Use of case reports and the Adverse Event Reporting System in systematic reviews: overcoming barriers to assess the link between Crohn’s disease medications and hepatosplenic T-cell lymphoma

BACKGROUND: To identify demographic and clinical characteristics associated with cases of hepatosplenic T-cell lymphoma (HSTCL) in patients with Crohn’s disease, and to assess strength of evidence for a causal relationship between medications and HSTCL in Crohn’s disease. METHODS: We identified cases of HSTCL in Crohn’s disease in studies included in a comparative effectiveness review of Crohn’s disease medications, through a separate search of PubMed and Embase for published case reports, and from the Food and Drug Administration (FDA) Adverse Event Reporting System (AERS). We used three causality assessment tools to evaluate the relationship between medication exposure and HSTCL. RESULTS: We found 37 unique cases of HSTCL in patients with Crohn’s disease. Six cases were unique to the published literature and nine were unique to AERS. Cases were typically young (<40 years of age) and male (86%). The most commonly reported medications were anti-metabolites (97%) and anti-tumor necrosis factor alpha (anti-TNFa) medications (76%). Dose and duration of therapy were not consistently reported. Use of aminosalicylates and corticosteroids were rarely reported, despite the high prevalence of these medications in routine treatment. Using the causality assessment tools, it could only be determined that anti-metabolite and anti-TNFa therapies were possible causes of HSTCL in Crohn’s disease based on the data contained in the case reports. CONCLUSION: Systematic reviews that incorporate case reports of rare lethal events should search both published literature and AERS, but consideration should be given to the limitations of case reports. In this study, establishing a causative effect other than ‘possible’ between anti-metabolite or anti-TNFa therapies and HSTCL was not feasible because case reports lacked data required by the causality assessments, and because of the limited applicability of causality assessment tools for rare irreversible events. We recommend minimum reporting requirements for case reports to improve causality assessment and routine reporting of rare life-threatening events, including their absence, in clinical trials to help clinicians determine whether rare adverse events are causally related to a medication

Heterologous immunity triggered by a single, latent virus in Mus musculus: combined costimulation- and adhesion- blockade decrease rejection.

The mechanisms underlying latent-virus-mediated heterologous immunity, and subsequent transplant rejection, especially in the setting of T cell costimulation blockade, remain undetermined. To address this, we have utilized MHV68 to develop a rodent model of latent virus-induced heterologous alloimmunity. MHV68 infection was correlated with multimodal immune deviation, which included increased secretion of CXCL9 and CXCL10, and with the expansion of a CD8(dim) T cell population. CD8(dim) T cells exhibited decreased expression of multiple costimulation molecules and increased expression of two adhesion molecules, LFA-1 and VLA-4. In the setting of MHV68 latency, recipients demonstrated accelerated costimulation blockade-resistant rejection of skin allografts compared to non-infected animals (MST 13.5 d in infected animals vs 22 d in non-infected animals, p<.0001). In contrast, the duration of graft acceptance was equivalent between non-infected and infected animals when treated with combined anti-LFA-1/anti-VLA-4 adhesion blockade (MST 24 d for non-infected and 27 d for infected, p = n.s.). The combination of CTLA-4-Ig/anti-CD154-based costimulation blockade+anti-LFA-1/anti-VLA-4-based adhesion blockade led to prolonged graft acceptance in both non-infected and infected cohorts (MST>100 d for both, p<.0001 versus costimulation blockade for either). While in the non-infected cohort, either CTLA-4-Ig or anti-CD154 alone could effectively pair with adhesion blockade to prolong allograft acceptance, in infected animals, the prolonged acceptance of skin grafts could only be recapitulated when anti-LFA-1 and anti-VLA-4 antibodies were combined with anti-CD154 (without CTLA-4-Ig, MST>100 d). Graft acceptance was significantly impaired when CTLA-4-Ig alone (no anti-CD154) was combined with adhesion blockade (MST 41 d). These results suggest that in the setting of MHV68 infection, synergy occurs predominantly between adhesion pathways and CD154-based costimulation, and that combined targeting of both pathways may be required to overcome the increased risk of rejection that occurs in the setting of latent-virus-mediated immune deviation

Combined Blockade of costimulation and adhesion pathways overcomes heterologous alloimmunity.

<p>A) Kaplan-Meier survival curves comparing skin graft survival between non-infected, CoB treated mice (solid black line, MST 22 d, n = 48, 8 independent experiments), non-infected mice treated with anti-LFA-1 and anti-VLA-4 antibodies (dotted black line, MST 24 d, n = 12, 2 independent experiments), MHV68-infected, CoB treated mice (solid red line, MST 13.5 d, n = 40, 7 independent experiments), and MHV68-infected mice treated with anti-LFA-1 and anti-VLA-4 antibodies (solid orange line, MST 27 d, n = 14, 2 experiments). When non-infected, CoB treated mice were compared to non-infected, anti-LFA-1+ anti-VLA-4 treated mice with the log-rank test, no significant difference was observed (p = n.s.). Comparison of MHV68-infected, anti-LFA-1+ anti-VLA-4 treated mice to non-infected mice treated with either regimen also yielded a non-significant p-value. MHV68-infected, anti-LFA-1+ anti-VLA-4 treated mice demonstrated significantly different skin graft survival compared to MHV68-infected, CoB-treated animals (p = .002). B) Kaplan-Meier survival curves comparing skin graft survival between the following groups: MHV68-infected mice treated with CoB (solid red line, MST 13.5, n = 40, 7 independent experiments); MHV68-infected mice treated with anti-LFA-1/anti-VLA4 (solid orange line, MST 27 d, n = 13, 2 independent experiments); MHV68-infected mice treated with CoB+anti-LFA1 (dotted grey line, MST 16, n = 12, 2 independent experiments); MHV68-infected mice treated with CoB+anti-VLA4 (dotted blue line, MST 20 d, n = 11, 2 independent experiments); MHV68-infected mice treated with CoB+anti-LFA1/anti-VLA4 (solid green line, MST>100 d, n = 25, 3 independent experiments). Statistical comparisons were made with the log-rank test. In infected animals, comparison of combined CoB and dual adhesion blockade to CoB alone yielded p<.0001. Comparison between MHV68-infected animals treated with CoB alone, CoB+anti-LFA-1, and CoB+anti-VLA-4 revealed no significant differences (p = 0.103). C) Kaplan-Meier survival curves comparing skin graft survival between the following groups: Non-infected mice treated with CoB (solid black line, MST 22 d, n = 48, 8 independent experiments); Non-infected mice treated with CoB+anti-LFA-1/anti-VLA-4 (dotted black line, MST>100 d, n = 26, 3 independent experiments); MHV68-infected mice treated with CoB (solid red line, MST 13.5, n = 40, 7 independent experiments); MHV68-infected mice treated with CoB+anti-LFA1/anti-VLA4 (solid green line, MST>100 d, n = 25, 3 independent experiments). Statistical comparisons of skin graft survival between groups were made with the log-rank test. In non-infected animals, comparison of combined CoB+dual adhesion blockade to CoB alone resulted in p<.0001. In MHV68-infected animals, comparison of CoB+anti-LFA-1/anti-VLA-4 treatment to CoB treatment yielded p<.0001. (D) Maintenance adhesion blockade (anti-LFA1/anti-VLA4) therapy was discontinued from 8 MHV68-infected mice (MHV68 WT or MHV68 M1.STOP) treated with CoB+anti-LFA1/anti-VLA4 and who had stable surviving allografts after 90-100 days of treatment with dual adhesion blockade. Following discontinuation of therapy, all grafts failed with a median time of 34 d. (E) Mice treated with combined costimulation and adhesion blockade demonstrated a decreased frequency of KLRG1+/CD127+ CD8+ T cells relative to mice treated with costimulation blockade alone. Gray columns: MHV68-infected mice treated with CoB. Red columns: MHV68-infected mice treated with CoB+antiLFA1/anti-VLA4. Y axis: % of KLRG1+/CD127+ CD8+ T cells expressed as a percentage of total CD8+ T cells. After accounting for multiple testing using the Holm method, this effect was significant at 28 days after skin grafting. In mice treated with CoB alone (n = 6), 33.3% of CD8+ T cells were KLRG1+/CD127+ versus 19.9% in mice with combined treatment (n = 4, p = .042*). Error bars represent the standard error of the mean.</p

Latent infection with MHV68 results in abbreviated skin allograft survival.

<p>A) Kaplan-Meier survival curves showing skin graft survival for the following cohorts: Black solid line - Non-infected B6 recipients of BALB/C skin allografts, receiving CTLA-4-Ig+anti-CD154 CoB. N = 48, 8 independent experiments, MST 22 d. Red solid line - MHV68-infected B6 recipients of BALB/c skin allografts receiving CTLA-4-Ig+anti-CD154 CoB. N = 40, 7 independent experiments, MST 13.5 d. Grey box - indicates MST from two historical control cohorts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071221#pone.0071221-Coley1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071221#pone.0071221-Reisman1" target="_blank">[23]</a> of non-infected B6 recipients receiving BALB/c skin grafts without any immunosuppression (MST 13 d). Statistical Analysis: Log-rank comparison of MHV68-infected/CoB treated versus non-infected/CoB treated yielded p<.0001. Log-rank comparison of MHV68-infected/CoB treated to non-infected/no immunosuppression yielded a non-significant p-value. B) Representative skin allografts from mice 12 days after graft placement. The graft on the non-infected animal remains healthy and pristine while the graft on the animal infected with latent MHV68 demonstrates necrosis, scarring, and erythema.</p

CD8^dim exhibit decreased expression of costimulatory molecules and increased expression of adhesion molecules.

<p>A) Histograms from flow-cytometry based-phenotyping at 6 weeks after infection (or age-matched, non-infected controls) compare the relative expression of the costimulatory molecules CD28, ICOS, and 4-1BB between CD8 T cells from non-infected animals (gray filled area), CD8^bright (solid black line) from MHV68-infected animals, and CD8^dim (solid red line) from MHV68-infected animals. Each cell population represents the combined results from 5 individual mice. Similar results (not shown) were observed 3 weeks after infection. B) Flow-cytometric phenotyping from mice 6 weeks after infection (or age-matched, non-infected controls) compare the relative expression of the adhesion molecules LFA-1 and VLA-4 between CD8 T cells from non-infected animals (gray filled area), CD8^bright (solid black line) from MHV68-infected animals, and CD8^dim (solid red line) from MHV68-infected animals. Each cell population represents the combined results from 5 individual mice. Similar results (not shown) were observed 3 weeks after infection. C) Comparison of gene expression profiles between CD8+ T cells from non-infected animals, CD8^bright from infected animals, and CD8^dim from infected animals (additional data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071221#pone.0071221.s002" target="_blank">Table S2</a>). Data shown from 3 independent experiments, with 1–3 biologic replicates and 1–4 technical replicates each. D) Flow cytometric analysis of CD8+ T cells from MHV68-infected animals at 6 weeks after infection identifies MHV68-specific CD8 T cells by the use of both MHC tetramers and peptide-stimulated IFN-γ release. The plots show MHV68-specificity versus CD8 surface expression. Each plot represents combined results from 5 mice. E) Flow cytometric analysis of CD8+ T cells from MHV68-infected mice at 6 weeks after infection demonstrating TCR Vβ4 staining versus CD8 staining. Each plot represents combined results from 5 mice. F) Longitudinal analysis of total CD8^dim cells (dotted red line), CD8^dim Vβ4- (solid green line), and CD8^dim Vβ4+ (dashed gray line) from MHV68-infected mice 6 weeks after infection (n = 10, except for week 1; n = 5). Error bars represent 1 standard deviation.</p