Céline Bossard

Mucosal IL-10 and TGF-β play crucial roles in preventing LPS-driven, IFN-γ–mediated epithelial damage in human colon explants

IL-10 is an immunomodulatory cytokine that plays an obligate role in preventing spontaneous enterocolitis in mice. However, little is known about IL-10 function in the human intestinal mucosa. We showed here that IL-10 was constitutively expressed and secreted by the human normal colonic mucosa, including epithelial cells. Depletion of IL-10 in mucosal explants induced both downregulation of the IL-10-inducible, immunosuppressive gene BCL3 and upregulation of IFN-gamma, TNF-alpha, and IL-17. Interestingly, TGF-beta blockade also strongly induced IFN-gamma production. In addition, the high levels of IFN-gamma produced upon IL-10 depletion were responsible for surface epithelium damage and crypt loss, mainly by apoptosis. Polymyxin B, used as a scavenger of endogenous LPS, abolished both IFN-gamma production and epithelial barrier disruption. Finally, adding a commensal bacteria strain to mucosa explant cultures depleted of both IL-10 and LPS reproduced the ability of endogenous LPS to induce IFN-gamma secretion. These findings demonstrate that IL-10 ablation leads to an endogenous IFN-gamma-mediated inflammatory response via LPS from commensal bacteria in the human colonic mucosa. We also found that both IL-10 and TGF-beta play crucial roles in maintaining human colonic mucosa homeostasis.

Specific Clinical and Biological Features Characterize Inflammatory Bowel Disease–Associated Colorectal Cancers Showing Microsatellite Instability

PURPOSE Microsatellite instability (MSI) due to mismatch repair (MMR) deficiency has been reported to occur at variable frequencies in inflammatory bowel disease-associated intestinal neoplasias (IBD-Ns). We investigated a large series of IBD-N for associations between MSI and several biologic and clinical parameters related to tumors, patients, and their treatment. PATIENTS AND METHODS A total of 277 IBD-Ns in 205 patients were screened for MSI. Biologic and clinical variables of patients with high levels of DNA microsatellite instability high (MSI-H) were collected and compared with those associated with 33 MSI-H non-IBD colorectal cancers (CRCs). RESULTS A total of 27 IBD-Ns from 17 patients were found to be MSI-H. Compared with sporadic MSI-H CRCs, patients presented with a younger age at diagnosis, and there was no female predominance and no right-sided predominance. Unlike sporadic MSI-H CRCs, MSI-H IBD-Ns presented with heterogeneous mismatch repair defects involving MLH1, MSH2, MSH6, or PMS2, and a low frequency of MLH1 promoter methylation. They exhibited frequent BRAF mutations and frameshift mutations in genes containing coding repeat sequences. CONCLUSION The mechanisms underlying MMR deficiency in MSI-H IBD-Ns are different from those in sporadic MSI-H tumors and seem to be more related to those observed in hereditary MSI-H tumors. However, BRAF mutations were observed in MSI-H IBD-Ns, similar to sporadic MSI-H tumors, but unlike hereditary MSI-H tumors. Finally, the mutational events in target genes for instability are the same in MSI-H IBD-N tumors as in non-IBD sporadic and hereditary colorectal MSI-H cancers, indicating a colon-related repertoire of target gene alterations.

Phosphohistone H3 labelling for histoprognostic grading of breast adenocarcinomas and computer-assisted determination of mitotic index

Background: Microscopic evaluation of mitotic figures is a routine procedure in the assessment of the histoprognostic grade of tumours. Nevertheless, their count may be fraught with difficulties. As histone H3 phosphorylation at serine 10 is closely linked to chromosomal condensation, a new monoclonal antibody directed to phosphorylated histone H3 (PPH3) was recently proposed to detect mitotic cells. Aim: To test the reliability of this antibody in detecting and counting mitotic figures in sections of breast adenocarcinomas, because of the importance of mitotic count in histoprognostic grading. Methods: The pattern of PPH3 staining in formalin-fixed paraffin wax-embedded tissues, including normal tissues and a series of 39 breast adenocarcinomas, was examined. A new computer-assisted method was also developed for determining the mitotic index. Results and conclusions: In all tissues tested, PPH3-labelled mitotic figures were easily detected, allowing a rapid identification of the area of highest mitotic activity. In breast carcinomas, a strong correlation was observed between PPH3-stained and haematoxylin and eosin-stained mitotic counts (r = 0.86, p<0.0001). Counting of prophase nuclei that coexpress cyclin B1, a marker of the G2/M phase, was possible by PPH3 staining; its accuracy led us to reconsider the tumour grade in three cases. Finally, an automatic computer-assisted method was designed for assessing mitotic index with confocal microscopy and image-analysis software.

IL-22 deficiency in donor T cells attenuates murine acute graft-versus-host disease mortality while sparing the graft-versus-leukemia effect.

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation (allo-HCT), limiting the success of this therapy. Many proinflammatory cytokines secreted following the conditioning regimen have been linked to aGVHD initiation. Interleukin-22 (IL-22) is a cytokine related to IL-10 for its structure and is secreted by T helper type 17 (TH17) cells and innate immune cells. Given the paradoxical role of IL-22 in inflammation with both protective or proinflammatory functions, we investigated whether IL-22 could have a role in aGVHD pathophysiology in a mouse allo-HCT model. In this study, we show that IL-22 deficiency in donor T cells can decrease the severity of aGVHD, while limiting systemic and local inflammation in aGVHD target organs. In addition, we found that Foxp3+ regulatory T cells (Treg cells) were increased in recipient mice that received IL-22-deficient T cells, suggesting that Treg were involved in the reduced severity of GVHD. Finally, we found that the graft-versus-leukemia (GVL) effect mediated by donor T cells was preserved in the absence of IL-22. Overall, these data suggest that targeting of IL-22 may represent a valid approach towards decreasing aGVHD severity after allo-HCT while preserving the GVL effect.

HLA‐E/β2 microglobulin overexpression in colorectal cancer is associated with recruitment of inhibitory immune cells and tumor progression

The host immune response plays a major role in colorectal carcinoma (CRC) progression. A mechanism of tumor immune escape might involve expression of the human leucocyte antigen (HLA)‐E/β2m on tumor cells. The inhibitory effect of HLA‐E/β2m on CD8+ cytotoxic T lymphocytes and natural killer (NK) cells is mediated by the main HLA‐E receptor CD94/NKG2A. As the pathophysiological relevance of this mechanism in CRC remains unknown, this prompted us to examine, in situ, in a series of 80 CRC (i) the HLA‐E and β2m coexpression by tumor cells, (ii) the density of CD8+, cytotoxic, CD244+ and NKP46+ intraepithelial tumor‐infiltrating lymphocyte (IEL‐TIL) and (iii) the expression of CD94/NKG2 receptor on IEL‐TIL. These data were then correlated to patient survival. We provided (i) the in situ demonstration of HLA‐E/β2m overexpression by tumor cells in 21% of CRC characterized by an overrepresentation of signet ring cell carcinomas, mucinous carcinomas and medullary carcinomas, (ii) the significant association between HLA‐E/β2m overexpression by tumor cells and increased density of CD8+ cytotoxic, CD244+ and CD94+ IEL‐TIL and (iii) finally, the unfavorable prognosis associated with HLA‐E/β2m overexpression by tumor cells. Our findings show that HLA‐E/β2m overexpression is a surrogate marker of poor prognosis and point to a novel mechanism of tumor immune escape in CRC in restraining inhibitory IEL‐TIL.

Combination of romidepsin with cyclophosphamide, doxorubicin, vincristine, and prednisone in previously untreated patients with peripheral T-cell lymphoma: a non-randomised, phase 1b/2 study

BACKGROUND Romidepsin is a histone deacetylase inhibitor approved in the USA for patients with recurrent or refractory peripheral T-cell lymphoma and has shown activity in this setting with mainly haematological and gastrointestinal toxicity. Although it has limited efficacy, cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) therapy is widely used for treatment of de-novo peripheral T-cell lymphoma. We aimed to assess the safety, tolerability, and activity of romidepsin combined with CHOP in patients with previously untreated disease. METHODS We enrolled patients aged 18-80 years with histologically proven, previously untreated, peripheral T-cell lymphoma (Eastern Cooperative Oncology Group performance status ≤2) into a dose-escalation (phase 1b) and expansion (phase 2) study at nine Lymphoma Study Association centres in France. In the dose-escalation phase, we allocated consecutive blocks of three participants to receive eight 3 week cycles of CHOP (intravenous cyclophosphamide 750 mg/m(2), doxorubicin 50 mg/m(2), and vincristine 1.4 mg/m(2) [maximum 2 mg] on day 1 and oral prednisone 40 mg/m(2) on days 1-5) in association with varying doses of romidepsin. The starting dose was 10 mg/m(2) intravenously on days 1 and 8 of each cycle, and we used a 3 + 3 design. We assessed dose-limiting toxicities only during the first two cycles. The primary endpoint was to determine the recommended dose for the combination. For the phase 2 study, we aimed to increase the cohort of patients receiving the recommended dose to a total of 25 patients. Patients were assessed for safety outcomes at least twice per cycle according to the Common Terminology Criteria for Adverse Events, version 4.0. Safety analyses included all patients who received at least one dose of romidepsin and CHOP. This trial is registered at the European Clinical Trials Database (EudraCT), number 2010-020962-91 and ClinicalTrials.gov, number NCT01280526. FINDINGS Between Jan 13, 2011, and May 21, 2013, we enrolled 37 patients (18 treated in phase 1b and 19 patients in phase 2). Three of six patients initially treated at 10 mg/m(2) had a dose-limiting toxicity. The dose-escalation committee decided to modify the study protocol to redefine dose-limiting toxicities with regard to haematological toxicity. Three patients were treated with 8 mg/m(2) of romidepsin, an additional three at 10 mg/m(2) (one dose-limiting toxicity), and six patients at 12 mg/m(2) (three dose-limiting toxicities). We chose romidepsin 12 mg/m(2) as the recommended dose for phase 2. Of the 37 patients treated, three had early cardiac events (two myocardial infarctions and one acute cardiac failure). No deaths were attributable to toxicity. 25 (68%) of 37 patients had at least one serious adverse event. Overall, the most frequent serious adverse events were febrile neutropenia (five [14%] of 37 patients), physical health deterioration (five [14%]), lung infection (four [11%]), and vomiting (three [8%]). 33 (89%) of patients had grade 3-4 neutropenia, and 29 (78%) had grade 3-4 thrombocytopenia. INTERPRETATION Romidepsin can be combined with CHOP but this combination should now be tested in comparison to CHOP alone in a randomised trial. FUNDING Celgene.

Immunohistochemistry as a valuable tool to assess CD30 expression in peripheral T-cell lymphomas: high correlation with mRNA levels

The extended use of brentuximab-vedotin was reported for CD30(+) nonanaplastic peripheral T-cell lymphomas (PTCLs) with promising efficacy. CD30 status assessment is thus a critical factor for therapeutic decision, but the reliability of immunohistochemistry (IHC) in evaluating its expression remains to be defined. This prompted us to investigate the correlation between semiquantitative CD30 protein assessment by IHC and messenger RNA (mRNA) assessment by microarrays in a cohort of 376 noncutaneous PTCLs representative of the main entities. By IHC, CD30 expression was heterogeneous across and within entities and significantly associated with large tumor cell size. In addition to 100% anaplastic large-cell lymphomas, 57% of other PTCL entities were CD30-positive at a 5% threshold. CD30 protein expression was highly correlated to mRNA levels. mRNA levels were bimodal, separating high from low CD30-expressing PTCL cases. We conclude that IHC is a valuable tool in clinical practice to assess CD30 expression in PTCLs.

IL-22BP is produced by eosinophils in human gut and blocks IL-22 protective actions during colitis

Crohn’s disease and ulcerative colitis, the two major forms of inflammatory bowel diseases (IBDs), are characterized by high levels of IL-22 production. Rodent studies revealed that this cytokine is protective during colitis but whether this is true in IBDs is unclear. We show here that levels of the soluble inhibitor of IL-22, interleukin 22-binding protein (IL-22BP), are significantly enhanced during IBDs owing to increased numbers of IL-22BP-producing eosinophils, that we unexpectedly identify as the most abundant source of IL-22BP protein in human gut. In addition, using IL-22BP-deficient rats, we confirm that endogenous IL-22BP is effective at blocking protective actions of IL-22 during acute colitis. In conclusion, our study provides new important insights regarding the biology of IL-22 and IL-22BP in the gut and indicates that protective actions of IL-22 are likely to be suboptimal in IBDs thus making IL-22BP a new relevant therapeutic target.

Type II enteropathy-associated T-cell lymphoma features a unique genomic profile with highly recurrent SETD2 alterations.

Enteropathy-associated T-cell lymphoma (EATL), a rare and aggressive intestinal malignancy of intraepithelial T lymphocytes, comprises two disease variants (EATL-I and EATL-II) differing in clinical characteristics and pathological features. Here we report findings derived from whole-exome sequencing of 15 EATL-II tumour-normal tissue pairs. The tumour suppressor gene SETD2 encoding a non-redundant H3K36-specific trimethyltransferase is altered in 14/15 cases (93%), mainly by loss-of-function mutations and/or loss of the corresponding locus (3p21.31). These alterations consistently correlate with defective H3K36 trimethylation. The JAK/STAT pathway comprises recurrent STAT5B (60%), JAK3 (46%) and SH2B3 (20%) mutations, including a STAT5B V712E activating variant. In addition, frequent mutations in TP53, BRAF and KRAS are observed. Conversely, in EATL-I, no SETD2, STAT5B or JAK3 mutations are found, and H3K36 trimethylation is preserved. This study describes SETD2 inactivation as EATL-II molecular hallmark, supports EATL-I and -II being two distinct entities, and defines potential new targets for therapeutic intervention.

Angioimmunoblastic T-cell lymphoma is the most common T-cell lymphoma in two distinct French information data sets

Reliable information regarding the current prevalence of peripheral T-cell lymphoma (PTCL) entities is missing. Herein we report on the frequency of PTCL entities in France between 2010 and 2013. Using Lymphopath , a national lymphoma network established by the French National Cancer Agency, which