Steroid-Refractory Acute GVHD: Predictors and Outcomes

Patients with steroid-resistant acute graft versus host disease (aGVHD) have a dismal prognosis, with mortality rates in excess of 90%. We sought to identify a subgroup of patients less likely to benefit from initial therapy with corticosteroids as well as the impact of response on day 14 on outcome. Retrospective evaluation was performed of patients with biopsy-proven aGVHD treated with corticosteroids after allogeneic HSCT at M.D. Anderson Cancer Center from 1998 through 2002 (N = 287). Overall response to first-line therapy on day 14 was 56%. Grade III-IV aGVHD and hyperacute GVHD were the most significant factors predicting failure. Patients who fail to respond to steroids by day 14 should be considered for clinical trials. Severity of aGVHD, hyperacute GVHD, and sex mismatch could be integrated into prognostic scoring systems which may allow for pretreatment identification of patients unlikely to benefit from standard therapy with corticosteroids

Complement-Binding Donor-Specific Anti-HLA Antibodies and Risk of Primary Graft Failure in Hematopoietic Stem Cell Transplantation

AbstractDetection of donor-specific anti-HLA antibodies (DSA) has been associated with graft rejection in all forms of transplantation. The mechanism by which DSA increase the risk of graft failure remains unclear. We hypothesized that complement-binding DSA are associated with engraftment failure in hematopoietic stem cell transplantation (HSCT) and analyzed 122 haploidentical transplant recipients tested prospectively for DSA. Retrospective analysis to detect C1q binding DSA (C1q+DSA) was performed on 22 allosensitized recipients. Twenty-two of 122 patients (18%) had DSA, 19 of which were women (86%). Seven patients with DSA (32%) rejected the graft. Median DSA level at transplant for patients who failed to engraft was 10,055 mean fluorescence intensity (MFI) versus 2065 MFI for those who engrafted (P = .007). Nine patients with DSA were C1q positive in the initial samples with median DSA levels of 15,279 MFI (range, 1554 to 28,615), compared with 7 C1q-negative patients with median DSA levels of 2471 MFI (range, 665 to 12,254) (P = .016). Of 9 patients who were C1q positive in the initial samples, 5 patients remained C1q positive at time of transplant (all with high DSA levels [median, 15,279; range, 6487 to 22,944]) and experienced engraftment failure, whereas 4 patients became C1q negative pretransplant and all engrafted the donor cells (P = .008). In conclusion, patients with high DSA levels (>5000 MFI) and complement-binding DSA antibodies (C1q positive) appear to be at much higher risk of primary graft failure. The presence of C1q+DSA should be assessed in allosensitized patients before HSCT. Reduction of C1q+DSA levels might prevent engraftment failure in HSCT

Deletion of the Short Arm of Chromosome 1 (del 1p) is a Strong Predictor of Poor Outcome in Myeloma Patients Undergoing an Autotransplant

AbstractSeveral chromosomal abnormalities detected by conventional cytogenetic analysis have an adverse impact on the outcome in myeloma patients. A wide spectrum of abnormalities involving chromosomes 1, 13, 14, and 17 has been described. We analyzed the outcome of 83 patients with clonal cytogenetic abnormalities, who underwent high-dose therapy and autologous stem cell transplantation for multiple myeloma at our institution. Clonal abnormalities were detected at diagnosis by conventional cytogenetic analysis in 83 patients. Patients underwent a single autologous transplant between April 2000 and May 2005. Preparative regimen was high-dose melphalan alone (73), or a combination of topotecan, melphalan, and cyclophosphamide (TMC = 10). The most commonly observed chromosomal abnormalities were deletion of chromosome 13 (32%), hyperdiploidy (21%), deletion of chromosome 1p (18%), and t (11; 14) in 7% patients. Median follow-up among surviving patients was 25.5 months. Median interval from diagnosis to autotransplant was 7.7 months (range: 2.5-52). Median progression-free survival (PFS) for the entire group was 19 months and the median overall survival (OS) was 52 months. On univariate analysis, both PFS and OS were significantly shorter in patients with deletion 1p (P = .001 and <.0001, respectively). Thirty-two patients whose cytogenetic abnormalities returned to normal prior to autotransplant had longer PFS and OS than patients with persistent abnormalities (P = .02 and .08, respectively). Deletion 1p is associated with a significantly shorter remission and survival in patients undergoing high-dose therapy and a single autologous transplant for myeloma

Mucus-degrading Bacteroides link carbapenems to aggravated graft-versus-host disease

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Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy

Cutaneous Manifestations of Chronic Graft-versus-Host Disease

AbstractCutaneous chronic graft versus host disease has traditionally been classified into lichenoid and scleroderma-like forms. However, the initial presentation is sometimes subtle and a variety of less common cutaneous manifestation may be prevalent. This clinical review focuses on the lesional morphology of chronic graft versus host disease, and presents a classification system that may prove useful in early diagnosis. In addition, this approach may help to facilitate the correlation of different morphologic entities with outcome and response to therapy

High Prevalence of Vitamin D Deficiency in Allogeneic Stem Cell Transplant Recipients

Abstract Vitamin D deficiency has long been understood to place patients at increased risk for reduced bone density and the development of pathological fractures. In addition to these classical manifestations, a wide body of emerging evidence suggests that Vitamin D deficiency may also increase the risk of illnesses associated with inflammation, including cardiovascular disease, infectious disease, autoimmune disease, and many common cancers. Our own in vitro data has confirmed that Vitamin D receptors are sharply upregulated on activated T cells and that physiologic calcitriol concentrations appear to have direct immunomodulatory functions (Joseph and Komanduri, unpublished). Vitamin D sufficiency is estimated by measuring 25-hydroxyvitamin D (25-OH D or calcidiol), and although there is no consensus on the optimal levels, many clinicians define sufficiency as >30 ng/ml, insufficiency as 20–30 ng/ml, and deficiency as <20 ng/ml. As one of the main sources of Vitamin D is through exposure to sunlight, allogeneic stem cell transplant (SCT) patients are at especially high risk of developing Vitamin D deficiency secondary to prolonged hospitalizations and standard recommendations to avoid sun exposure due to their increased risk of photosensitivity. To our knowledge, there are no large studies describing the incidence of Vitamin D deficiency in allogeneic SCT patients. We initially conducted a pilot study to assess Vitamin D levels in a group of allogeneic SCT recipients approximately assessed at approximately post-SCT day +100 (n=71) and found that only 18% of patients were sufficient, while the incidence of deficiency and insufficiency were to be 62%, and 20% respectively. To further elucidate if the deficiency was present pre-transplant or occurred post-transplant because of hospitalization and avoidance of sun exposure, we then assessed pre-transplant 25-hydroxyvitamin D levels in a larger group of allogeneic SCT recipients (n=108), and found that only 8% had normal baseline levels, while the vast majority were deficient (71%) or demonstrated insufficiency (21%). The baseline characteristics of this population are shown in the table below. Although this pilot study was not designed to assess the clinical significance of Vitamin D deficiency in the allogeneic transplant population, we believe that the unexpectedly high incidence of Vitamin D deficiency in SCT recipients deserves further study. Given the increasing epidemiologic and laboratory data implicating the Vitamin D axis in inflammation, we plan to assess traditional clinical outcomes associated with deficiency (including bone density and fracture rates) as well as immunological endpoints (GVHD and infection incidence) in future longitudinal studies. In conclusion, the incidence of Vitamin D deficiency in the allogeneic transplant setting is very high and additional studies are warranted to determine the clinical consequences associated with deficiency, and the potential therapeutic benefits of Vitamin D repletion in SCT recipients

Demonstration of a Direct Immunomodulatory Role for Vitamin D in Human T Cells Using Flow Cytometry

Abstract Clinical and epidemiological studies have demonstrated an increasingly stronger link between Vitamin D deficiency and a broad array of illnesses characterized by inflammation, including autoimmune diseases, coronary artery disease, and cancers. Vitamin D is a steroid hormone that exerts the majority of its biologic effects via the binding of the intracellular Vitamin D receptor (VDR). While upregulation of VDR has been demonstrated in activated bulk T cells using traditional approaches (e.g., western blotting), such assays cannot precisely define VDR distribution and kinetics. To overcome these limitations, we developed what we believe to be the first flow cytometric assay to quantify VDR expression at a single-cell level. We used a primary antibody against VDR (mouse monoclonal IgG2a to human VDR) in permeabilized T cells followed by a labeled secondary antibody. We detected a positive cell population using flow cytometry that was sharply increased following activation, consistent with upregulation of VDR confirmed by immunoblotting of sorted cells. We then applied this validated assay to define the kinetics of VDR upregulation in activated T cells. We stimulated PBMC with PMA:Ionomycin (P:I) for varying intervals and assessed intracellular VDR using flow cytometry. VDR is significantly upregulated by 15 min after stimulation, reaches a plateau after 6 hr, and may remain elevated for up to 7 d. We compared VDR to classical early and late T cell activation markers (CD69 and CD25, respectively), and we found that VDR was upregulated as consistently as (but even earlier than) CD69, and that VDR and CD25 were both consistently upregulated at later intervals (p<0.0001). To examine the association between VDR expression and proliferation, we stimulated CFSE-labeled T cells with OKT3 (2mg/ml) for 5 d and found that proliferating T cells expressed a significantly higher level of VDR than resting T cells, which maintained baseline VDR expression (p<0.0001). To assess the association between T cell cytokine production and VDR expression, we stimulated T cells with (P:I) for 6 hr in the presence of brefeldin A, and we confirmed that all cytokine-producing cells (TNFα, IL-2, IFNγ) were contained within the VDR-high population. We then assessed whether physiologic concentrations of Vitamin D could inhibit T cell proliferation in vitro. We stimulated CFSE-labeled PBMC with either OKT3 or irradiated allogeneic dendritic cells (DC) in the presence or absence of physiologic concentrations of calcitriol (50 nm) for 5 to 7 d. The presence of calcitriol during OKT3 stimulation resulted in significantly reduced cell division (p=0.004, n=5). Using a previously validated phenotype to demarcate activated alloreactive CD4+ T cells (CD4hiCD38+), we demonstrated that physiologic calcitriol supplementation decreased alloreactive activation following 7 d stimulation with allogeneic DC (p=0.0003, n=10). In conclusion, VDR is a consistent and specific early and late marker of T cell activation, suggesting a direct role for the Vitamin D axis in immunoregulation. Furthermore, physiological concentrations of Vitamin D can inhibit T cell proliferation induced by polyclonal stimuli, including allogeneic DC. These data provide confirmation for a direct immunoregulatory role for Vitamin D and suggest that further mechanistic and clinical studies may yield novel therapeutic strategies for inflammatory conditions, including graft-versus-host disease