Alternative donor bone marrow transplant for children with Philadelphia chromosome ALL.

Children with Philadelphia chromosome positive (Ph+) acute lymphocytic leukemia (ALL) have only a 20% event-free survival when treated with chemotherapy alone. Bone marrow transplant (BMT) for patients with matched siblings has been associated with significantly better long-term survival. We asked whether children who lack a matched sibling donor would do as well if an alternative donor was utilized. Between 1987 and 2002, we transplanted 29 children and adolescents using either an unrelated donor (23) or a mismatched family member (six). The conditioning regimen included cytosine-arabinoside, cyclophosphamide and total body irradiation. Graft-versus-host disease (GVHD) prophylaxis consisted of T-cell depletion (antibody T10B9 or OKT3 and complement) with post transplant cyclosporine (CSA). All patients engrafted. Four developed grades III-IV acute GVHD. Three of 24 evaluable patients developed extensive chronic GVHD. Two patients died of relapse (7%). Two long-term survivors (>6 years) died of malignant glioblastoma multiforme. Event-free survival at 3, 5, and 10 years is 56, 51, and 46%, respectively. Five of six patients in >CR2 or relapse at the time of transplant died. Our data should encourage the use of alternative donor transplants early in the course of disease for children with Ph+ ALL.

Evaluation of histogenesis of B-lymphocytes in pediatric EBV-related post-transplant lymphoproliferative disorders.

Post-transplant lymphoproliferative disorders (PTLD) are morphologically/clinically heterogeneous. The main goal of this study was to define the histogenesis of PTLD (B-cell phenotype, EBV-related) in seven pediatric patients after allogeneic T-cell-depleted bone marrow transplantation. Immunohistochemical stains using histogenetic markers, including Bcl-6 (expressed by germinal center (GC) B cells), MUM1/IFR4 (late GC and post GC B cells), and CD138 (post GC B cells), were performed on paraffin-embedded tissue. By morphology, four cases were classified as polymorphic PTLD and three as monomorphic PTLD, according to the WHO classification. By the expression pattern of histogenetic markers, five cases (two polymorphic, three monomorphic PTLD) were of late GC/early post GC B-cell origin expressing only MUM1/IRF4. The remaining two cases (one monomorphic, one polymorphic PTLD) were of post GC B-cell origin expressing MUM1/IRF4 and CD138, but not Bcl-6. Our study indicates that histogenesis of PTLD may be defined by histogenetic markers using immunohistochemistry. The results suggest that most pediatric PTLD are of late GC/early post GC B-cell origin, and a minor group is of post GC B-cell origin. The histogenesis of PTLD appears independent of morphologic appearance. Further studies are warranted to confirm our observation and to evaluate the clinical significance of histogenetic pattern of PTLD.

Protection from lethal murine graft-versus-host disease without compromise of alloengraftment using transgenic donor T cells expressing a thymidine kinase suicide gene.

Donor T cells play a pivotal role in facilitating alloengraftment but also cause graft-versus-host disease (GVHD). Ex vivo T-cell depletion (TCD) of donor marrow is the most effective strategy for reducing GVHD but can compromise engraftment. This study examined an approach whereby donor T cells are selectively eliminated in vivo after transplantation using transgenic mice in which a thymidine kinase (TK) suicide gene is targeted to the T cell using a CD3 promoter/enhancer construct. Lethally irradiated B10.BR mice transplanted with major histocompatibility complex (MHC)-incompatible TCD C57BL/6 (B6) bone marrow (BM) plus TK(+) T cells were protected from GVHD after treatment with ganciclovir (GCV) in a schedule-dependent fashion. To examine the effect of GCV treatment on alloengraftment, sublethally irradiated AKR mice underwent transplantation with TCD B6 BM plus limiting numbers (5 x 10(5)) of B6 TK(+) T cells. Animals treated with GCV had comparable donor engraftment but significantly reduced GVHD when compared with untreated mice. These mice also had a significantly increased number of donor splenic T cells when assessed 4 weeks after bone marrow transplantation. Thus, the administration of GCV did not render recipients T-cell deficient, but rather enhanced lymphocyte recovery. Adoptive transfer of spleen cells from GCV-treated chimeric mice into secondary AKR recipients failed to cause GVHD indicating that donor T cells were tolerant of recipient alloantigens. These studies demonstrate that administration of TK gene-modified donor T cells can be used as an approach to mitigate GVHD without compromising alloengraftment.

Impact of donor type on outcome of bone marrow transplantation for Wiskott-Aldrich syndrome: collaborative study of the International Bone Marrow Transplant Registry and the National Marrow Donor Program.

Human leukocyte antigen (HLA)-identical sibling bone marrow transplantation is an effective treatment for Wiskott-Aldrich syndrome. However, most children with this disease lack such donors and many patients receive transplants from alternative donors. This study compared outcomes of HLA-identical sibling, other related donor, and unrelated donor transplantation for Wiskott-Aldrich syndrome. The outcome of 170 transplantations for Wiskott-Aldrich syndrome, from 1968 to 1996, reported to the International Bone Marrow Transplant Registry and/or National Marrow Donor Program were assessed. Fifty-five were from HLA-identical sibling donors, 48 from other relatives, and 67 from unrelated donors. Multivariate proportional hazards regression was used to compare outcome by donor type and identify other prognostic factors. Most transplant recipients were younger than 5 years (79%), had a pretransplantation performance score greater than or equal to 90% (63%), received pretransplantation preparative regimens without radiation (82%), and had non-T-cell-depleted grafts (77%). Eighty percent received their transplant after 1986. The 5-year probability of survival (95% confidence interval) for all subjects was 70% (63%-77%). Probabilities differed by donor type: 87% (74%-93%) with HLA-identical sibling donors, 52% (37%-65%) with other related donors, and 71% (58%-80%) with unrelated donors (P =.0006). Multivariate analysis indicated significantly lower survival using related donors other than HLA-identical siblings (P =.0004) or unrelated donors in boys older than 5 years (P =.0001), compared to HLA-identical sibling transplants. Boys receiving an unrelated donor transplant before age 5 had survivals similar to those receiving HLA-identical sibling transplants. The best transplantation outcomes in Wiskott-Aldrich syndrome are achieved with HLA-identical sibling donors. Equivalent survivals are possible with unrelated donors in young children.

Analysis of risk factors for the development of GVHD after T cell-depleted allogeneic BMT: effect of HLA disparity, ABO incompatibility, and method of T-cell depletion.

Multivariate analysis was performed to determine the independent factors affecting the risk of acute GVHD (aGVHD) grades II to IV and extensive chronic GVHD (cGVHD) and the rate of survival in 481 recipients of T cell-depleted (TCD) marrow allografts who received transplants at a single center between 1991 and 2000. All patients received grafts partially depleted of CD3+ T cells by complement-mediated lysis using 2 narrow-specificity monoclonal antibodies (MoAbs), T10B9.1A-31 (n = 400) or Muromonab-Orthoclone OKT3 (n = 81). Factors considered in the analysis included patient/donor sex, age, cytomegalovirus (CMV) status, and ABO blood group along with T-cell dose, disease, and disease status, donor relationship, HLA antigen (Ag)mismatch (MM), growth-factor use, anti-thymocyte globulin use, year of transplantation, and the MoAb used for TCD. The results showed an association of HLA with an increased relative risk (RR) of aGVHD for recipients of grafts from relateddonors that were > or =2 Ag MM (n = 73, RR = 2.09, P = .005), matched unrelated (UR) donors (n = 130, RR = 1.98, P = .004), and > or =2 Ag MM UR donors (n = 34, RR = 2.68, P = .003) compared with the baseline matched-sibling group (n = 121). No increased risk of aGVHD was seen for 0 to 1 Ag MM family donors (n = 24) or 1 Ag MM UR donors (n = 99). aGVHD risk was increased with minor, but not major or major-minor, ABO disparity (RR = 2.0, P = .003) compared with that of ABO-identical pairs. We found less effective TCD and resultant higher T-cell dose for recipients of grafts that were T cell depleted using OKT3. However, the use of OKT3 and not the T-cell dose was associated with increased aGVH-D risk (RR of 1.84, P = .001). Increased risk of extensive cGVHD was associated with patient age of >20 years (RR = 2.2, P < .0001) and with CMV status (positive patient/negative donor, RR = 1.9, P = .002). Decreased survival was associated with older age (>20 years), a > or =2 Ag MM related donor, a 1 or > or =2 Ag MM UR donor, risk group, and a CMV-positive patient/-negative donor pair. There was no difference in survival for 0 to 1 Ag MM related or matched UR donors compared with the baseline group. These data indicate that there are quantitative as well as potential qualitative differences in outcome depending on the TCD method. Expected and unexpected risk factors for GVHD and survival were associated with partial TCD. Our data support the consideration of ABO match in donor selection, the preferential selection of CMV-positive donors for CMV-positive recipients, and the acceptance of 1 but not > or =2 Ag HLA MM donors.

Engraftment and survival after unrelated-donor bone marrow transplantation: a report from the national marrow donor program.

We analyzed engraftment of unrelated-donor (URD) bone marrow in 5246 patients who received transplants facilitated by the National Marrow Donor Program between August 1991 and June 1999. Among patients surviving at least 28 days, 4% had primary graft failure (failure to achieve an absolute neutrophil count > 5 x 10(8)/L before death or second stem-cell infusion). Multivariate logistic regression analysis showed that engraftment was associated with marrow matched at HLA-A, HLA-B, and DRB1; higher cell dose; younger recipient; male recipient; and recipient from a non-African American ethnic group. More rapid myeloid engraftment was associated with marrow serologically matched at HLA-A and HLA-B, DRB1 match, higher cell dose (in non-T-cell-depleted cases), younger recipient, recipient seronegativity for cytomegalovirus (CMV), male donor, no methotrexate for graft-versus-host disease prophylaxis, and transplantation done in more recent years. A platelet count higher than 50 x 10(9)/L was achieved by 47% of patients by day 100. Conditional on survival to day 100, survival at 3 years was 61% in those with platelet engraftment at day 30, 58% in those with engraftment between day 30 and day 100, and 33% in those without engraftment at day 100 (P <.0001). Factors favoring platelet engraftment were higher cell dose, DRB1 allele match, recipient seronegativity for CMV, HLA-A and HLA-B serologically matched donor, and male donor. Secondary graft failure occurred in 10% of patients achieving initial engraftment, and 18% of those patients are alive. These data demonstrate that quality of engraftment is an important predictor of survival after URD bone marrow transplantation.

Infiltrating T cells during liver graft-versus-host disease show a restricted T-cell repertoire.

Data from animal models have shown that hepatic graft-versus-host disease (GVHD) may be mediated by donor T cells interacting with liver adhesion molecules, other minor histocompatibility antigens, or both. We hypothesized that T-cell infiltrates within a liver biopsy during clinical GVHD would show a restricted T-cell response because the T cells would be responding to a limited number of antigens. We studied the peripheral T-cell repertoire and the liver-infiltrating T-cell repertoire of a patient who developed skin GVHD and subsequent liver GVHD after a matched sibling bone marrow transplantation for acute myeloid leukemia. Spectratype analysis of peripheral blood at the time of liver GVHD revealed that the patient had reconstituted a complex peripheral T-cell repertoire as evidenced by the presence of complementarity-determining region 3 (CDR3) length heterogeneity in most of the T-cell families. The repertoire complexity was skewed in variable gene beta (VB) 5.3, VB4, VB7, VB8, and VB15. Spectratype analysis on the liver biopsy sample revealed a limited infiltrate with an oligoclonal expansion in VBs 4, 7, and 8. We evaluated the T-cell infiltrate in more detail by sequencing the relevant expansions noted by spectratype and developing probes for the predominant CDR3 sequences. These clonotype probes were hybridized to peripheral blood and liver samples from the patient, a T-cell line developed from the patient's peripheral blood at the time of the initial skin GVHD, the donor's blood and marrow, and control samples. The results showed that the T-cell infiltrate during liver GVHD is mediated by a limited number of T cells, and that those cells are mostly different from the ones expanded from the peripheral blood during an acute skin GVHD reaction. These data support the concept that liver GVHD is a response to tissue-specific minor histocompatibility antigens.

Alternative-donor hematopoietic stem-cell transplantation for severe aplastic anemia.

Bone marrow transplantation (BMT) from a human leukocyte antigen (HLA)-matched sibling is the treatment of choice in children and young adults with severe aplastic anemia (SAA). However, because only 30% of patients have a suitable donor, more aggressive nontransplant immunosuppressive regimens have been used, with reasonable results. The use of mismatched family member and unrelated donors, initially fraught with problems of nonengraftment and severe graft-versus-host disease (GVHD), has improved markedly over the past 10 years. The establishment of donor registries, more precise HLA typing methods, and better supportive care are significant factors in the improved outcomes. The challenge for the future is to assemble the optimal combination of donor selection, conditioning regimen, and GVHD prophylaxis to enhance disease-free survival. These better outcomes should encourage the treating physician to consider stem-cell transplant at an earlier stage of disease.

Clinical pharmacokinetics of the CD19 receptor-directed tyrosine kinase inhibitor B43-Genistein in patients with B-lineage lymphoid malignancies.

The authors examined the pharmacokinetics of the CD19 receptor-directed tyrosine kinase inhibitor B43-Genistein in 17 patients (4 children, 13 adults) with B-lineage lymphoid malignancies, including 12 patients with acute lymphoblastic leukemia (ALL) and 5 patients with non-Hodgkin's lymphoma (NHL). The immunoconjugate was administered intravenously as a 1-hour continuous infusion at a dose level of either 0.1 mg/kg (N = 12) or 0.18 mg/kg (N = 5), and the plasma concentration-time data were modeled by using the WinNonlin program to estimate the pharmacokinetic parameters. Pharmacokinetic analyses revealed a plasma half-life of 19 +/- 4 hours, mean residence time of 22 +/- 4 hours, and a systemic clearance of 18 +/- 2 mL/h/kg. The average (mean +/- SEM) values for the maximum plasma concentration Cmax, volume of distribution at steady state (Vss), and area under curve (AUC) were 1092 +/- 225 ng/ml, 291 +/- 37 mL/kg, and 9987 +/- 2021 micrograms x h/L, respectively. The AUC values were higher at the 0.18 mg/kg dose level than at the 0.1 mg/kg dose level (16,848 +/- 5118 micrograms x h/L vs. 7128 +/- 1156 micrograms x h/L, p = 0.009). Patients with ALL had a significantly larger volume of distribution at steady state (332 +/- 47 mL/kg vs. 191 +/- 12 mL/kg, p = 0.04), faster clearance (21 +/- 3 mL/h/kg vs. 11 +/- 2 mL/h/kg, p = 0.03), and lower dose-corrected AUC than patients with NHL (6010 +/- 836 micrograms x h/L vs. 12,044 +/- 2707 micrograms x h/L, p = 0.006). There was a trend toward faster clearance rates (23 +/- 4 mL/h/kg vs. 16 +/- 3 mL/h/kg, p = 0.1), shorter elimination half-lives (5.7 +/- 3.6 hours vs. 13 +/- 8.8 hours, p = 0.1), and shorter mean residence times (11 +/- 3 hours vs. 25 +/- 5 hours, p = 0.08) for non-Caucasian patients as compared to Caucasian patients. When compared to adult patients, pediatric patients showed a significantly larger volume of distribution at steady state (418 +/- 82 mL/kg vs. 252 +/- 34 mL/kg, p = 0.02) and a longer elimination half-lives (18.4 +/- 13.6 hours vs. 8.7 +/- 6.7 hours, p = 0.04). The pharmacokinetics of B43-Genistein was not affected by the gender of the patients or by bone marrow transplantation in past medical history. Overall, B43-Genistein showed favorable pharmacokinetics in this heavily pretreated leukemia/lymphoma patient population, which is reminiscent of its recently reported favorable pharmacokinetics in cynomolgus monkeys. To our knowledge, this is the first clinical pharmacokinetics study of a tyrosine kinase inhibitor containing immunoconjugate.

Detection of Epstein-Barr virus EBER sequence in post-transplant lymphoma patients with DNA dendrimers.

The EBER RNAs are the most numerous viral transcripts in latently infected lymphocytes in healthy individuals and also in the tumor cells of Epstein Barr virus (EBV)-associated malignancies. A rise in EBV load in peripheral blood has been associated with the onset of post-transplant lymphoproliferative disease (PTLD) in immunocompromised patients. Treatment of PTLD with adoptive immunotherapy has made the rapid and accurate determination of EBV load essential. The relationship between EBV load and other EBV-associated malignancies, like Hodgkin's disease or AIDS-associated lymphoma, is unknown. In order to define viral load based on the number of EBV-infected cells in the peripheral blood, we developed a method which combines cellular dilution of peripheral blood mononuclear cells with the direct detection of EBER-1 RNA with DNA dendrimers. DNA dendrimers are large scaffolds of DNA which give at least a 500 1000-fold increase in detection of membrane bound nucleic acid over oilgonucleotide probes. The use of a novel class of these nucleic acid superstructures is described as a specific probe for EBER-1 detection. When two PTLD patients were analyzed for viral load with DNA dendrimers, at least one in 250000 peripheral blood mononuclear cells were shown to be infected with EBV.

Treatment of therapy-refractory B-lineage acute lymphoblastic leukemia with an apoptosis-inducing CD19-directed tyrosine kinase inhibitor.

Seven children and eight adults with CD19+ B-lineage acute lymphoblastic leukemia, as well as one adult with chronic lymphocytic leukemia, were treated with the CD19 receptor-directed tyrosine kinase inhibitor B43-Genistein. All patients had failed previous chemotherapy regimens, and six patients had relapsed after bone marrow transplantation. B43-Genistein was administered as a 1-hour i.v. infusion at 0.1-0.32 mg/kg/day dose levels for 10 consecutive days or 3 consecutive days weekly for a total of nine doses. B43-Genistein was well tolerated by all patients with no life-threatening side effects. There were six episodes of grade 2-3 fever, two of which were clearly drug related, one episode each of grade 3 myalgia, grade 2 sinus tachycardia, and grade 2 vascular leak syndrome. There was one durable complete remission and two transient responses. Pharmacokinetic analyses in 12 patients revealed a plasma half-life of 20 +/- 5 h, mean residence time of 24 +/- 5 h, and a systemic clearance rate of 20 +/- 3 ml/h/kg. Moderate levels of human antimouse antibody (HAMA) ranging from 20-87 ng/ml were detected in the day 28 blood samples from three of nine cases examined. Treatment of these three HAMA-positive patients with a second course of B43-Genistein did not yield measurable immunoconjugate levels in the plasma, indicating that the administered B43-Genistein molecules were rapidly cleared from circulation due to the HAMA. On the basis of its acceptable toxicity profile and its ability to elicit objective responses at nontoxic dose levels, B43-Genistein may provide the basis for an effective treatment strategy for B-lineage acute lymphoblastic leukemia patients who have failed standard therapy.

Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings.

PURPOSE: To compare outcomes of bone marrow transplants for leukemia from HLA-identical siblings, haploidentical HLA-mismatched relatives, and HLA-matched and mismatched unrelated donors. PATIENTS: A total of 2,055 recipients of allogeneic bone marrow transplants for chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) were entered onto the study. Transplants were performed between 1985 and 1991 and reported to the International Bone Marrow Transplant Registry (IBMTR). Donors were HLA-identical siblings (n = 1,224); haploidentical relatives mismatched for one (n = 238) or two (n = 102) HLA-A, -B, or -DR antigens; or unrelated persons who were HLA-matched (n = 383) or mismatched for one HLA-A, -B, or -DR antigen (n = 108). HLA typing was performed using serologic techniques. RESULTS: Transplant-related mortality was significantly higher after alternative donor transplants than after HLA-identical sibling transplants. Among patients with early leukemia (CML in chronic phase or acute leukemia in first remission), 3-year transplant-related mortality (+/-SE) was 21% +/- 2% after HLA-identical sibling transplants and greater than 50% after all types of alternative donor transplants studied. Among patients with early leukemia, relative risks of treatment failure (inverse of leukemia-free survival), using HLA-identical sibling transplants as the reference group, were 2.43 (P < .0001) with 1-HLA-antigen-mismatched related donors, 3.79 (P < .0001) with 2-HLA-antigen-mismatched related donors, 2.11 (P < .0001) with HLA-matched unrelated donors, and 3.33 (P < .0001) with 1-HLA-antigen-mismatched unrelated donors. For patients with more advanced leukemia, differences in treatment failure were less striking: 1-HLA-antigen-mismatched relatives, 1.22 (P = not significant [NS]); 2-HLA-antigen-mismatched relatives, 1.81 (P < .0001); HLA-matched unrelated donors, 1.39 (P = .002); and 1-HLA-antigen-mismatched unrelated donors, 1.63 (P = .002). CONCLUSION: Although transplants from alternative donors are effective in some patients with leukemia, treatment failure is higher than after HLA-identical sibling transplants. Outcome depends on leukemia state, donor-recipient relationship, and degree of HLA matching. In early leukemia, alternative donor transplants have a more than twofold increased risk of treatment failure compared with HLA-identical sibling transplants. This difference is less in advanced leukemia.

Detection of adenovirus DNA in peripheral blood mononuclear cells by polymerase chain reaction assay.

Adenovirus can establish persistent infections which may reactivate and cause disease in immunocompromised hosts. Lymphocytes have been postulated to serve as a site of adenoviral persistence based upon the ability to isolate adenovirus from tonsils and to detect adenovirus DNA by Southern blot hybridization in peripheral blood mononuclear cells (PBMC). To test this hypothesis, a more sensitive and specific polymerase chain reaction (PCR) assay was developed to detect adenovirus DNA. Two sets of nested primers were designed to conserved sequences in the adenovirus E1A and hexon genes. The E1A and hexon primers amplified DNA from representative adenoviral serotypes in all six adenoviral groups (A-F). Both primers detected a single copy of the adenovirus type 2 genome but were less sensitive for the group B type 35. None of 33 PBMC specimens from healthy adults and only one of 40 pediatric samples was positive (at a low level) for adenovirus DNA by nested PCR assay. In comparison, PBMC from two children with fatal adenoviral infection were both strongly positive for adenovirus DNA. It is concluded that, in contrast to a previous study, PBMC are not a common site of persistent group C adenoviral infection. In addition, assay of PBMC by the adenovirus-specific PCR may help detect early invasive disease and warrants further evaluation.

Long-term results of selective renal shielding in patients undergoing total body irradiation in preparation for bone marrow transplantation.

The purpose of this study was to evaluate the effect of partial renal shielding used in conjunction with total body irradiation (TBI) on the incidence of bone marrow transplantation nephropathy (BMT Np) seen as a late sequelae after transplantation. Of 402 patients who have undergone bone marrow transplantation (BMT) at the Medical College of Wisconsin (MCW) 157 were greater than 18 years of age, received 14 Gy TBI and survived at least 100 days post-transplant. The incidence of BMT nephropathy was evaluated in these patients by dose to the kidneys. In the 72 patients who received 14 Gy TBI with no renal shielding, the actuarial risk of developing BMT Np at 2 1/2 years (30 months) post-BMT was 29 +/- 7%. Sixty-eight patients received 14 Gy TBI with partial renal shielding of 15% (renal dose = 11.9 Gy), the actuarial risk of developing BMT Np was 14 +/- 5% at 2 1/2 years. Seventeen patients received 14 Gy TBI with renal shielding of 30% (renal dose = 9.8 Gy); none of this group have developed BMT Np despite a median follow-up of over 2 1/2 years (985 days). The trend of decreasing BMT Np with increasing shielding is statistically significant (P = 0.012). Prognostic factors such as age, type of transplant and good-risk vs poor-risk disease status were evaluated and were similar in each cohort of patients described above. We conclude that given the statistically significant benefit seen here in the reduced incidence of BMT Np by the use of selective renal shielding, this should be seriously considered for all patients who receive TBI, but especially for patients whose renal doses exceed 10 Gy.

Human adenovirus-specific CD8+ T-cell responses are not inhibited by E3-19K in the presence of gamma interferon.

Adenovirus has considerable potential as a gene therapy vector, but recent animal data suggest that transduced cells are destroyed by adenovirus-specific cytotoxic T-lymphocyte (CTL) responses. Therefore, it will be important to develop strategies to evade adenovirus-specific CTL responses in humans. As a first step, an assay was developed to detect and characterize human CTLs directed against adenovirus. Adenovirus-specific CTL responses were demonstrated to be present in four of five healthy adults by in vitro stimulation of peripheral blood mononuclear cells with autologous fibroblasts infected with the adenovirus type 2 (Ad2) E3 deletion mutant Ad2+ND1. Killing by adenovirus-specific CTLs was major histocompatibility complex class I restricted and was documented to be mediated by CD8+ T cells. Wild-type-Ad2-infected cells were poor CTL targets compared with cells infected with the E3 deletion mutant because of the expression of E3-19K, an early viral glycoprotein which prevents transport of major histocompatibility complex class I antigens out of the endoplasmic reticulum to the cell surface. However, preincubation of targets with gamma interferon resulted in enhanced killing of wild-type-Ad2-infected cells, to levels comparable to those obtained with Ad2+ ND1-infected cells. Radioimmunoprecipitation analysis revealed that gamma interferon not only increased the synthesis of class I antigens but also allowed excess molecules to escape from the endoplasmic reticulum. It is concluded that E3-19K expression in adenovirus-infected cells inhibits human CTL recognition in vitro but that gamma interferon may help overcome the E3-19K effect during acute infection in vivo.

Spontaneous, persistent infection of a B-cell lymphoma with adenovirus.

An adenovirus culture-positive lymphoblastoid cell line was derived from a bone marrow transplant recipient with fatal B-cell lymphoproliferative disease and adenovirus pneumonia. At autopsy, focal areas of the lymphoma infiltrating the patient's lung were positive for adenovirus proteins by immunohistochemical staining. The Epstein-Barr virus-transformed B-cell line Mk, established from pleural fluid cells, contained adenovirus virions in both the nucleus and the cytoplasm by electron microscopy. The majority of Mk cells expressed adenovirus proteins and produced a high level of infectious adenovirus by plaque assay analysis. However, in contrast to the rapid cell death induced by adenovirus in other permissive cell lines, Mk was maintained stably in tissue culture for 6 months. These data indicate that adenoviral replication is not sufficient for cell lysis and confirm that adenovirus can cause persistent infection in human lymphoid cells in vivo.

Use of unrelated marrow grafts compensates for reduced graft-versus-leukemia reactivity after T-cell-depleted allogeneic marrow transplantation for chronic myelogenous leukemia.

The effect of donor/recipient histocompatibility on relapse in patients receiving T-cell-depleted (TCD) grafts for chronic myelogenous leukemia (CML) was evaluated. Specifically, we sought to determine whether TCD results in an attenuation of the graft-versus-leukemia (GVL) effect on recipients of unrelated marrow grafts similar to that observed in HLA-identical sibling marrow transplantations. This question was addressed by comparative analysis of the relapse rates in marrow grafts who otherwise received identical preparative regimens and graft-versus-host disease (GVHD) prophylaxis schedules (T-cell depletion with T10B9 monoclonal antibody and complement plus posttransplant cyclosporine) and by serial molecular analyses using the polymerase chain reaction (PCR) to detect the bcr/abl RNA transcript in patients transplanted with unrelated donor grafts. Patients transplanted with advanced disease (accelerated phase or blast crisis) had equally high relapse rates, regardless of whether they received HLA-identical sibling (56%;95% confidence interval [CI], 29% to 82%) or unrelated marrow grafts (8%; 95% CI, 0% to 28%) had a significantly lower incidence of relapse than did patients transplanted with HLA-identical marrow grafts (47%; 95% CI, 23% to 71%; P = .002). Because all patients were similarly treated, these data indicate that the lower relapse rate in these unrelated patients was caused by an augmented GVL effect that was most likely attributable to increased HLA disparity between donor and recipient. The probability of developing both acute and chronic GVHD was significantly increased in chronic-phase recipients of unrelated marrow grafts, suggesting that the enhanced GVL effect was at least partly GVHD-associated. The lack of such a finding in advanced disease patient receiving unrelated marrow grafts raises the possibility that clinically significant GVL effect after TCD marrow transplantation was limited and confined to patients with more indolent disease. Serial PCR analyses for the presence of the bcr/abl RNA transcript showed that the vast majority of patients transplanted in chronic phase with unrelated marrow grafts were persistently PCR-negative, indicating that the GVL effect was durable in these patients. Most of these patients were observed to become PCR negative within 1 to 2 months after transplantation, showing that early eradication of leukemia was possible with TCD marrow grafts. This study shows that the use of unrelated marrow grafts compensates for reduced GVL reactivity associated with TCD in patients transplanted for CML. Furthermore, these data indicate that, in selected patient populations with CML, TCD can be used to reduce GVHD without a commensurate compromise in the GVL effect.

Therapeutic plasma exchange does not appear to be effective in the management of thrombotic thrombocytopenic purpura/hemolytic uremic syndrome following bone marrow transplantation.

Recognition of thrombotic thrombocytopenic purpura (TTP)/hemolytic uremic syndrome (HUS) following BMT has increased in recent years. The pathogenesis and etiology may be related to endothelial cell damage secondary to irradiation and/or CsA. Optimal management of this condition remains unclear. Due to similarity between this syndrome and classical TTP, patients with TTP/HUS following BMT are commonly treated with therapeutic plasma exchange (TPE). We describe our experience with 9 such patients who were treated with TPE (8 cases) and immunoadsorption with a Staphylococcal Protein A column (1 case). The exchanges were done with fresh frozen plasma and/or cryoprecipitate-depleted frozen plasma. Out of 8 patients treated with TPE, 6 died within 2 months of TPE due to secondary infections, metabolic disturbances and progression of TTP/HUS. Of these 6 patients, 5 had no hematological response, while 1 had hematological improvement. Two patients are alive 4 and 3 years later, however, they had shown only minimal hematological response at the end of 28 and 20 TPE, respectively. Their renal function remains stable but severely reduced. The ninth patient who received Staphylococcal Protein A column treatment died within 5 days of treatment without hematological improvement. Thus, in contrast to its effectiveness in classical TTP, TPE does not appear to be as effective in the management of well established TTP/HUS following BMT.

Characterization of human proliferative T cell responses to adenovirus.

Although cellular immune responses are likely important for recovery from acute adenovirus infection, they have not been studied in humans. As a first step, a sensitive assay for the detection of adenovirus-specific proliferative T cell responses was developed. Peripheral blood mononuclear cells from 29 of 30 healthy adults exhibited specific proliferative responses to adenovirus type 2 antigen. Adenovirus-specific proliferation was found to be mediated by CD4+ T cells. Specific proliferative responses were detected to purified Ad2 virions and by Ad2-infected cell lysates, indicating that adenovirus structural proteins are important targets. In addition, specific proliferative responses to the uncommon adenovirus type 35 were found in persons without serologic evidence of prior Ad35 infection. These data suggest that adenovirus-specific CD4+ T cells recognize conserved antigens among different serotypes and that a majority of persons develop long-lived CD4+ T cell responses to adenovirus.

Effect of T-cell depletion as graft-versus-host disease prophylaxis on engraftment, relapse, and disease-free survival in unrelated marrow transplantation for chronic myelogenous leukemia.

Between January 1988 and March 1993, 48 patients received T-cell-depleted marrow grafts from unrelated donors as treatment for chronic myelogenous leukemia (CML). The median age of the population was 31.7 years (range 5.4 to 53) with 17 of 48 patients greater than 40 years of age. Twenty-seven patients were transplanted in chronic phase, 17 in accelerated phase, and 4 in blast crisis. All patients received a standardized preparative regimen of cyclophosphamide, high-dose cytosine arabinoside, methylprednisolone, and total body irradiation. Marrow grafts were depleted of mature T cells with the alpha beta T-cell receptor antibody T10B9 as graft-versus-host disease (GVHD) prophylaxis. All patients also received posttransplant cyclosporine therapy. Twenty-eight of 48 patients were mismatched with their donors for one or more HLA-A, B, DR, or DQ loci by either serology or high-resolution oligonucleotide genotyping. Nine of 28 were mismatched at multiple HLA loci. Durable engraftment was achieved in 94% (45/48) of patients. The actuarial probability of developing grades II to IV and grades III to IV acute GVHD were 39.6% (95% confidence interval (CI) 26.9 to 53.0) and 8.3% (95% CI 6.1 to 10.9) for the entire cohort. There was no difference in the incidence of grades II to IV acute GVHD between patients receiving matched (36.8%) or mismatched (41.4%) marrow grafts (P = .77). The actuarial probability of relapse at 2 years was 8.8% (95% CI 2.1 to 21.6) for the entire cohort and 18% (95% CI 4 to 41) for patients transplanted in either the accelerated or blast crisis phase (advanced disease). One cytogenetic relapse has occurred among patients transplanted in the chronic phase. The probability of disease-free survival at 2 years was 52% (95% CI 24 to 70) for patients transplanted in chronic phase and 46% (95% CI 25 to 73) for patients transplanted with advanced disease. No difference in disease-free survival was observed between patients receiving matched (49%) or mismatched (51%) marrow grafts (P = .90). This study shows that patients receiving unrelated T-cell-depleted marrow grafts for CML can achieve durable engraftment with a low incidence of severe GVHD and apparent preservation of graft-versus-leukemia reactivity. These data also suggest that T-cell depletion may allow patients who might otherwise experience unacceptable toxicity from GVHD-related complications caused by older age or increased HLA disparity to benefit from unrelated marrow grafts.