Long-term prognosis for 1-year relapse-free survivors of CD34+ cell-selected allogeneic hematopoietic stem cell transplantation: a landmark analysis.

CD34+ cell selection significantly improves GvHD-free survival in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, specific information regarding long-term prognosis and risk factors for late mortality after CD34+ cell-selected allo-HSCT is lacking. We conducted a single-center landmark analysis in 276 patients alive without relapse 1 year after CD34+ cell-selected allo-HSCT for AML (n=164), ALL (n=33) or myelodysplastic syndrome (n=79). At 5 years' follow-up after the 1-year landmark (range 0.03-13 years), estimated relapse-free survival (RFS) was 73% and overall survival (OS) 76%. The 5-year cumulative incidence of relapse and non-relapse mortality (NRM) were 11% and 16%, respectively. In multivariate analysis, Hematopoietic Cell Transplantation Comorbidity Index score⩾3 correlated with marginally worse RFS (hazard ratio (HR) 1.78, 95% confidence interval (CI) 0.97-3.28, P=0.06) and significantly worse OS (HR 2.53, 95% CI 1.26-5.08, P=0.004). Despite only 24% of patients with acute GvHD within 1 year, this also significantly correlated with worse RFS and OS, with increasing grades of acute GvHD associating with increasingly poorer survival on multivariate analysis (P<0.0001). Of 63 deaths after the landmark, GvHD accounted for 27% of deaths and was the most common cause of late mortality, followed by relapse and infection. Although prognosis is excellent for patients alive without relapse 1 year after CD34+ cell-selected allo-HSCT, risks of late relapse and NRM persist, particularly due to GvHD.

Soluble and membrane-bound interleukin (IL)-15 Rα/IL-15 complexes mediate proliferation of high-avidity central memory CD8+ T cells for adoptive immunotherapy of cancer and infections.

The lack of persistence of infused T cells is a principal limitation of adoptive immunotherapy in man. Interleukin (IL)-15 can sustain memory T cell expansion when presented in complex with IL-15Rα (15Rα/15). We developed a novel in-vitro system for generation of stable 15Rα/15 complexes. Immunologically quantifiable amounts of IL-15 were obtained when both IL-15Rα and IL-15 genes were co-transduced in NIH 3T3 fibroblast-based artificial antigen-presenting cells expressing human leucocyte antigen (HLA) A:0201, ß2 microglobulin, CD80, CD58 and CD54 [A2-artificial antigen presenting cell (AAPC)] and a murine pro-B cell line (Baf-3) (A2-AAPC15Rα/15 and Baf-315Rα/15 ). Transduction of cells with IL-15 alone resulted in only transient expression of IL-15, with minimal amounts of immunologically detectable IL-15. In comparison, cells transduced with IL-15Rα alone (A2-AAPCRα ) demonstrated stable expression of IL-15Rα; however, when loaded with soluble IL-15 (sIL-15), these cells sequestered 15Rα/15 intracellularly and also demonstrated minimal amounts of IL-15. Human T cells stimulated in vitro against a viral antigen (CMVpp65) in the presence of 15Rα/15 generated superior yields of high-avidity CMVpp65 epitope-specific T cells [cytomegalovirus-cytotoxic T lymphocytes (CMV-CTLs)] responding to ≤ 10- 13 M peptide concentrations, and lysing targets cells at lower effector : target ratios (1 : 10 and 1 : 100), where sIL-15, sIL-2 or sIL-7 CMV-CTLs demonstrated minimal or no activity. Both soluble and surface presented 15Rα/15, but not sIL-15, sustained in-vitro expansion of CD62L+ and CCR7+ central memory phenotype CMV-CTLs (TCM ). 15Rα/15 complexes represent a potent adjuvant for augmenting the efficacy of adoptive immunotherapy. Such cell-bound or soluble 15Rα/15 complexes could be developed for use in combination immunotherapy approaches.

Virus-specific T-cell banks for 'off the shelf' adoptive therapy of refractory infections.

Adoptive immunotherapy with transplant donor-derived virus-specific T cells has emerged as a potentially curative approach for the treatment of drug-refractory EBV+lymphomas as well as CMV and adenovirus infections complicating allogeneic hematopoietic cell transplants. Adoptive transfer of HLA partially matched virus-specific T cells from healthy third party donors has also shown promise in the treatment of these conditions, with disease response rates of 50-76% and strikingly low incidences of toxicity or GVHD recorded in initial trials. In this review, we examine the reported experience with transplant donor and third party donor-derived virus-specific T cells, identifying characteristics of the viral pathogen, the T cells administered and the diseased host that contribute to treatment response or failure. We also describe the characteristics of virus-specific T-cell lines in our center's bank and the frequency with which in vitro culture promotes expansion of immunodominant T cells specific for epitopes that are presented by a limited array of prevalent HLA alleles, which facilitates their broad applicability for treatment.

Late effects in patients with Fanconi anemia following allogeneic hematopoietic stem cell transplantation from alternative donors.

Hematopoietic stem cell transplantation (HSCT) is curative for hematological manifestations of Fanconi anemia (FA). We performed a retrospective analysis of 22 patients with FA and aplastic anemia, myelodysplastic syndrome or acute myelogenous leukemia who underwent a HSCT at Memorial Sloan Kettering Cancer Center and survived at least 1 year post HSCT. Patients underwent either a TBI- (N=18) or busulfan- (N=4) based cytoreduction followed by T-cell-depleted transplants from alternative donors. Twenty patients were alive at time of the study with a 5- and 10-year overall survival of 100 and 84% and no evidence of chronic GvHD. Among the 18 patients receiving a TBI-based regimen, 11 (61%) had persistent hemochromatosis, 4 (22%) developed hypothyroidism, 7 (39%) had insulin resistance and 5 (27%) developed hypertriglyceridemia after transplant. Eleven of 16 evaluable patients (68%), receiving TBI, developed gonadal dysfunction. Two patients who received a TBI-based regimen died of squamous cell carcinoma. One patient developed hemochromatosis, hypothyroidism and gonadal dysfunction after busulfan-based cytoreduction. TBI appears to be a risk factor for malignant and endocrine late effects in the FA host. Multidisciplinary follow-up of patients with FA (including cancer screening) is essential for early detection and management of late complications, and improving long-term outcomes.

T-cell depleted allogeneic hematopoietic cell transplants as a platform for adoptive therapy with leukemia selective or virus-specific T-cells.

Allogeneic hematopoietic cell transplants adequately depleted of T-cells can reduce or prevent acute and chronic GVHD in both HLA-matched and haplotype-disparate hosts, without post-transplant prophylaxis with immunosuppressive drugs. Recent trials indicate that high doses of CD34+ progenitors from G-CSF mobilized peripheral blood leukocytes isolated and T-cell depleted by immunoadsorption to paramagnetic beads, when administered after myeloablative conditioning with TBI and chemotherapy or chemotherapy alone can secure consistent engraftment and abrogate GVHD in patients with acute leukemia without incurring an increased risk of a recurrent leukemia. Early clinical trials also indicate that high doses of in vitro generated leukemia-reactive donor T-cells can be adoptively transferred and can induce remissions of leukemia relapse without GVHD. Similarly, virus-specific T-cells generated from the transplant donor or an HLA partially matched third party, have induced remissions of Rituxan-refractory EBV lymphomas and can clear CMV disease or viremia persisting despite antiviral therapy in a high proportion of cases. Analyses of treatment responses and failures illustrate both the advantages and limitations of donor or banked, third party-derived T-cells, but underscore the potential of adoptive T-cell therapy in the absence of ongoing immunosuppression.

Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential.

WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2)-restricted peptide derived from Wilms tumor protein 1 (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single-chain variable fragment (scFv) antibody specific for the T-cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant (KD)=3 nm) and exquisite specificity towards its targeted epitope or HLA-A2(+)/WT1(+) tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD=2 pm) binding to the T-cell epitope and to tumor targets and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor-expressing human T- or NK-92-MI-transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T-cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high-affinity TCR-like antibodies could be rapidly explored for potential clinical development.

Comparison of outcomes at two institutions of patients with ALL receiving ex vivo T-cell-depleted or unmodified allografts.

We compared outcomes of adult patients receiving T-cell-depleted (TCD) hematopoietic SCT (HCT) without additional GVHD prophylaxis at Memorial Sloan Kettering Cancer Center (MSKCC, N=52), with those of patients receiving conventional grafts at MD Anderson Cancer Center (MDACC, N=115) for ALL in CR1 or CR2. Patients received myeloablative conditioning. Thirty-nine patients received anti-thymocyte globulin at MSKCC and 29 at MDACC. Cumulative incidence of grades 2-4 acute (P=0.001, 17.3% vs 42.6% at 100 days) and chronic GVHD (P=0.006, 13.5% vs 33.4% at 3 years) were significantly lower in the TCD group. The non-relapse mortality at day 100, 1 and 3 years was 15.4, 25.0 and 35.9% in the TCD group and 9.6, 23.6 and 28.6% in the unmodified group (P=0.368). There was no difference in relapse (P=0.107, 21.3% vs 35.5% at 3 years), OS (P=0.854, 42.6% vs 43.0% at 3 years) or RFS (P=0.653, 42.8% vs 35.9% at 3 years). In an adjusted model, age >50, cytogenetics and CR status were associated with inferior RFS (hazard ratio (HR)=2.16, P=0.003, HR=1.77, P=0.022, HR=2.47, P<0.001), whereas graft type was NS (HR=0.90, P=0.635). OS and RFS rates are similar in patients undergoing TCD or conventional HCT, but TCD effectively reduces the rate of GVHD.

Artificial Antigen Presenting Cells: An Off the Shelf Approach for Generation of Desirable T-Cell Populations for Broad Application of Adoptive Immunotherapy.

Adoptive transfer of antigen specific T-cells can lead to eradication of cancer and viral infections. The broad application of this approach has further been hampered by the limited availability of adequate numbers of T-cells for treatment in a timely manner. This has led to efforts for the development of efficient methods to generate large numbers of T-cells with specificity for tumor or viral antigens that can be harnessed for use in cancer therapy. Recent studies have demonstrated that during encounter with tumor antigen, the signals delivered to T-cells by professional antigen-presenting cells can affect T-cell programming and their subsequent therapeutic efficacy. This has stimulated efforts to develop artificial antigen-presenting cells that allow optimal control over the signals provided to T-cells. In this review, we will discuss the cellular artificial antigen-presenting cell systems and their use in T-cell adoptive immunotherapy for cancer and infections.

Graft-versus-host disease after double-unit cord blood transplantation has unique features and an association with engrafting unit-to-recipient HLA match.

Manifestations of and risk factors for graft-versus-host disease (GVHD) after double-unit cord blood transplantation (DCBT) are not firmly established. We evaluated 115 DCBT recipients (median age, 37 years) who underwent transplantation for hematologic malignancies with myeloablative or nonmyeloablative conditioning and calcineurin inhibitor/mycophenolate mofetil immunosuppression. Incidence of day 180 grades II to IV and III to IV acute GVHD (aGVHD) were 53% (95% confidence interval, 44 to 62) and 23% (95% confidence interval, 15 to 31), respectively, with a median onset of 40 days (range, 14 to 169). Eighty percent of patients with grades II to IV aGVHD had gut involvement, and 79% and 85% had day 28 treatment responses to systemic corticosteroids or budesonide, respectively. Of 89 engrafted patients cancer-free at day 100, 54% subsequently had active GVHD, with 79% of those affected having persistent or recurrent aGVHD or overlap syndrome. Late GVHD in the form of classic chronic GVHD was uncommon. Notably, grades III to IV aGVHD incidence was lower if the engrafting unit human leukocyte antigen (HLA)-A, -B, -DRB1 allele match was >4/6 to the recipient (hazard ratio, 0.385; P = .031), whereas engrafting unit infused nucleated cell dose and unit-to-unit HLA match were not significant. GVHD after DCBT was common in our study, predominantly affected the gut, and had a high therapy response, and late GVHD frequently had acute features. Our findings support the consideration of HLA- A,-B,-DRB1 allele donor-recipient (but not unit-unit) HLA match in unit selection, a practice change in the field. Moreover, new prophylaxis strategies that target the gastrointestinal tract are needed.

Second-line age-adjusted International Prognostic Index in patients with advanced non-Hodgkin lymphoma after T-cell depleted allogeneic hematopoietic SCT.

T-cell depleted allogeneic hematopoietic SCT (TCD-HSCT) have shown durable disease-free survival with a low risk of GVHD in patients with AML. We investigated this approach in 61 patients with primary refractory or relapsed non-Hodgkin lymphoma (NHL), who underwent TCD-HSCT from January 1992 through September 2004. Patients received myeloablative cytoreduction consisting of hyperfractionated total body irradiation, followed by either thiotepa and cyclophosphamide (45 patients) or thiotepa and fludarabine (16 patients). We determined the second-line age-adjusted International Prognostic Index score (sAAIPI) before transplant transplant. Median follow-up of surviving patients is 6 years. The 10-year OS and EFS were 50% and 43%, respectively. The relapse rate at 10 years was 21% in patients with chemosensitive disease and 52% in those with resistant disease at time of HSCT. Nine of the 18 patients who relapsed entered a subsequent CR. OS (P=0.01) correlated with the sAAIPI. The incidence of grades II-IV acute GVHD was 18%. We conclude that allogeneic TCD-HSCT can induce high rates of OS and EFS in advanced NHL with a low incidence of GVHD. Furthermore, the sAAIPI can predict outcomes and may be used to select the most appropriate patients for this type of transplant.

Long-term follow-up of patients treated with daclizumab for steroid-refractory acute graft-vs-host disease.

Daclizumab has been shown to have activity in acute GVHD, but appears to be associated with an increased risk of infection. To investigate further the long-term effects of daclizumab, we performed a retrospective review of 57 patients who underwent an allogeneic hematopoietic stem cell transplant from January 1993 through June 2000 and were treated with daclizumab for steroid-refractory acute GVHD. The median number of daclizumab doses given was 5 (range 1-22). GVHD was assessed at baseline, days 15, 29 and 43. By day 43, 54% patients had an improvement in their overall GVHD score, including 76% patients aged < or =18. Opportunistic infections developed in 95% patients. Forty-three patients (75%) died following treatment with daclizumab. The causes of death included active GVHD and infection (79%), active GVHD (5%), chronic GVHD (2%) and relapse (14%). Patients with grade 3-4 GVHD had a significantly shorter median survival than patients with grade 1-2 GVHD (2.0 vs 5.1 months, P=0.001). Daclizumab has no infusion-related toxicity, is active in steroid-refractory GVHD, especially among pediatric patients, but is associated with significant morbidity and mortality due to infectious complications. Careful patient selection and aggressive prophylaxis against viral and fungal infections are recommended.

Predictors for persistent cytomegalovirus reactivation after T-cell-depleted allogeneic hematopoietic stem cell transplantation.

UNLABELLED: Cytomegalovirus (CMV) reactivation occurs in up to 60% of CMV-seropositive recipients after allogeneic hematopoietic stem cell transplantation (HSCT). The incidence of CMV disease among T-cell-depleted HSCT patients has been reported from 5-15%. The incidence of reactivation refractory to antivirals in this population is not well studied. METHODS: In this retrospective study we characterized the outcome of CMV reactivation in a cohort of 255 adult and pediatric patients who underwent T-cell-depleted HSCT at Memorial Sloan-Kettering Cancer Center from September 1999 through August 2004. CMV infection was monitored by the pp65 antigenemia assay (CMV Ag). Persistent reactivation was defined as antigenemia positivity >21 days on antiviral therapy. RESULTS: Of 118 CMV-seropositive recipients, 69 (58.4%) had reactivated CMV. Twenty of 69 (29%) developed persistent reactivation at first episode of reactivation, and 7 (10%) in subsequent episode. All patients with persistent reactivation received >/=2 antivirals and CMV hyperimmune globulin; 45% received combination antiviral therapy. The median duration of persistent reactivation was 98 days, range 31-256 days. In multivariate analysis, maximum CMV Ag >25 cells/slide was associated with persistent reactivation (odds ratio 16.2%, 95% confidence interval 4-64, P<0.0001). CMV disease occurred in 6/27 (22%) patients with persistent reactivation. Patients with persistent reactivation had lower CD4(+) and CD8(+) lymphocyte counts compared with those with non-persistent reactivation at day +90 post HSCT (P=0.01 and 0.02, respectively). CONCLUSIONS: Persistent reactivation occurred in 39% of T-cell-depleted HSCT despite treatment with currently available antivirals. Maximum CMV Ag >25 cells/slide was associated with persistent CMV reactivation. More effective treatment modalities are needed for this high-risk population to reduce CMV-associated morbidity and mortality.

Final height in pediatric patients after hyperfractionated total body irradiation and stem cell transplantation.

Impaired linear growth has been shown to occur in individuals treated during childhood with single-dose and fractionated total body irradiation (TBI) before stem cell transplantation. Our objective was to describe the final heights attained and patient/treatment factors correlating with final height in a cohort of childhood cancer survivors treated with hyperfractionated TBI (total dose 1375 or 1500 cGy). Thirty individuals (18 men) were included in the study. The mean final height standard deviation score (s.d.s.) was -1.9 +/- 0.2, significantly lower than height s.d.s. at TBI (-0.2 +/- 0.2, P < 0.001). Final height s.d.s. was significantly correlated with age at diagnosis, age at TBI and target height (P = 0.04, P < 0.001, P < 0.001, respectively). Treatment with growth hormone (GH) (n = 7) maintained mean height s.d.s. at -2.0 from the onset of GH therapy until attainment of final height. The mean final sitting height s.d.s. was -2.2 +/- 0.2 (n = 16), significantly shorter than mean final standing height s.d.s. (P < 0.01). In conclusion, treatment with hyperfractionated TBI is associated with a reduction in standing height and an even greater reduction in sitting height. Final height after hyperfractionated TBI was similar to that reported after fractionated TBI.

Invasive adenoviral infections in T-cell-depleted allogeneic hematopoietic stem cell transplantation: high mortality in the era of cidofovir.

BACKGROUND: Adenovirus (ADV) infection occurs in 5-21% of allogeneic hematopoietic stem cell transplants (HSCT). Symptomatic enteritis and hemorrhagic cystitis may be encountered but are seldom fatal. In contrast, mortality rates of up to 75% are reported for adenoviral pneumonia or hepatitis. Cidofovir is currently being increasingly used for treatment of adenoviral infections after HSCT. The efficacy of cidofovir in patients with invasive adenoviral infection is not established. FINDINGS: We reviewed 687 adult and pediatric patients who received allogeneic HSCT at our institution from 1998 through June 2005. ADV was isolated from 64 (9.3%) patients. Eleven patients received cidofovir for invasive disease occurring at median 39 days (range 3-145) post HSCT. The median age was 40 (range 6-61) years. Seventy-three percent received a T-cell-depleted graft and 18% had grade 3-4 graft-versus-host disease (GVHD) of the gut. Three out of 3 (100%) patients with adenoviral pneumonia died. One patient with hepatitis, cholecysitis, and viremia cleared the infection after 3 months. Two out of 7 (28.6%) patients with hemorrhagic colitis or cystitis died of ADV (1 with extensive GVHD). CONCLUSION: Mortality rates of ADV pneumonitis after allogeneic HSCT remain high in the era of cidofovir. Clinical trials are needed to evaluate management strategies for this life-threatening infection.

Cytoreduction with iodine-131-anti-CD33 antibodies before bone marrow transplantation for advanced myeloid leukemias.

The monoclonal antibodies M195 and HuM195 target CD33, a glycoprotein found on myeloid leukemia cells. When labeled with iodine-131 ((131)I), these antibodies can eliminate large disease burdens and produce prolonged myelosuppression. We studied whether (131)I-labeled M195 and HuM195 could be combined safely with busulfan and cyclophosphamide (BuCy) as conditioning for allogeneic BMT. A total of 31 patients with relapsed/refractory acute myeloloid leukemia (AML) (n=16), accelerated/myeloblastic chronic myeloid leukemia (CML) (n=14), or advanced myelodysplastic syndrome (n=1) received (131)I-M195 or (131)I-HuM195 (122-437 mCi) plus busulfan (16 mg/kg) and cyclophosphamide (90-120 mg/kg) followed by infusion of related-donor bone marrow (27 first BMT; four second BMT). Hyperbilirubinemia was the most common extramedullary toxicity, occurring in 69% of patients during the first 28 days after BMT. Gamma camera imaging showed targeting of the radioisotope to the bone marrow, liver, and spleen, with absorbed radiation doses to the marrow of 272-1470 cGy. The median survival was 4.9 months (range 0.3-90+ months). Three patients with relapsed AML remain in complete remission 59+, 87+, and 90+ months following bone marrow transplantation (BMT). These studies show the feasibility of adding CD33-targeted radioimmunotherapy to a standard BMT preparative regimen; however, randomized trials will be needed to prove a benefit to intensified conditioning with radioimmunotherapy.

N7: a novel multi-modality therapy of high risk neuroblastoma (NB) in children diagnosed over 1 year of age.

BACKGROUND: The N7 protocol for poor-risk neuroblastoma uses dose-intensive chemotherapy (as in N6 protocol [Kushner et al.: J Clin Oncol 12:2607-2613, 1994] but with lower dosing of vincristine) for induction, surgical resection and 2100 cGy hyperfractionated radiotherapy for local control, and for consolidation, targeted radioimmunotherapy with 131I-labeled anti-GD2 3F8 monoclonal antibody and immunotherapy with unlabeled/unmodified 3F8 (400 mg/m2). PROCEDURE: The chemotherapy consists of: cyclophosphamide 70 mg/kg/d x 2 and a 72-hr infusion of doxorubicin 75 mg/m2 plus vincristine 2 mg/m2, for courses 1, 2, 4, and 6; and cisplatin 50 mg/m2/d x 4 and etoposide 200 mg/m2/d x 3, for courses 3, 5, and 7. 131I-3F8 is dosed at 20 mCi/kg, which is myeloablative and therefore necessitates stem-cell support. RESULTS: Of the first 24 consecutive previously untreated patients more than 1 year old at diagnosis, 22 were stage 4 and two were unresectable stage 3 with MYCN amplification. Chemotherapy achieved CR/VGPR in 21 of 24 patients. Twenty patients to date have completed treatment with 131I-3F8, and 15 patients have completed all treatment. With a median follow-up of 19 months, 18 of 24 patients remain progression-free. CONCLUSIONS: Major toxicities were grade 4 myelosuppression and mucositis during chemotherapy, and self-limited pain and urticaria during antibody treatment. Late effects include hearing deficits and hypothyroidism.