Distinct Biomarker Profiles in Ex Vivo T Cell Depletion Graft Manipulation Strategies: CD34+ Selection versus CD3+/19+ Depletion in Matched Sibling Allogeneic Peripheral Blood Stem Cell Transplantation.

Various approaches have been developed for ex vivo T cell depletion in allogeneic stem cell transplantation to prevent graft-versus-host disease (GVHD). Direct comparisons of T cell depletion strategies have not been well studied, however. We evaluated cellular and plasma biomarkers in 2 different graft manipulation strategies, CD3+CD19+ cell depletion (CD3/19D) versus CD34+ selection (CD34S), and their associations with clinical outcomes. Identical conditions, including the myeloablative preparative regimen, HLA-identical sibling donor, GVHD prophylaxis, and graft source, were used in the 2 cohorts. Major clinical outcomes were similar in the 2 groups in terms of overall survival, nonrelapse mortality, and cumulative incidence of relapse; however, the cumulative incidence of acute GVHD trended to be higher in the CD3/19D cohort compared with the CD34S cohort. A distinct biomarker profile was noted in the CD3/19D cohort: higher levels of ST2, impaired Helios- FoxP3+Treg reconstitution, and rapid reconstitution of naïve, Th2, and Th17 CD4 cells in the early post-transplantation period. In vitro graft replication studies confirmed that CD3/19D disproportionately depleted Tregs and other CD4 subset repertoires in the graft. This study confirms the utility of biomarker monitoring, which can be directly correlated with biological consequences and possible future therapeutic indications.

Acquired RhD mosaicism identifies fibrotic transformation of thrombopoietin receptor-mutated essential thrombocythemia.

BACKGROUND: Acquired copy-neutral loss of heterozygosity has been described in myeloid malignant progression with an otherwise normal karyotype. CASE REPORT: A 65-year-old woman with MPL-mutated essential thrombocythemia and progression to myelofibrosis was noted upon routine pretransplant testing to have mixed field reactivity with anti-D and an historic discrepancy in RhD type. The patient had never received transfusions or transplantation. RESULTS: Gel immunoagglutination revealed group A red blood cells and a mixed-field reaction for the D phenotype, with a predominant D-negative population and a small subset of circulating red blood cells carrying the D antigen. Subsequent genomic microarray single nucleotide polymorphism profiling revealed copy-neutral loss of heterozygosity of chromosome 1 p36.33-p34.2, a known molecular mechanism underlying fibrotic progression of MPL-mutated essential thrombocythemia. The chromosomal region affected by this copy-neutral loss of heterozygosity encompassed the RHD, RHCE, and MPL genes. We propose a model of chronological molecular events that is supported by RHD zygosity assays in peripheral lymphoid and myeloid-derived cells. CONCLUSION: Copy-neutral loss of heterozygosity events that lead to clonal selection and myeloid malignant progression may also affect the expression of adjacent unrelated genes, including those encoding for blood group antigens. Detection of mixed-field reactions and investigation of discrepant blood typing results are important for proper transfusion support of these patients and can provide useful surrogate markers of myeloproliferative disease progression.

Bone marrow mesenchymal stromal cells to treat tissue damage in allogeneic stem cell transplant recipients: correlation of biological markers with clinical responses.

Bone marrow mesenchymal stromal cells (BMSCs) have been used to treat acute graft-versus-host disease (GVHD) and other complications following allogeneic hematopoietic stem cell transplantation (SCT). We conducted a phase I trial using third party, early passage BMSCs for patients with steroid-refractory GVHD, tissue injury, or marrow failure following SCT to investigate safety and efficacy. To identify mechanisms of BMSC immunomodulation and tissue repair, patients were serially monitored for plasma GVHD biomarkers, cytokines, and lymphocyte phenotype. Ten subjects were infused a fixed dose of 2 × 10(6) BMSCs/kg intravenously weekly for three doses. There was no treatment-related toxicity (primary endpoint). Eight subjects were evaluable for response at 4 weeks after the last infusion. Five of the seven patients with steroid-refractory acute GVHD achieved a complete response, two of two patients with tissue injury (pneumomediastinum/pneumothorax) achieved resolution but there was no response in two subjects with delayed marrow failure. Rapid reductions in inflammatory cytokines were observed. Clinical responses correlated with a fall in biomarkers (Reg 3α, CK18, and Elafin) relevant for the site of GVHD or tissue injury. The GVHD complete responders survived significantly longer and had higher baseline absolute lymphocyte and central memory CD4 and CD8 counts. Cytokine changes also segregated with survival. These results confirm that BMSCs are associated with rapid clinical and biomarker responses in GVHD and tissue injury. However, BMSCs were ineffective in patients with prolonged GVHD with lower lymphocyte counts, which suggest that effective GVHD control by BMSCs requires a relatively intact immune system.

Repair of impaired pulmonary function is possible in very-long-term allogeneic stem cell transplantation survivors.

Both early- and late-onset noninfectious pulmonary injury are important contributors to the nonrelapse mortality seen after allogeneic stem cell transplantation (allo-SCT), particularly in subjects conditioned with high-dose total body irradiation (TBI). To characterize the kinetics of recovery from pulmonary injury in long-term survivors, we collected data on 138 subjects who survived > 3 years (median survival, 10.2 years) after predominantly TBI-based allo-SCT from their HLA-matched siblings. Baseline pulmonary function tests served as the reference for subsequent measurements at 3, 5, 10, and 15 years for each survivor. The only parameter showing a clinically and statistically significant decline post-transplant was adjusted diffusion capacity of lung for carbon monoxide (DLCO), which reached a nadir at 5 years but surprisingly normalized at the 10-year mark. Multivariable modeling identified chronic graft-versus-host disease (P < .02) and abnormal baseline-adjusted DLCO (P < .03) as the only significant factors associated with the decline in adjusted DLCO at 5 years but excluded smoking, conditioning intensity, baseline C-reactive protein level, TBI dose to the lungs, disease, and demographic variables. In conclusion, pulmonary injury as monitored by the adjusted DLCO continues to deteriorate in the first 5 years after allo-SCT but recovers at 10 years.

Evolution of the donor T-cell repertoire in recipients in the second decade after allogeneic stem cell transplantation.

After allogeneic stem cell transplantation (SCT), T lymphocyte function is reestablished from the donor's postthymic T cells and through thymic T-cell neogenesis. The immune repertoire and its relation to that of the donor have not been characterized in detail in long-term adult SCT survivors. We studied 21 healthy patients in their second decade after a myeloablative SCT for hematologic malignancy (median follow-up, 12 years). Immune profiles were compared with donor samples cryopreserved at transplant and beyond 10 years from SCT. Only one recipient was on continuing immunosuppression. Compared with the donor at transplant, there was no significant difference in CD4, CD8, natural killer, and B-cell blood counts. However, compared with donors, recipients had significantly fewer naive T cells, lower T-cell receptor excision circle levels, fewer CD4 central memory cells, more effector CD8(+) cells, and more regulatory T cells. TCR repertoire analysis showed no significant difference in complexity of TCRVß spectratype between recipients and donors, although spectratype profiles had diverged with both gain and loss of donor repertoire peaks in the recipient. In conclusion, long-term allogeneic SCT survivors have subtle defects in their immune profile consistent with defective thymic function but compatible with normal health. This study is registered at http://www.clinicaltrials.gov as NCT00106925.

Selectively T cell-depleted allografts from HLA-matched sibling donors followed by low-dose posttransplantation immunosuppression to improve transplantation outcome in patients with hematologic malignancies.

We evaluated a photodepletion technique to selectively deplete host-reacting T cells from human leukocyte antigen (HLA)-matched sibling stem cell transplantations with the goal of reducing posttransplantation immunosuppression to improve antimalignancy effects postallografting. Donor lymphocytes were stimulated with irradiated expanded recipient T lymphocytes in an ex vivo mixed lymphocyte reaction. Alloactivated T cells preferentially retaining the photosensitizer 4,5-dibromorhodamine 123 (TH9402) were eliminated by exposure to visible light. Twenty-four patients with hematologic malignancies (16 high risk) conditioned with fludarabine, cyclophosphamide, and totalbody irradiation received a CD34-selected stem cell allograft from an HLA-matched sibling along with 5 × 10(6)/kg selectively depleted donor T cells. Low-dose cyclosporine was used for posttransplantation immunosuppression. Eleven patients survived at a median of 30 months. Probabilities (± SEM) for overall and disease-free survival are 39% ± 12% and 30% ± 12%, respectively, whereas grade III-IV acute graft-versus-host disease (aGVHD) was 13% ± 7%. Six patients relapsed, with a relapse probability of 27% ± 10%. These results suggest that selectively photodepleted allografts in matched sibling transplantations followed by low-dose immunosuppression may protect against severe aGVHD but is associated with delayed immune recovery.

Favorable outcomes in patients surviving 5 or more years after allogeneic hematopoietic stem cell transplantation for hematologic malignancies.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for some hematologic malignancies. As the overall number of survivors continues to increase, studies systematically examining outcomes in long-term survivors are needed. We studied the clinical and quality-of-life outcomes in HSCT recipients surviving 5 or more years from HSCT. Since 1993, 262 patients with hematologic malignancies received a T cell-depleted myeloablative HSCT from an HLA-identical sibling at a single center. Ninety-two survived beyond 5 years from HSCT (median follow-up 9.4 years, range: 5.1-15.3). Median age at transplantation was 35 years (range: 10-56). Twenty-two (24%) received a bone marrow transplant, and 70 (76%) received a peripheral blood HSCT. Of the 92 survivors, 60 completed quality-of-life measures. The main outcomes examined were chronic graft-versus-host-disease, disease relapse, survival, health-related quality-of-life (HRQL) (Functional Assessment of Cancer Therapy-General), physical and mental health (SF-36), and symptom experience (Rotterdam Symptom Checklist). Seventy-five (82%) of 92 survivors no longer required systemic immunosuppressive treatment. Four (4.3%) relapsed with leukemia at a median of 8.5 years (range: 6.2-14.0) after HSCT. Four (4.3%) died between 7.4 and 13.4 years post-HSCT (1 relapse, 1 lung cancer, 1 pneumonia, 1 brain hemorrhage). Most survivors beyond 5 years had an excellent performance status with no difference in physical and mental health and higher HRQL scores (P = .02) compared with population norms. Although physical and psychologic symptom distress was low, those with higher symptom distress experienced inferior HRQL. These results show that 5 or more years after T cell-depleted HSCT for hematologic malignancy most individuals survive disease free with an excellent performance status, preserved physical and psychological health, and excellent HRQL.

Framingham Risk Score Is an Ineffective Screening Strategy for Coronary Heart Disease in Long-Term Allogeneic Hematopoietic Cell Transplant Survivors.

Long-term allogeneic hematopoietic cell transplant (allo-HCT) survivors suffer an elevated risk of coronary heart disease (CHD). We conducted a prospective, nonrandomized, cross-sectional study to screen asymptomatic survivors at a single allo-HCT center using cardiac computed tomography (CT) involving coronary CT angiography (CCTA) and the coronary artery calcium (CAC) score. Seventy-nine subjects with a median age of 39 years at allo-HCT and a median follow-up interval of 8 years were evaluated for CHD by Framingham Risk Score (FRS) and cardiac CT. CHD was detected in 33 of 79 (42%) subjects; 91% of lesions were nonobstructive, 19.5% of were noncalcified and 30% had associated valvular calcification. Overall, CAC was significantly superior to FRS in detecting early CHD in allo-HCT survivors [∆C = 0.25; P < 0.0001]. While both FRS and CAC were highly, >95% specific, FRS had a sensitivity, positive and negative predictive values of only 28% (95% CI, 14%-47%), 90% (95% CI, 55%-100%) and 60% (95% CI, 47%-73%), respectively. In contrast, the sensitivity, positive and negative predictive values of CAC were 78% (95% CI, 60%-91%), 96% (95% CI, 80%-100%) and 83% (95% CI, 69%-93%), respectively. Significantly, cardiac CT detected CHD in 23 of the 68 (34%) survivors deemed to have a low Framingham risk. Radiation exposure during cardiac CT was negligible, and there were no adverse events. In conclusion, CAC score with or without CCTA is a safe, feasible and sensitive screening technique for CHD. The FRS greatly underestimates CHD in allo-HCT survivors.

Prolonged chronic graft-versus-host disease is a risk factor for thyroid failure in long-term survivors after matched sibling donor stem cell transplantation for hematologic malignancies.

We studied thyroid function in 81 long-term survivors of allogeneic stem cell transplantation (allo-SCT), with a median follow-up of 84 months (range, 45 to 166 months). Median age at transplantation was 35 years (range, 6 to 66). Seventy-two of the patients received a total body irradiation (TBI)-containing conditioning regimen (n = 23, 12 Gy; n = 49, 13 Gy). Twenty-one of the patients (25.9%) had subclinical hypothyroidism, and 9 (11.1%) developed overt hypothyroidism at a median of 28 months (range, 3 to 78 months) after allo-SCT. Multivariate logistic regression analysis demonstrated that prolonged immunosuppressive therapy (IST) was significantly associated with subclinical hypothyroidism (odds ratio [OR] = 3.8) and overt hypothyroidism (OR = 2.6). Antithyroglobulin and thyroid peroxidase antibody were detected in 12 of 60 patients tested (20%). No correlation was found between the occurrence of thyroid antibodies and hypothyroidism (P = .13) or chronic graft-versus-host disease (cGVHD) (P = .55). In conclusion, thyroid dysfunction is relatively common after allo-SCT and is more likely to occur in patients receiving prolonged IST for cGVHD; however, thyroid dysfunction does not appear to be related to an antibody-mediated autoimmune process.

Cellular immune profiling after sequential clofarabine and lenalidomide for high risk myelodysplastic syndromes and acute myeloid leukemia.

Patients with high risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) are commonly older with multiple co-morbidities, rendering them unsuitable for intensive induction chemotherapy or transplantation. We report preliminary cellular immune profiling of four cases receiving sequential clofarabine and lenalidomide for high risk MDS and AML in a phase I study. Our results highlight the potential of immune profiling for monitoring immune-modifying agents in high risk MDS and AML.

Clinical and biological predictors of outcome following relapse of CML post-allo-SCT.

Although there are now fewer allo-SCTs performed for CML, leukemic relapse post transplant remains a persistent problem. To better define clinical and biological parameters determining postrelapse outcome, we studied 59 patients with CML relapsing after HLA-identical sibling allo-SCT between 1993 and 2008. Eighteen (30.5%) were transplanted in advanced phase and 41 (69.5%) in chronic phase. With a median follow-up from relapse of 7.9 years, 5-year post relapse survival (PRS) was 62%. Multivariate analysis found disease status at transplant, time to diagnosis of relapse from transplant and pretransplant tyrosine kinase inhibitor (TKI) use as significant factors associated with PRS. Analysis of BCR-ABL transcript expression in the hematopoietic progenitor compartment was performed in 36 patients (22 relapsed, 8 non-relapsed and 6 TKI alone controls). Patients with BCR-ABL expression in their early hematopoietic stem cell compartment (Lineage(-)CD34(+)CD38(-)CD90(+)) had worse survival irrespective of the disease status. We conclude that disease status remains the strongest clinical prognostic factor for PRS in CML following allo-SCT. The persistence of BCR-ABL expression in the progenitor cell compartment in some patients after SCT emphasizes the need to target CML-leukemia stem cells.

Male survivors of allogeneic hematopoietic stem cell transplantation have a long term persisting risk of cardiovascular events.

Long-term survivors of allogeneic stem cell transplantation (SCT) have increased risk of cardiovascular disease. We retrospectively studied cardiovascular risk factors (CVRFs) in 109 SCT survivors (62 males, 47 females; median age 34 years) five years or more after bone marrow (15) or T cell-depleted peripheral blood (94) SCT for CML (56), acute leukemia (29), MDS (13), and others (11). One death and two cardiovascular events were reported. At five and ten years after SCT, respectively, 44% and 52% had abnormal lipid profiles; 23% of 5-year survivors met the Adult Treatment Panel III threshold for dyslipidemia treatment, which is substantially higher than the age-matched general population. There were significant increases in prevalence of hypertension (p < 0.001), diabetes (p = 0.018), and body mass index (p = 0.044) after SCT compared with baseline. The Framingham general cardiovascular risk score (FGCRS) in males at five years after SCT projected a doubling (median 10.4% vs. 5.4%) in the 10-year risk of cardiovascular events. Females received HRT after SCT, and none had increased FGCRS. Chronic GVHD and C-reactive protein were not associated with CVRF at any time. All CVRFs stabilized between five and ten years after SCT. Thus, SCT survivors have sustained elevations in CVRFs. Males have a significantly increased risk of cardiovascular events in their second and third decade after SCT.