR3 n=1; relapse n=31; chemorefractory disease n=24). All patients were prepared by the NST protocol that included IV fludarabine 30mg/m2/day (on days -10 to -5) and oral busulfan 4mg/kg/d or IV busulfex at a dose of 3.2 mg\\kg\\d (on days -6 and -5), with IV ATG (on days -4 and -1).Results: Engraftment of neutrophils was achieved in 70/73 (96%) patients within a median of 15 (range 9–31) days, while engraftment of platelets was achieved in 69/73 patients (95%) within a median of 12 (range 0–86) days. Chimerism analysis data were available for 68 patients. Complete donor chimerism in the peripheral blood was achieved in 58/68 (85%) patients. 42/68 patients (62%) who survived at least one month and had a sustained engraftment, developed aGVHD. The cumulative incidence of severe aGVHD grade III-IV was 27% (18/68 patients). 35/65 (54%) patients who survived more than 100 days developed cGVHD, which was extensive in 22 (34%) cases. The cumulative incidence of NRM at 100d and 1y was 12% and 28% respectively. With a median follow up of 38m (range 10 – 118), 24/73 patients survived. Twenty-one patients survive in continuous CR, while 3 patients who relapsed post-transplant survive in CR after a second allo-SCT. The estimated long-term OS for the whole group is 30% (figure 1). 27/73 patients underwent transplantation while in CR1. Two out of these 27 patients relapsed and died, while another 8 patients died due to transplant associated complications. The estimated long-term OS for this subgroup is 63% (17/27 patients surviving in continuous CR) (figure 2).In conclusion, the results of our study showed that our NST approach is associated with a potent anti-leukemic efficacy. The results of our study look comparable to those obtained after standard myeloablative conditioning even when stratified into disease status subgroups. Prospective randomized trial comparing myeloablative vs nonmyeloablative regimens low-risk patients is warranted. [Display omitted] [Display omitted]

Lymphocyte Count on Day 56 Predicts for Reduced Risk of Infection Related Mortality Following Non-Myeloablative Allogeneic Haematopoietic Stem Cell Transplantation.

In vivo T-cell depletion using Alemtuzumab containing conditioning regimens are commonly employed in high risk patients undergoing non-myeloablative allogeneic transplants (NMT). The effect of lymphocyte count on mortality due to infection following NMT has not been previously reported. We examined lymphocyte counts as a surrogate marker of immune reconstitution following NMT. Between August 2000 and August 2006, 70 patients (M:48, F: 22, median age:49, range: 17–63) underwent NMTs for haematological malignancies [ALL, n=6; AML, n=24; CML, n=2; HD, n=5; NHL, n=16 and MM, n=17]. Donors were matched siblings in 25 patients and 45 received matched unrelated transplants. All patients received standard induction and consolidation therapy and 39 patients had previously received autologous transplant (31 had 1 procedure and 8 had 2 procedures). Three patients received NMT as a part of tandem transplantation. Fifty two transplants were from HLA identical donors, 16 received single antigen class I mismatched grafts and 2 patients received single antigen class II mismatched transplants. Thirty two patients (32/70, 46%) and 51 donors (51/70, 73%) were CMV sero-negative. Conditioning therapy was Alemtuzumab based in 48 patients (69%), low dose TBI in 14 cases (20%) and 8 patients (11%) received other combinations. All patients received cyclosporine with or without mycophenolate as GVHD prophylaxis. At the time of transplantation 19 patients were in CR1 (27%), 26 were in subsequent CR (37%), 14 had partial remission (20%) and 11 had active disease (16%). Overall 73% patients had high risk disease. With a median follow-up of 10 months (range: 0–46), the probability of overall survival (OS) at 2 yr. was 24% (95% CI: 10.8–36.8, median OS: 10 months). Forty-three patients died and death was attributed to infection in 23/43 (54%), progressive disease in 14/43 (32%), GVHD in 5/43(12%) and secondary cancer in 1/43 (2%). Twenty-seven patients relapsed (38.6%) and the probability of relapse at 2yr. was (37%, 95% CI: 18–56). The median lymphocyte count on day 28 (L28) post transplant was significantly lower with use of Alemtuzumab (0.1 vs. 0.6, p<0.001) but this effect was not seen on the day 56 lymphocyte count (0.2 vs. 0.5, p=0.8). Recursive partitioning of L28 and L56 identified L56 count of 0.4×109/L to have a significant impact on OS. Patients with L56 >0.4 (n=26) had a significantly better survival (median OS: 15 vs. 8 months, p=0.033). This survival advantage was related to reduction in infectious deaths. There was no effect on mortality due to relapse or GVHD. The incidence of CMV reactivation within the first 100 days was independent of L56 (39% vs. 36%, p=0.7). In conclusion, this study shows that lymphocyte count >0.4 on day 56 post-NMT is associated with better overall survival due to reduction in infection related deaths. This finding warrants further investigation in a larger cohort of patients.

Clinical relevance of recipient leukocyte infusion as antitumor therapy following nonmyeloablative allogeneic hematopoietic cell transplantation

Graft-versus-leukemia effects of donor lymphocytes have been considered to be central to the therapeutic benefit of nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) for malignant diseases. Surprisingly, some patients who reject donor grafts following nonmyeloablative HCT have sustained remissions of advanced, chemorefractory hematologic malignancies. In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLIs) induce loss of donor chimerism and mediate antitumor responses against host-type tumors. We assessed the clinical relevance of our mouse model. Mixed chimeric mice were generated by a nonmyeloablative protocol and some of them received host-derived tumor cells and/or RLIs or donor lymphocyte infusion (DLI). We examined chimerism, graft-versus-host disease (GVHD), and tumor survival. RLI is still effective when the leukocytes are obtained from tumor-bearing mice. Established mixed chimerism is required prior to the induced rejection to achieve maximum antitumor effects. The antitumor effects of RLI are not dependent on a specific donor strain or conditioning protocol. In contrast to DLI, RLI leads to donor cell rejection without the risk of GVHD. Together, these data reinforce the clinical potential of RLI therapy as a new HCT strategy that does not carry the risk of GVHD.

Allogeneic nonmyeloablative hematopoietic cell transplantation in metastatic colon cancer: tumor-specific T cells directed to a tumor-associated antigen are generated in vivo during GVHD

A pilot study was conducted to evaluate safety and activity of nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) in colorectal carcinoma (CRC) and to determine whether a T-cell response to a tumor-associated antigen (TAA) was induced. Fifteen patients with metastatic CRC underwent HCT from human leukocyte antigen (HLA)-matched siblings after a nonmyeloablative conditioning regimen. All patients engrafted with a median donor T-cell chimerism of 72% at day +56. Eight patients experienced grades II to IV acute graft-versus-host disease (GVHD). Despite progressive disease before HCT, partial remission and disease stabilization longer than 90 days were observed in 1 and 3 patients, respectively. Induction of TAA-specific T cells was evaluated with a carcinoembryonic antigen (CEA)-specific HLA-A(*)0201 pentamer in 6 patients with CRC. CEA-specific CD8(+) T cells were detected in 3 of 3 patients concomitant with GHVD onset, but not in 3 of 3 patients without GVHD. They were also not detected in 9 of 9 control patients with GVHD who received transplants for diagnoses other than CRC. Antitumor activity of CEA-specific T cells was also validated in vitro. In one patient, the induction of CEA-specific T cells was associated with a decrease of serum CEA levels and a partial response. Thus, graft-versus-host reactions associated with allogeneic HCT can trigger the generation of T cells specific for CEA, and this may be associated with a clinical response.

Extending Postgrafting Cyclosporine Decreases the Risk of Severe Graft-versus-Host Disease after Nonmyeloablative Hematopoietic Cell Transplantation

It is unknown whether the duration of systemic immunosuppressive treatment after allogeneic nonmyeloablative hematopoietic cell transplantation (HCT) might influence the incidence, severity, timing, and/or corticosteroid-responsiveness of graft-versus-host disease (GVHD). We retrospectively analyzed outcomes among 185 patients with hematologic malignancies who were given grafts from HLA-matched related donors following conditioning with 2 Gy total body irradiation alone or in combination with fludarabine between December 1998 and March 2003. Postgrafting immunosuppression consisted of mycophenolate mofetil (days 0-27) in combination with 3 different cyclosporine (CSP) regimens: taper from (A) days 35 to 56 (n=107), (B) days 56 to 77 (n=35), and (C) days 56 to 180 (n=43). The overall incidences of grades II-IV and III-IV acute GVHD, and extensive chronic GVHD were 52%, 13%, and 56%, respectively. The duration of CSP prophylaxis did not significantly influence the overall rate of acute GVHD (grade II-IV), extensive chronic GVHD, or non-relapse mortality. However, prolonged administration of CSP (group C) was associated with a significantly decreased hazard of grades III-IV acute GVHD (HR 0.2, 95% CI [0.04, 0.9]) and with an increased likelihood of discontinuing all systemic immunosuppression (HR 2.4, 95% CI [1.1, 5.2]) when compared to the shortest course of CSP (group A). Longer CSP duration decreased the risk of severe GVHD and increased the likelihood of discontinuing all systemic immunosuppression after nonmyeloablative HCT with HLA-matched related grafts.

Ex vivo expanded T regulatory (T reg) cells block the breaking of tolerance in mixed chimeras

Nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) consisting of 2 Gray (Gy) total body irradiation (TBI), dog leukocyte antigen-identical marrow, and short-term postgrafting immunosuppression with cyclosporine and mycophenolate mofetil was developed in the dog model as an effective way to establish long-term, stable mixed hematopoietic chimerism. We hypothesized that Treg cells are crucial to maintaining stable mixed chimerism. All dogs in the current study were stable mixed chimeras established as described.

Comparison of 2 GVHD prophylaxis regimens in nonmyeloablative allogeneic hematopoietic stem cell transplantation (HSCT) with fludarabine and low dose TBI as conditioning: Favorable outcome with addition of short course of methotrexate

Comparison of 2 GVHD prophylaxis regimens in nonmyeloablative allogeneic hematopoietic stem cell transplantation (HSCT) with fludarabine and low dose TBI as conditioning: Favorable outcome with addition of short course of methotrexate

Kaposi's sarcoma triggered by endogenous HHV‐8 reactivation after non‐myeloablative allogeneic haematopoietic transplantation

Human herpesvirus 8 (HHV-8) is causally associated with Kaposi's sarcoma (KS). KS is most frequently observed in HIV patients and in solid organ transplant recipients. The role of HHV-8 in allogeneic haematopoietic cell transplantation (HCT) remains to be determined. Here we describe a case in which KS concomitantly occurred with CMV reactivation after a non-myeloablative allogeneic HCT and presented with skin lesions, but not visceral involvement. Skin biopsy confirmed the diagnosis and ruled out graft versus host disease or disease recurrence. Molecular findings indicated viral reactivation of the recipient's primary infection. Tumour lesions completely receded when immunosuppression was tapered. Prevalence studies in donors and recipients are needed to determine the clinical impact of HHV-8 in HCT.

A pilot study of nonmyeloablative allogeneic hematopoietic stem cell transplant for advanced systemic mastocytosis

Systemic mastocytosis (SM) is a disease characterized by tissue infiltration of neoplastic mast cells originating from hematopoietic stem cells. Patients with advanced SM have a poor prognosis, and there is no mast cell ablative therapy available for most patients who carry an activating point mutation in the c-kit gene. We report results of a prospective study evaluating the safety, engraftment, and possibility of inducing a graft-versus-mast cell (GvMC) effect after allogeneic nonmyeloablative hematopoietic cell transplantation (HCT) from an HLA-identical sibling. Three patients with advanced SM were transplanted. All achieved complete donor T cell chimerism followed by clinical evidence for GvMC effect. However, all patients experienced disease progression with the longest response duration of 39 months. The GvMC effect can be observed after nonmyeloablative HCT with limited efficacy. Effective cytoreductive therapy prior to HCT may be required for long-term disease control and cure.

Chronic Kidney Disease Following Non-Myeloablative Hematopoietic Cell Transplantation

Chronic kidney disease (CKD) following myeloablative allogeneic hematopoietic cell transplantation (HCT) occurs in 20% of survivors at 1 year and is believed to be due to radiation nephritis. Non-myeloablative allogeneic HCT is a recent procedure that employs significantly lower doses of chemoradiotherapy, however, incidence and risk factors for CKD following non-myleoablative HCT have not been defined. We performed a retrospective cohort study of 122 patients from three institutions who were available for analysis at 6 months following non-myeloablative HCT. Patients received two Gy of radiation; 62% received fludarabine as preconditioning. CKD was defined as at least a 25% reduction in glomerular filtration rate (GFR) from baseline using the abbreviated modified diet in renal disease (MDRD) equation. Eighty-one of 122 patients (66%) showed evidence of CKD at follow-up. Multivariate analysis revealed that acute renal failure (ARF) during the first 100 days post-transplant was associated with development of CKD (Adjusted OR 32.8 with 95% CI 4.3-250) after controlling for other variables. Previous autologous HCT, long-term calcineurin inhibitor use and extensive chronic GVHD were independently associated with CKD. CKD following non-myeloablative HCT appears to be a distinct clinical entity and likely not related to radiation nephritis. Future research should focus on possible mechanisms for alleviating chronic injury and decreasing use of calcineurin inhibitors.

A Polymorphic Oncofetal Antigen Recognized by CD8+ CTL from Two Patients Experiencing Regression of Metastatic Renal Cell Carcinoma after Allogeneic HCT.

Regression of metatstatic renal cell carcinoma (RCC) has been observed in 8–57% of patients undergoing nonmyeloablative allogeneic hematopoietic cell transplant (HCT) from HLA-matched donors. Tumor regression in this setting typically occurs after complete donor T cell engraftment is established, and is correlated with the development of GVHD. We previously isolated from two patients experiencing regression of metastatic RCC after nonmyeloablative allogeneic HCT CD8+ CTL clones that recognized a RCC-associated minor histocompatibility (H) antigen presented by HLA-A*0201, and localized the gene encoding this antigen to chromosome 19q. To identify the gene that encodes this antigen, a cDNA expression library made from a HLA-A*0201+ minor H antigen-positive tumor cell line was screened using a transfection assay in COS-7 cells. After two rounds of screening, a 1.4 kb cDNA was identified that stimulated HLA-A*0201-dependent cytokine release from minor H antigen-specific CTL. Sequencing showed that this cDNA was derived from the MGC13170 gene on chromosome 19q, within the region of significant linkage. Sequencing of MGC13170 alleles in minor H antigen-positive and minor H antigen-negative cells revealed six single nucleotide polymorphisms (SNPs), inherited as a haplotype and all previously identified, that determined susceptibility or resistance to lysis by minor H antigen-specific CTL. Analysis of 5′ and 3′ truncation constructs derived from the MGC13170 cDNA identified an interval encoding a 48-residue open reading frame that contained the epitope and spanned one of the six SNPs (dbSNP rs3745526) that had been shown to correlate with CTL recognition. Further analysis of minigenes spanning this nonsynonymous T↔A SNP identified an interval containing T at the polymorphic position and encoding a predicted 11-residue peptide that stimulated maximal HLA-A2-dependent cytokine release from minor H antigen-specific CTL. The T↔A SNP is predicted to create a Ser↔Thr polymorphism in the encoded peptide. A synthetic 11-mer peptide containing Ser at the polymorphic residue, but not the homologous peptide containing Thr at this position, was recognized by minor H antigen-specific CTL when pulsed onto minor H antigen-negative HLA-A*0201+ cells, thus demonstrating that the T allele of MGC13170 encodes the minor H antigenic peptide. The function of MGC13170 is unknown, but a previous study (Zhou et al., Ai Zheng 21:341–345, 2002) suggested a role in resistance of tumor cells to chemotherapeutic agents. The range of tissues in which the epitope-encoding MGC13170 sequence is expressed was evaluated in silico by probing the translated human EST database with the predicted sequence of the 48-residue polypeptide encoded by the epitope-containing ORF. Of the several hundred ESTs identified that encoded the Ser- or Thr-containing peptide sequence, 72% were derived from tumor cells or cell lines (including RCC), 5% from embryonic or fetal tissues, 9% from embryonic stem cells, and 11% from normal tissues. Thus, MGC13170 encodes a novel HLA-A*0201-restricted minor H antigen that is expressed in a wide variety of neoplastic, embryonic, and fetal cells. Further studies of MGC13170 as a possible target for antitumor immune responses after allogeneic HCT are underway.

Non-Myeloablative Allogeneic Hematopoietic Stem Cell Transplantation (SCT) with Pre-Transplant Targeted Immune Depletion Results in Rapid Full Donor Engraftment in Pediatric Patients with Malignancy.

Background: Although myeloablative SCT is curative for a number of pediatric malignancies, it is associated with significant acute toxicity and late effects. Non-myeloablative SCT can result in successful engraftment of adults with diminished toxicity, but there is limited experience in pediatric pts where increased thymic function might confer a greater risk of graft rejection or mixed chimerism. Prolonged periods of mixed chimerism might in turn impair the graft-vs-tumor response and increase the risk of relapse in highly proliferative pediatric cancers.Methods: Twenty-one pediatric pts (age 7–21, median 14 years) with high-risk malignancies (Hodgkins, ALL, AML, CML, rhabdomyosarcoma, Ewings sarcoma) were enrolled. One to three cycles (C#) of fludarabine-based induction chemotherapy were administered for disease control and host immune depletion (targeted CD4 count reduction). Pre-transplant conditioning consisted of cyclophosphamide (1,200 mg/m2/day) and fludarabine (30 mg/m2/day) x 4 days plus melphalan (100 mg/m2 x 1 dose in sarcoma pts). Unmodified, G-CSF mobilized peripheral blood stem cells collected from 5–6/6 HLA-matched first degree relatives were infused (median CD34 dose 11.4 x 106/kg, range 4.44–19.12; median CD3 dose 414 x 106/kg, range 228–815). Cyclosporine was used as GVHD prophylaxis.Results: CD4 counts decreased from 332 +/− 65 at entry to 176 +/− 30 (mean +/− SEM/μL) after induction (Fig 1). Treatment was well tolerated and there was no transplant-related mortality. Median time to ANC > 500 cells/μL was post-transplant day (D+) 9 (range 7–11). Complete donor lymphoid chimerism (> 95% by VNTR-PCR on CD3 sorted peripheral blood) was achieved in all pts (18/22 by D+14 and 21/22 by D+28) (Fig 2). Acute GVHD developed in 21/22 patients, but was low grade (18/21 grades 1–2, 3/21 grade 3) and responsive to immunosuppression.Conclusions: Rapid, complete donor engraftment and chimerism can be achieved in pediatric pts with cancer using targeted pre-transplant immune depletion and a non-myeloablative preparative regimen. This approach may reduce regimen-related toxicity, and thus has the potential to improve the long-term outlook for childhood cancer survivors. [Display omitted] [Display omitted]

Development of Late over Early Full Donor Chimerism (FDC) Results in Improved Progression-Free and Overall Survival in Patients with Advanced Malignant Lymphomas Receiving Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplantation (HSCT).

Mixed chimerism (MC) can be induced in mice after non-myeloablative conditioning and allogeneic HSCT. Full donor hematopoiesis conversion from MC can occur when nontolerant donor lymphocyte infusions (DLI) are given weeks following the transplant as a result of lymphohematopoietic GVH reactions that eradicate leukemia/lymphoma without GVHD. Based on this murine model, we initiated a trial in patients with advanced hematologic malignancies of non-myeloablative therapy with HLA-matched related donor HSCT with intentional induction of MC followed by prophylactic DLI (pDLI) at day +35 post-HSCT in those without clinical GVHD. This analysis examines the influence of the kinetics of sustained donor lymphoid (CD3+ cells) chimerism on transplant outcomes. Fifty-four patients with advanced hematologic malignancies received nonmyeloablative preparative therapy (cyclophosphamide, equine antithymocyte globulin, thymic irradiation and a brief course of cyclosporine) and HLA-matched HSCT (bone marrow, BM, n=42; GCSF-mobilized peripheral blood stem cells, PBSC, n=12). Chimerism analyses were performed weekly until day +100 post-HSCT and then once monthly for a year. Twenty-nine patients with aggressive malignant lymphomas (NHL, n=21; HD, n=8) were selected out of 50 evaluable patients in an effort to analyze a relatively uniform group in terms of their disease and sustained chimerism; patients who lost the allograft were removed from this analysis. Twenty five patients attained stable FDC, defined as > or = 90% donor T cells, and 4 maintained stable MC. Using 42 days post-HSCT as the defining point for chimerism conversion 13 patients achieved early FDC and 16 patients achieved late FDC. Median time to achieve FDC in the early and late groups was 14 days (range, 7–42) and 70 days (range, 49–91) respectively. Separation produced fairly equal distribution by disease characteristics as follows (early to late): NHL, 10 to 11 and HD, 3 to 5. The early group included 10 PBSC recipients who did not receive pDLI because of the spontaneous conversion to FDC; in contrast, the majority of the late group received BMT and 13 of them received DLI (pDLI, n=10, therapeutic DLI, n=3). The incidence of acute GVHD (grades II–IV) was similar between the early and late groups with rates of 77% (n=10 of 13) and 69% (n=11 of 16) respectively; for chronic GVHD, there was a tendency toward an increased rate in the early group of 75% (3/4) compared to the late group rate of 63% (5/8). Overall disease response rates in the early and late groups were 62% and 69%, but there was a trend toward improved complete remission rates in the late group relative to the early group (50% vs 31%). Progression-free survival (PFS) and overall survival (OS) at 1 year were improved in the late compared to the early group [31% (5/16) vs 15% (2/13) and 50% (8/16) vs 38% (5/13), respectively]. At 5 years, there was a statistically significant improved PFS and OS rate in the late group over the early group [31% (5/16) vs 0% (0/13), p =0.037, for both PFS and OS]. In conclusion, these data support the observation from our mouse models that the most potent graft-vs-tumor effect occurs in mixed chimeras with late chimerism conversion, often after delayed pDLI.

Relationship of mycophenolic acid exposure to clinical outcome after hematopoietic cell transplantation

Mycophenolate mofetil is used increasingly to provide immunosuppression after nonmyeloablative allogeneic hematopoietic cell transplantation. There is wide variability in the pharmacokinetics of mycophenolic acid (MPA), the active metabolite, and low concentrations are associated with rejection after organ transplantation. We hypothesized that low MPA was associated with poorer engraftment and a higher incidence of acute graft versus host disease. We evaluated the pharmacokinetics in 87 adult subjects undergoing nonmyeloablative-related and nonmyeloablative-unrelated hematopoietic cell transplantation who were receiving 1 g mycophenolate mofetil orally or intravenously every 12 hours plus cyclosporine (INN, ciclosporin). Subjects with an unbound MPA area under the curve (AUC) from 0 to 6 hours of less than 150 ng . h/mL had a higher cumulative incidence of grade II-IV acute graft versus host disease than subjects with a greater AUC (68% versus 40%, P = .02). An unbound AUC from 0 to 12 hours of less than 300 ng . h/mL was also associated with more frequent acute graft versus host disease (58% versus 35%, P = .05). There was no association between graft versus host disease and trough concentrations (P < or = .62). A higher cumulative incidence of engraftment was associated with total MPA trough concentrations greater than 1 microg/mL (P < .01). All engraftment failures occurred in the cord blood recipients. About one half of subjects were below the unbound AUC target after oral dosing with nearly a 5-fold variability in AUC. Intravenous dosing achieved unbound targets better than oral dosing. The current practice of dosing with 1 g twice daily provides inadequate plasma concentrations in many patients, and doses of at least 3 g/d are likely necessary. Therapeutic monitoring of MPA concentrations with dose adjustment into the therapeutic target appears to be necessary for the most effective use of mycophenolate mofetil.

Severe thrombocytopenia due to host‐derived anti‐HPA‐1a after non‐myeloablative allogeneic haematopoietic stem cell transplantation for multiple myeloma: a case report

Host- or donor-derived alloimmune thrombocytopenia can develop after non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT). We report the first case of host-derived HPA-1a antibodies. A 52-year-old male patient received HSCT from his human leucocyte antigen (HLA)-A, -B, -C, -DR identical brother after reduced intensity conditioning. Bilinear engraftment around day 12 was accompanied by a continuous decrease of platelet counts. We investigated for platelet antibodies because of a progressive decline of platelet counts and refractoriness to platelet transfusions. The patient's serum was tested by enzyme-linked immunosorbent assay (ELISA), a solid phase assay and monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay. Recipient's DNA from the time before HSCT and donor's DNA were genotyped for human platelet antigens. Serum obtained on day 15 after HSCT reacted strongly with the donor's platelets due to host-derived anti-HPA-1a- and anti-HLA I antibodies. Serum samples from days 39, 45 and 65 after HSCT contained only anti-HLA I; no antibodies were detectable on day 149. Platelet counts increased on day 20 spontaneously. The decrease of the antibodies accompanied by the increase of the platelet counts suggests progressive elimination of residual host cells. The HPA-1a antibodies affected thrombopoietic engraftment and the success of platelet transfusions.

Engraftment Syndrome after Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplantation: Incidence and Effects on Survival

Engraftment syndrome (ES) encompasses a constellation of symptoms that occur during neutrophil recovery after both autologous and allogeneic hematopoietic stem cell transplantation (HCT). Although it is well characterized after conventional myeloablative procedures, limited data exist on this complication after nonmyeloablative allogeneic HCT. The clinical manifestations, incidence, and risk factors associated with ES were investigated in a consecutive series of patients undergoing cyclophosphamide/fludarabine-based nonmyeloablative allogeneic HCT from a related HLA-compatible donor. Fifteen (10%) of 149 patients (median age, 53 years; range, 27–66 years) developed ES; the onset of symptoms occurred at a median of 10 days (range, 3–14 days), and they consisted of fever (100%), cough (53%), diffuse pulmonary infiltrates (100%), rash (13%), and room air hypoxia (87%). ES was more likely to develop in patients who received empiric amphotericin formulations after transplant conditioning (Fisher exact test; P = .007). In a multivariate analysis, older patient age, female sex, and treatment with amphotericin were predictors for the development of ES. Intravenous methylprednisolone led to the rapid resolution of ES; however, transplant-related mortality was significantly higher (cumulative incidence, 49% versus 16%; P = .0005), and median survival was significantly shorter (168 versus 418 days; P = .005) in patients with ES compared with non-ES patients. In conclusion, ES occurs commonly after cyclophosphamide/fludarabine-based nonmyeloablative transplantation and responds rapidly to corticosteroid treatment, but it is associated with a higher risk of nonrelapse mortality and with shorter overall survival.