The Biology of Chronic Graft-versus-Host Disease: A Task Force Report from the National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease

Chronic graft-versus-host disease (GVHD) is the leading cause of late, nonrelapse mortality and disability in allogeneic hematopoietic cell transplantation recipients and a major obstacle to improving outcomes. The biology of chronic GVHD remains enigmatic, but understanding the underpinnings of the immunologic mechanisms responsible for the initiation and progression of disease is fundamental to developing effective prevention and treatment strategies. The goals of this task force review are as follows: • Summarize the current state of the science regarding pathogenic mechanisms of chronic GVHD and critical knowledge gaps. • Develop working hypotheses/overriding concepts for chronic GVHD development. • Define the usefulness of current preclinical models to test working hypotheses and ultimately discover and develop new therapeutic strategies. • Identify shortcomings of preclinical models, and define criteria for the creation of additional models to address these limitations. This document is intended as a review of our understanding of chronic GVHD biology and therapies resulting from preclinical studies, and as a platform for developing innovative clinical strategies to prevent and treat chronic GVHD

Innovative Analyses Support a Role for DNA Damage and an Aberrant Cell Cycle in Myelodysplastic Syndrome Pathogenesis

We used flow cytometry to analyze the cell cycle, DNA damage, and apoptosis in hematopoietic subsets in MDS marrow. Subsets were assigned using CD45, side scatter, CD34, and CD71. Cell cycle fractions were analyzed using DRAQ 5 (DNA content) and MPM-2 (mitoses). DNA damage was assessed using p-H2A.X, and apoptosis using Annexin V. Compared to controls, MDS patients demonstrated no increased mitoses in erythroid, myeloid, or CD34+ cells. Myeloid progenitors demonstrated increased G2 cells, which with no increased mitoses suggested delayed passage through G2. Myeloid progenitors demonstrated increased p-H2A.X, consistent with DNA damage causing this delay. Annexin V reactivity was equivalent in MDS and controls. Results for each parameter varied among hematopoietic compartments, demonstrating the need to analyze compartments separately. Our results suggest that peripheral cytopenias in MDS are due to delayed cell cycle passage of marrow progenitors and that this delayed passage and leukemic progression derive from excessive DNA damage

National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-Versus-Host Disease: III. The 2014 Biomarker Working Group Report

Biology-based markers to confirm or aid in the diagnosis or prognosis of chronic GVHD after allogeneic hematopoietic cell transplantation (HCT) or monitor its progression are critically needed to facilitate evaluation of new therapies. Biomarkers have been defined as any characteristic that is objectively measured and evaluated as an indicator of a normal biological or pathogenic process, a pharmacologic response to a therapeutic intervention. Applications of biomarkers in chronic GVHD clinical trials or patient management include: a) diagnosis and assessment of chronic GVHD disease activity, including distinguishing irreversible damage from continued disease activity, b) prognostic risk to develop chronic GVHD, and c) prediction of response to therapy. Sample collection for chronic GVHD biomarkers studies should be well-documented following established quality control guidelines for sample acquisition, processing, preservation and testing, at intervals that are both calendar- and event-driven. The consistent therapeutic treatment of subjects and standardized documentation needed to support biomarker studies are most likely to be provided in prospective clinical trials. To date, no chronic GVHD biomarkers have been qualified for utilization in clinical applications. Since our previous chronic GVHD Biomarkers Working Group report in 2005, an increasing number of chronic GVHD candidate biomarkers are available for further investigation. This paper provides a four-part framework for biomarker investigations: identification, verification, qualification, and application with terminology based on Food and Drug Administration and European Medicines Agency guidelines

Rituximab-containing reduced-intensity conditioning improves progression-free survival following allogeneic transplantation in B cell non-Hodgkin lymphoma

Background: In B cell non-Hodgkin lymphoma (B-NHL), rituximab-containing reduced-intensity conditioning regimens (R-RIC) have been shown to provide favorable outcomes in single-arm studies; however, large multicenter studies comparing R-RIC and non-rituximab-containing reduced-intensity conditioning regimens (nonR-RIC) have not been performed. Using the CIBMTR database, we report the outcomes of R-RIC versus nonR-RIC regimens in B-NHL. Methods: We evaluated 1401 adult B-NHL patients undergoing allogeneic hematopoietic cell transplantation (alloHCT) who received nonR-RIC (n = 1022) or R-RIC (n = 379) regimens. Graft-versus-host disease (GVHD) prophylaxis was limited to calcineurin inhibitor-based approaches. Results: Median follow-up of survivors in the R-RIC and nonR-RIC groups was 47 and 37 months, respectively. On multivariate analysis, no difference was seen between the R-RIC and nonR-RIC cohorts in terms of acute GVHD grade II-IV (RR = 1.14, 95% CI = 0.83-1.56, p = 0.43) or grade III-IV (RR = 1.16, 95% CI = 0.72-1.89, p = 0.54), chronic GVHD (RR = 1.15, 95% CI = 0.92-1.46, p = 0.22), non-relapse mortality (RR = 0.90; 95% CI = 0.67-1.22; p = 0.51), relapse/progression (RR = 0.79; 95% CI = 0.63-1.01; p = 0.055), and mortality (RR = 0.84, 95% CI = 0.69-1.02, p = 0.08) risk. However, R-RIC was associated with a significantly improved progression-free survival (RR = 0.76; 95% CI 0.62-0.92; p = 0.006). On subgroup analysis, mortality benefit was noted in the R-RIC group patients not receiving busulfanbased RIC (RR = 0.76; 95% CI = 0.60-0.96; p = 0.02) and with the use of a higher cumulative rituximab dose (RR = 0.43; 95% CI = 0.21-0.90; p = 0.02). Conclusion: Our analysis shows that inclusion of rituximab in RIC regimens improves progression-free survival in patients with B cell NHL. These data supports the use of R-RIC in B-NHL patients undergoing allo-HCT

International, multicenter standardization of acute graft-versus-host disease clinical data collection: a report from the Mount Sinai Acute GVHD International Consortium

Acute graft-versus-host disease (GVHD) remains a leading cause of morbidity and nonrelapse mortality after allogeneic hematopoietic cell transplantation. The clinical staging of GVHD varies greatly between transplant centers and is frequently not agreed on by independent reviewers. The lack of standardized approaches to handle common sources of discrepancy in GVHD grading likely contributes to why promising GVHD treatments reported from single centers have failed to show benefit in randomized multicenter clinical trials. We developed guidelines through international expert consensus opinion to standardize the diagnosis and clinical staging of GVHD for use in a large international GVHD research consortium. During the first year of use, the guidance followed discussion of complex clinical phenotypes by experienced transplant physicians and data managers. These guidelines increase the uniformity of GVHD symptom capture, which may improve the reproducibility of GVHD clinical trials after further prospective validation

Biomarker Panel for Chronic Graft-Versus-Host Disease

PURPOSE: To identify diagnostic and prognostic markers of chronic graft-versus-host disease (cGVHD), the major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). PATIENTS AND METHODS: Using a quantitative proteomics approach, we compared pooled plasma samples obtained at matched time points after HCT (median, 103 days) from 35 patients with cGVHD and 18 without cGVHD (data are available via ProteomeXchange with identifier PXD002762). Of 105 proteins showing at least a 1.25-fold difference in expression, 22 were selected on the basis of involvement in relevant pathways and enzyme-linked immunosorbent assay availability. Chemokine (C-X-C motif) ligand 9 (CXCL9) and suppression of tumorigenicity 2 (ST2) also were measured on the basis of previously determined associations with GVHD. Concentrations of the four lead biomarkers were measured at or after diagnosis in plasma from two independent verification cohorts (n = 391) to determine their association with cGVHD. Their prognostic ability when measured at approximately day +100 after HCT was evaluated in plasma of a second verification cohort (n = 172). RESULTS: Of 24 proteins measured in the first verification cohort, nine proteins were associated with cGVHD, and only four (ST2, CXCL9, matrix metalloproteinase 3, and osteopontin) were necessary to compose a four-biomarker panel with an area under the receiver operating characteristic curve (AUC) of 0.89 and significant correlation with cGVHD diagnosis, cGVHD severity, and nonrelapse mortality. In a second verification cohort, this panel distinguished patients with cGVHD (AUC, 0.75), and finally, the panel measured at day +100 could predict cGVHD occurring within the next 3 months with an AUC of 0.67 and 0.79 without and with known clinical risk factors, respectively. CONCLUSION: We conclude that the biomarker panel measured at diagnosis or day +100 after HCT may allow patient stratification according to risk of cGVHD

Tear fluid biomarkers in ocular and systemic disease: potential use for predictive, preventive and personalised medicine

In the field of predictive, preventive and personalised medicine, researchers are keen to identify novel and reliable ways to predict and diagnose disease, as well as to monitor patient response to therapeutic agents. In the last decade alone, the sensitivity of profiling technologies has undergone huge improvements in detection sensitivity, thus allowing quantification of minute samples, for example body fluids that were previously difficult to assay. As a consequence, there has been a huge increase in tear fluid investigation, predominantly in the field of ocular surface disease. As tears are a more accessible and less complex body fluid (than serum or plasma) and sampling is much less invasive, research is starting to focus on how disease processes affect the proteomic, lipidomic and metabolomic composition of the tear film. By determining compositional changes to tear profiles, crucial pathways in disease progression may be identified, allowing for more predictive and personalised therapy of the individual. This article will provide an overview of the various putative tear fluid biomarkers that have been identified to date, ranging from ocular surface disease and retinopathies to cancer and multiple sclerosis. Putative tear fluid biomarkers of ocular disorders, as well as the more recent field of systemic disease biomarkers, will be shown

Selective modulation of subtype III IP3R by Akt regulates ER Ca2+ release and apoptosis

Ca2+ transfer from endoplasmic reticulum (ER) to mitochondria can trigger apoptotic pathways by inducing release of mitochondrial pro-apoptotic factors. Three different types of inositol 1,4,5-trisphosphate receptor (IP3R) serve to discharge Ca2+ from ER, but possess some peculiarities, especially in apoptosis induction. The anti-apoptotic protein Akt can phosphorylate all IP3R isoforms and protect cells from apoptosis, reducing ER Ca2+ release. However, it has not been elucidated which IP3R subtypes mediate these effects. Here, we show that Akt activation in COS7 cells, which lack of IP3R I, strongly suppresses IP3-mediated Ca2+ release and apoptosis. Conversely, in SH-SY 5Y cells, which are type III-deficient, Akt is unable to modulate ER Ca2+ flux, losing its anti-apoptotic activity. In SH-SY 5Y-expressing subtype III, Akt recovers its protective function on cell death, by reduction of Ca2+ release. Moreover, regulating Ca2+ flux to mitochondria, Akt maintains the mitochondrial integrity and delays the trigger of apoptosis, in a type III-dependent mechanism. These results demonstrate a specific activity of Akt on IP3R III, leading to diminished Ca2+ transfer to mitochondria and protection from apoptosis, suggesting an additional level of cell death regulation mediated by Akt

Mitochondrial fusion is regulated by Reaper to modulate Drosophila programmed cell death

In most multicellular organisms, the decision to undergo programmed cell death in response to cellular damage or developmental cues is typically transmitted through mitochondria. It has been suggested that an exception is the apoptotic pathway of Drosophila melanogaster, in which the role of mitochondria remains unclear. Although IAP antagonists in Drosophila such as Reaper, Hid and Grim may induce cell death without mitochondrial membrane permeabilization, it is surprising that all three localize to mitochondria. Moreover, induction of Reaper and Hid appears to result in mitochondrial fragmentation during Drosophila cell death. Most importantly, disruption of mitochondrial fission can inhibit Reaper and Hid-induced cell death, suggesting that alterations in mitochondrial dynamics can modulate cell death in fly cells. We report here that Drosophila Reaper can induce mitochondrial fragmentation by binding to and inhibiting the pro-fusion protein MFN2 and its Drosophila counterpart dMFN/Marf. Our in vitro and in vivo analyses reveal that dMFN overexpression can inhibit cell death induced by Reaper or γ-irradiation. In addition, knockdown of dMFN causes a striking loss of adult wing tissue and significant apoptosis in the developing wing discs. Our findings are consistent with a growing body of work describing a role for mitochondrial fission and fusion machinery in the decision of cells to die