Differential expression of interferon-induced genes and other tissue-based biomarkers in acute graft-versus-host disease vs. lupus erythematosus in skin.

BACKGROUND: In both acute graft-versus-host disease (GVHD) and lupus erythematosus (LE), the patient's own tissues are subjected to immunological assault via complex mechanisms influenced by interferon (IFN) and other cytokines. Although not typically confused clinically, these entities have overlapping histopathological findings in the skin. AIM: To assess whether GVHD can be differentiated from LE using molecular methods on skin specimens. METHODS: We developed a quantitative reverse transcription PCR assay based on previously identified tissue-based biomarkers of cutaneous GVHD, and compared gene expression in GVHD with that in LE. RESULTS: Both entities showed robust expression of IFN-induced genes and of genes encoding proteins involved in antigen presentation, cell signalling and tissue repair. Levels of gene expression differed significantly in GVHD compared with LE, particularly those of IFN-induced genes such as MX1, OAS3, TAP1 and STAT3 (P < 0.01). Three logistic regression models could differentiate the two entities with a high degree of certainty (receiver operating characteristic area under the curve of 1.0). CONCLUSION: The study demonstrates the feasibility of distinguishing between microscopically similar inflammatory dermatoses using tissue-based molecular techniques.

Shorter long-term post-transplant life expectancy may be due to prior chemotherapy for the underlying disease: analysis of 3012 patients with acute myeloid leukemia enrolled on 9 consecutive ECOG-ACRIN trials.

Several studies reported that patients with acute myeloid leukemia (AML) who remain in long-term remission after allogeneic or autologous transplant have a shorter life expectancy, compared to the general population. However, little is known about the life expectancy of adult long-term survivors of AML who were treated with chemotherapy alone without a transplant and there have been no comparisons with survival among the general population. The current study indicates that the life expectancy of AML patients who achieved and maintained CR for at least 3 years is shorter than expected for age in the US population. This was observed also in patients who did not undergo a transplant including those who have not relapsed during the entire long follow-up period. Thus, late relapse does not explain why patients without transplants have a shortened life expectancy. Taken together, these data strongly suggest that prior chemotherapy for the underlying AML is at least a major contributing factor for the known shortened life expectancy post-transplant.

Growth characteristics of virus-transformed cells. Maximum population density, inhibition by normal cells, serum requirement, growth in soft agar, and xenogeneic transplantability.

Virus transformants (like cancer cells, cells transformed by X-ray or carcinogens, or those which have transformed spontaneously) exhibit a number of phenotypic changes which are usually associated, and which may be lost concurrently. That association is, however, not invariable. More particularly, the altered characteristics here studied (escape from contact inhibition of growth and susceptibility to inhibition by other cells, decreased serum requirement, and ability to grow in soft agar) do not, in and of themselves, endow the cell with the capacity to produce a tumor, at least as judged by the methods of assay here used. Although the question as to whether the tumorigenicity of virus transformants is causally linked to any of these associated changes cannot be answered definitively, the evidence suggests a close linkage, rather than identity, between the determinants of oncogenicity and the other properties here studied.

Detection, isolation, and functional characterization of two human T-cell subclasses bearing unique differentiation antigens.

A heterologous antihuman T-cell serum (anti-TH1), raised against purified peripheral T cells, and absorbed with an autologous Ig+ line, was shown to bind specifically to T- but not to B-lymphoid cells by both a complement-dependent cytotoxic assay and indirect immunofluorescence. Whereas 90% fetal thymocytes and thymocytes were killed by anti-TH1 and complement, a consistently restricted population (50-60%) of peripheral T cells from several normal donors were lysed, indicating that anti-TH1 is directed against one or more thymus-specific antigens which are lost or reduced on a subpopulation of human T cells in the periphery. Functional analysis of the unreactive (TH1-) and reactive (TH1+) T-cell subclasses demonstrated that TH1- cells mounted a good proliferative response to a battery of specific soluble antigens (mumps, PPD, tetanus toxoid) but neither responded in MLC, nor elaborated LMF in response to tetanus toxoid. In contrast TH1+ cells proliferated in MLC and elaborated LMF but did not respond by 3H-incorporation to soluble antigens. The relevance of these findings to human T-cell functions in vivo and to previously described functional subclasses of murine T cells is discussed.

Pre-Transplant Marital Status and Hematopoietic Cell Transplantation Outcomes

Background: Evidence about the impact of marital status before hematopoietic cell transplantation (hct) on outcomes after hct is conflicting. Methods: We identified patients 40 years of age and older within the Center for International Blood and Marrow Transplant Research registry who underwent hct between January 2008 and December 2015. Marital status before hct was declared as one of: married or living with a partner, single (never married), separated or divorced, and widowed. We performed a multivariable analysis to determine the association of marital status with outcomes after hct. Results: We identified 10,226 allogeneic and 5714 autologous hct cases with, respectively, a median follow-up of 37 months (range: 1-102 months) and 40 months (range: 1-106 months). No association between marital status and overall survival was observed in either the allogeneic (p = 0.58) or autologous (p = 0.17) setting. However, marital status was associated with grades 2-4 acute graft-versus-host disease (gvhd), p < 0.001, and chronic gvhd, p = 0.04. The risk of grades 2-4 acute gvhd was increased in separated compared with married patients [hazard ratio (hr): 1.13; 95% confidence interval (ci): 1.03 to 1.24], and single patients had a reduced risk of grades 2-4 acute gvhd (hr: 0.87; 95% ci: 0.77 to 0.98). The risk of chronic gvhd was lower in widowed compared with married patients (hr: 0.82; 95% ci: 0.67 to 0.99). Conclusions: Overall survival after hct is not influenced by marital status, but associations were evident between marital status and grades 2-4 acute and chronic gvhd. To better appreciate the effects of marital status and social support, future research should consider using validated scales to measure social support and patient and caregiver reports of caregiver commitment, and to assess health-related quality of life together with health care utilization.

Iron overload in hematopoietic cell transplantation.

Iron overload, primarily related to RBC transfusions, is a relatively common complication in hematopoietic cell transplant (HCT) recipients. Iron overload increases the risk of infections, veno-occlusive disease and hepatic dysfunction post transplant. Elevated pretransplant ferritin levels have been reported to increase the risk of nonrelapse mortality following HCT and might influence the risk of acute and chronic GVHD. Serum ferritin is sensitive but not specific for iron overload and is a poor predictor of body iron burden. Estimation of hepatic iron content with a liver biopsy or magnetic resonance imaging should be considered prior to initiating therapy for post transplant iron overload. A subgroup of transplant survivors with mild iron overload and no end-organ damage may not need therapy. Phlebotomy is the treatment of choice with iron-chelation therapy reserved for patients not eligible for phlebotomy. Natural history, evolution and treatment of iron overload in transplant survivors have not been adequately investigated and more studies are needed to determine its impact on short-term and long-term morbidity and mortality.

Autologous graft-versus-host disease: harnessing anti-tumor immunity through impaired self-tolerance.

The absence of a graft-versus-malignancy (GVM) effect may be responsible for the higher relapse rate seen after autologous hematopoietic cell transplantation (auto-HCT) compared with allogeneic hematopoietic cell transplantation (allo-HCT). Acute GVHD developing after allo-HCT, however, is associated with significant morbidity and mortality. An autoimmune syndrome similar to acute GVHD has been reported to occur after auto-HCT and has been termed the 'auto-aggression' syndrome or autologous GVHD (auto-GVHD). Auto-GVHD tends to be milder than classical GVHD, most commonly involves the skin (rarely the gastrointestinal tract, liver or both) and often is self-limited. Auto-GVHD has been reported to occur both spontaneously and in subjects receiving post transplant immune modulation with CsA, IFN-gamma or the combination. The development of auto-GVHD depends upon the derangement of self tolerance either through administration of post transplant CsA, depletion of regulatory T cells following the preparative chemoradiotherapy or both. Self-reactive CD8(+) T cells paradoxically are able to recognize a self peptide antigen presented by MHC class II molecules and appear to mediate the syndrome. Many clinical trials have been performed using CsA with or without IFN-gamma in an attempt to induce auto-GVHD. While many patients do indeed develop the syndrome, any associated anti-tumor effect remains questionable to date. New strategies to exploit auto-GVHD and enhance a GVM effect such as through the depletion of regulatory T cells or through use of newer immunomodulatory agents may improve the efficacy of auto-HCT.

Myeloablative therapy with autologous stem cell rescue for patients with Ewing sarcoma.

The aim of this study was to identify risk factors associated with PFS in patients with Ewing sarcoma undergoing ASCT; 116 patients underwent ASCT in 1989-2000 and reported to the Center for International Blood and Marrow Transplant Research. Eighty patients (69%) received ASCT as first-line therapy and 36 (31%), for recurrent disease. Risk factors affecting ASCT were analyzed with use of the Cox regression method. Metastatic disease at diagnosis, recurrence prior to ASCT and performance score <90 were associated with higher rates of disease recurrence/progression. Five-year probabilities of PFS in patients with localized and metastatic disease at diagnosis who received ASCT as first-line therapy were 49% (95% CI 30-69) and 34% (95% CI 22-47) respectively. The 5-year probability of PFS in patients with localized disease at diagnosis, and received ASCT after recurrence was 14% (95% CI 3-30). PFS rates after ASCT are comparable to published rates in patients with similar disease characteristics treated with conventional chemotherapy, surgery and irradiation suggesting a limited role for ASCT in these patients. Therefore, ASCT if considered should be for high-risk patients in the setting of carefully controlled clinical trials.

Long-term outcomes of adults with acute lymphoblastic leukemia after autologous or unrelated donor bone marrow transplantation: a comparative analysis by the National Marrow Donor Program and Center for International Blood and Marrow Transplant Research.

For adults with high-risk or recurrent ALL who lack a suitable sibling donor, the decision between autologous (Auto) and unrelated donor (URD) hematopoietic stem cell transplantation (HSCT) is difficult due to variable risks of relapse and treatment-related mortality (TRM). We analysed data from two transplant registries to determine outcomes between Auto and URD HSCT for 260 adult ALL patients in first (CR1) or second (CR2) CR. All patients received a myeloablative conditioning regimen. The median follow-up was 77 (range 12-170) months. TRM at 1 year post transplant was significantly higher with URD HSCT; however, there were minimal differences in TRM according to disease status. Relapse was higher with Auto HSCT and was increased in patients transplanted in CR2. Five-year leukemia-free (37 vs 39%) and overall survival (OS) rates (38 vs 39%) were similar for Auto HSCT vs URD HSCT in CR1. There were trends favoring URD HSCT in CR2. The long-term follow-up in this analysis demonstrated that either Auto or URD HSCT could result in long-term leukaemia-free survival and OS for adult ALL patients. The optimal time (CR1 vs CR2) and technique to perform HSCT remains an important clinical question for adult ALL patients.

Allogeneic hematopoietic cell transplantation for metastatic breast cancer.

We reviewed 66 women with poor-risk metastatic breast cancer from 15 centers to describe the efficacy of allogeneic hematopoietic cell transplantation (HCT). Median follow-up for survivors was 40 months (range, 3-64). A total of 39 patients (59%) received myeloablative and 27 (41%) reduced-intensity conditioning (RIC) regimens. More patients in the RIC group had poor pretransplant performance status (63 vs 26%, P=0.002). RIC group developed less chronic GVHD (8 vs 36% at 1 year, P=0.003). Treatment-related mortality rates were lower with RIC (7 vs 29% at 100 days, P=0.03). A total of 9 of 33 patients (27%) who underwent immune manipulation for persistent or progressive disease had disease control, suggesting a graft-vs-tumor (GVT) effect. Progression-free survival (PFS) at 1 year was 23% with myeloablative conditioning and 8% with RIC (P=0.09). Women who developed acute GVHD after an RIC regimen had lower risks of relapse or progression than those who did not (relative risk, 3.05: P=0.03), consistent with a GVT effect, but this did not affect PFS. These findings support the need for preclinical and clinical studies that facilitate targeted adoptive immunotherapy for breast cancer to explore the benefit of a GVT effect in breast cancer.

Time-dependent effect of non-Hodgkin's lymphoma grade on disease-free survival of relapsed/refractory patients treated with high-dose chemotherapy plus autotransplantation.

Evaluation of time to event outcomes usually is examined by the Kaplan-Meier method and Cox proportional hazards models. We developed a modified statistical model based on histologic grade and other variables to describe the time-dependent outcome for autologous stem cell transplant (autotransplant) performed for non-Hodgkin's lymphoma (NHL) based on histologic grade and other variables. One hundred and fourteen relapsed or refractory NHL patients were treated using BCNU 600 mg/m2, etoposide 2400 mg/m2, and cisplatin 200 mg/m2 IV followed by autotransplant. Median age was 53.5 (range: 25-70) years, 78 patients had aggressive NHL and 36 indolent NHL. Seventy-five patients received involved-field radiotherapy just prior to transplant. At a median follow-up of 33 (range: 3 to 118) months, the estimated 5-year Kaplan-Meier probabilities of overall survival and disease-free survival were 61% and 51%, respectively. Cox proportional hazards model analysis showed that proportionality did not hold for lymphoma grade, indicating that the relationship between the grade and disease-free survival differed over time. By piece-wise Cox model, the relative risk for experiencing relapse or death after 1 year in patients with indolent compared with patients with aggressive NHL was 2.81 (p=0.019) with 95% confidence interval (1.19, 6.65). The time-dependent effect of lymphoma grade on disease-free survival suggests the need for early (within first year) incorporation of novel therapeutic approaches in management of patients with indolent NHL undergoing autotransplant.

Early relapse after autologous hematopoietic cell transplantation remains a poor prognostic factor in multiple myeloma but outcomes have improved over time.

Duration of initial disease response remains a strong prognostic factor in multiple myeloma (MM) particularly for upfront autologous hematopoietic cell transplant (AHCT) recipients. We hypothesized that new drug classes and combinations employed prior to AHCT as well as after post-AHCT relapse may have changed the natural history of MM in this population. We analyzed the Center for International Blood and Marrow Transplant Research database to track overall survival (OS) of MM patients receiving single AHCT within 12 months after diagnosis (N=3256) and relapsing early post-AHCT (<24 months), and to identify factors predicting for early vs late relapses (24-48 months post-AHCT). Over three periods (2001-2004, 2005-2008, 2009-2013), patient characteristics were balanced except for lower proportion of Stage III, higher likelihood of one induction therapy with novel triplets and higher rates of planned post-AHCT maintenance over time. The proportion of patients relapsing early was stable over time at 35-38%. Factors reducing risk of early relapse included lower stage, chemosensitivity, transplant after 2008 and post-AHCT maintenance. Shorter post-relapse OS was associated with early relapse, IgA MM, Karnofsky <90, stage III, >1 line of induction and lack of maintenance. Post-AHCT early relapse remains a poor prognostic factor, even though outcomes have improved over time.

Bone marrow-derived lymphoid cell lines from patients with agammaglobulinemia.

In vitro infection with Epstein-Barr virus of bone marrow-derived (B) lymphocytes from blood of patients with X-linked or common varied agammaglobulinemia resulted in the establishment of long-term B-lymphoid cell lines (LCL). LCL were established only with B lymphocytes from patients whose B cells failed to respond to in vitro mitogenic stimulation. In contrast, lymphocytes from patients without small B lymphocytes or with B cells that synthesized but failed to secrete Ig failed uniformly to establish LCL. Analysis of the "nonsecretory" B lymphocytes demonstrated the presence of receptors for C3b and C3d as well as surface Ig, but absence of detectable receptors for Epstein-Barr virus. All of the six cell lines that were established formed rosettes with EAC3 but not with sheep erythrocytes. Four of the six LCL were principally surface IgD bearing and did not synthesize detectable Ig for secretion. Two of the cell lines were indistinguishable from cell lines from normal individuals: they had surface IgG, A, M, and D and synthesized and secreted Ig.

Extracellular adenosine triphosphate activates calcium mobilization in human phagocytic leukocytes and neutrophil/monocyte progenitor cells.

We have examined the ability of extracellular ATP to elicit intracellular Ca2+ mobilization in a broad range of human leukocytes at particular stages of hematopoietic differentiation. The average cytosolic [Ca2+] in various leukocyte populations was measured in Fura 2-loaded cell suspensions while the cytosolic [Ca2+] in individual, Indo 1-loaded leukocytes was assayed by flow cytometric methods. Utilizing normal blood- and marrow-derived cells, human leukemic cell lines, and mononuclear cell fractions derived from the blood of patients with various leukemias, we have found that ATP-induced Ca2+ mobilization appears restricted to leukocytes of neutrophil/monocyte ontogeny. Significant ATP-induced increases in cytosolic [Ca2+] were observed in neutrophils, monocytes, and myeloid progenitor cells as immature as myeloblasts, but not in lymphocytes. Extensive characterization of the ATP-induced changes in [Ca2+] observed in the HL-60 promyelocytic cell line have indicated these Ca2+-mobilizing effects of ATP can be correlated with an activation of inositol phospholipid breakdown via the occupation of P2-purinergic receptors Significantly, of the various agonists (FMLP, platelet-activating factor, LTB4, and ATP) which elicit equivalent and maximal Ca2+ mobilization in mature neutrophils and monocytes, ATP was the most efficacious stimulant of Ca2+ mobilization in immature neutrophil/monocyte precursors. Thus, expression of putative P2-purinergic receptors for ATP appears to precede expression of other receptor types known to activate the inositol phospholipid signaling cascades in terminally differentiated phagocytes.

Reactivity of a monoclonal antibody with human ovarian carcinoma.

A murine monoclonal antibody (OC125) has been developed that reacts with each of six epithelial ovarian carcinoma cell lines and with cryopreserved tumor tissue from 12 of 20 ovarian cancer patients. By contrast, the antibody does not bind to a variety of nonmalignant tissues, including adult and fetal ovary. OC125 reacts with only 1 of 14 cell lines derived from nonovarian neoplasms and has failed to react with cryostat sections from 12 nonovarian carcinomas.

Impaired growth and elevated fas receptor expression in PIGA(+) stem cells in primary paroxysmal nocturnal hemoglobinuria.

The genetic defect underlying paroxysmal nocturnal hemoglobinuria (PNH) has been shown to reside in PIGA, a gene that encodes an element required for the first step in glycophosphatidylinositol anchor assembly. Why PIGA-mutated cells are able to expand in PNH marrow, however, is as yet unclear. To address this question, we compared the growth of affected CD59(-)CD34(+) and unaffected CD59(+)CD34(+) cells from patients with that of normal CD59(+)CD34(+) cells in liquid culture. One hundred FACS-sorted cells were added per well into microtiter plates, and after 11 days at 37 degrees C the progeny were counted and were analyzed for their differentiation pattern. We found that CD59(-)CD34(+) cells from PNH patients proliferated to levels approaching those of normal cells, but that CD59(+)CD34(+) cells from the patients gave rise to 20- to 140-fold fewer cells. Prior to sorting, the patients' CD59(-) and CD59(+)CD34(+) cells were equivalent with respect to early differentiation markers, and following culture, the CD45 differentiation patterns were identical to those of control CD34(+) cells. Further analyses of the unsorted CD59(+)CD34(+) population, however, showed elevated levels of Fas receptor. Addition of agonist anti-Fas mAb to cultures reduced the CD59(+)CD34(+) cell yield by up to 78% but had a minimal effect on the CD59(-)CD34(+) cells, whereas antagonist anti-Fas mAb enhanced the yield by up to 250%. These results suggest that expansion of PIGA-mutated cells in PNH marrow is due to a growth defect in nonmutated cells, and that greater susceptibility to apoptosis is one factor involved in the growth impairment.

Randomized trial of peribulbar triamcinolone acetonide with and without focal photocoagulation for mild diabetic macular edema: a pilot study.

OBJECTIVE: To provide pilot data on the safety and efficacy of anterior and posterior sub-Tenon injections of triamcinolone either alone or in combination with focal photocoagulation in the treatment of mild diabetic macular edema (DME). DESIGN: Prospective, phase II, multicenter, randomized clinical trial. PARTICIPANTS: One hundred nine patients (129 eyes) with mild DME and visual acuity 20/40 or better. METHODS: The participants were assigned randomly to receive either focal photocoagulation (n = 38), a 20-mg anterior sub-Tenon injection of triamcinolone (n = 23), a 20-mg anterior sub-Tenon injection followed by focal photocoagulation after 4 weeks (n = 25), a 40-mg posterior sub-Tenon injection of triamcinolone (n = 21), or a 40-mg posterior sub-Tenon injection followed by focal photocoagulation after 4 weeks (n = 22). Follow-up visits were performed at 4, 8, 17, and 34 weeks. MAIN OUTCOME MEASURES: Change in visual acuity and retinal thickness measured with optical coherence tomography (OCT). RESULTS: At baseline, mean visual acuity in the study eyes was 20/25 and mean OCT central subfield thickness was 328 mum. Changes in retinal thickening and in visual acuity were not significantly different among the 5 groups at 34 weeks (P = 0.46 and P = 0.94, respectively). There was a suggestion of a greater proportion of eyes having a central subfield thickness less than 250 mum at 17 weeks when the peribulbar triamcinolone was combined with focal photocoagulation. Elevated intraocular pressure and ptosis were adverse effects attributable to the injections. CONCLUSIONS: In cases of DME with good visual acuity, peribulbar triamcinolone, with or without focal photocoagulation, is unlikely to be of substantial benefit. Based on these results, a phase III trial to evaluate the benefit of these treatments for mild DME is not warranted.

Diagnosis and treatment of transplantation-associated thrombotic microangiopathy: real progress or are we still waiting?

Transplantation-associated thrombotic microangiopathy (TA-TMA) is an infrequent but devastating syndrome that occurs in allogeneic hematopoietic stem cell transplant recipients, and is associated with a variety of transplantation-related factors, including conditioning regimens, immunosuppressive agents, GVHD and opportunistic infections. Progress in managing this condition has been hampered by lack of a consensus definition and poor understanding of the pathophysiology of the disorder. Two different groups recently have proposed consensus definitions, yet they fail to distinguish the primary syndrome from the secondary causes, such as a variety of infections, medication exposure or other conditions. Increasing evidence suggests that TA-TMA is a multifactorial disorder that is distinct from thrombotic thrombocytopenic purpura (TTP), and likely represents the final common pathway of a number of endothelial cell insults. TA-TMA responds poorly to conventional treatment for TTP, including plasma exchange, but newer agents, including daclizumab and defibrotide show promise. In addition, other agents known to modify endothelial responses to injury, including statins, prostacyclin analogues, endothelin-receptor antagonists and free radical scavengers, may lead to improved outcomes for patients affected by this disorder.