Tjalling Bosse

Pretreatment with interferon-γ enhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis

Mesenchymal stromal cells (MSCs) are currently under investigation for the treatment of inflammatory disorders, including Crohns disease. MSCs are pluripotent cells with immunosuppressive properties. Recent data suggest that resting MSCs do not have significant immunomodulatory activity, but that the immunosuppressive function of MSCs has to be elicited by interferon‐γ (IFN‐γ). In this article, we assessed the effects of IFN‐γ prestimulation of MSCs (IMSCs) on their immunosuppressive properties in vitro and in vivo. To this end, we pretreated MSCs with IFN‐γ and assessed their therapeutic effects in dextran sodium sulfate (DSS)‐ and trinitrobenzene sulfonate (TNBS)‐induced colitis in mice. We found that mice treated with IMSCs (but not MSCs) showed a significantly attenuated development of DSS‐induced colitis. Furthermore, IMSCs alleviated symptoms of TNBS‐induced colitis. IMSC‐treated mice displayed an increase in body weight, lower colitis scores, and better survival rates compared with untreated mice. In addition, serum amyloid A protein levels and local proinflammatory cytokine levels in colonic tissues were significantly suppressed after administration of IMSC. We also observed that IMSCs showed greater migration potential than unstimulated MSCs to sites within the inflamed intestine. In conclusion, we show that prestimulation of MSCs with IFN‐γ enhances their capacity to inhibit Th1 inflammatory responses, resulting in diminished mucosal damage in experimental colitis. These data demonstrate that IFN‐γ activation of MSCs increases their immunosuppresive capacities and importantly, their therapeutic efficacy in vivo. STEM CELLS 2011;29:1549–1558

ESMO-ESGO-ESTRO Consensus Conference on Endometrial Cancer: Diagnosis, Treatment and Follow-up

Abstract The first joint European Society for Medical Oncology (ESMO), European SocieTy for Radiotherapy & Oncology (ESTRO) and European Society of Gynaecological Oncology (ESGO) consensus conference on endometrial cancer was held on 11–13 December 2014 in Milan, Italy, and comprised a multidisciplinary panel of 40 leading experts in the management of endometrial cancer. Before the conference, the expert panel prepared three clinically-relevant questions about endometrial cancer relating to the following four areas: prevention and screening, surgery, adjuvant treatment and advanced and recurrent disease. All relevant scientific literature, as identified by the experts, was reviewed in advance. During the consensus conference, the panel developed recommendations for each specific question and a consensus was reached. Results of this consensus conference, together with a summary of evidence supporting each recommendation, are detailed in this article. All participants have approved this final article.

Prognostic Significance of POLE Proofreading Mutations in Endometrial Cancer

Background: Current risk stratification in endometrial cancer (EC) results in frequent over- and underuse of adjuvant therapy, and may be improved by novel biomarkers. We examined whether POLE proofreading mutations, recently reported in about 7% of ECs, predict prognosis. Methods: We performed targeted POLE sequencing in ECs from the PORTEC-1 and -2 trials (n = 788), and analyzed clinical outcome according to POLE status. We combined these results with those from three additional series (n = 628) by meta-analysis to generate multivariable-adjusted, pooled hazard ratios (HRs) for recurrence-free survival (RFS) and cancer-specific survival (CSS) of POLE-mutant ECs. All statistical tests were two-sided. Results: POLE mutations were detected in 48 of 788 (6.1%) ECs from PORTEC-1 and-2 and were associated with high tumor grade (P < .001). Women with POLE-mutant ECs had fewer recurrences (6.2% vs 14.1%) and EC deaths (2.3% vs 9.7%), though, in the total PORTEC cohort, differences in RFS and CSS were not statistically significant (multivariable-adjusted HR = 0.43, 95% CI = 0.13 to 1.37, P = .15; HR = 0.19, 95% CI = 0.03 to 1.44, P = .11 respectively). However, of 109 grade 3 tumors, 0 of 15 POLE-mutant ECs recurred, compared with 29 of 94 (30.9%) POLE wild-type cancers; reflected in statistically significantly greater RFS (multivariable-adjusted HR = 0.11, 95% CI = 0.001 to 0.84, P = .03). In the additional series, there were no EC-related events in any of 33 POLE-mutant ECs, resulting in a multivariable-adjusted, pooled HR of 0.33 for RFS (95% CI = 0.12 to 0.91, P = .03) and 0.26 for CSS (95% CI = 0.06 to 1.08, P = .06). Conclusion: POLE proofreading mutations predict favorable EC prognosis, independently of other clinicopathological variables, with the greatest effect seen in high-grade tumors. This novel biomarker may help to reduce overtreatment in EC.

POLE Proofreading Mutations Elicit an Antitumor Immune Response in Endometrial Cancer

Purpose: Recent studies have shown that 7% to 12% of endometrial cancers are ultramutated due to somatic mutation in the proofreading exonuclease domain of the DNA replicase POLE. Interestingly, these tumors have an excellent prognosis. In view of the emerging data linking mutation burden, immune response, and clinical outcome in cancer, we investigated whether POLE-mutant endometrial cancers showed evidence of increased immunogenicity. Experimental Design: We examined immune infiltration and activation according to tumor POLE proofreading mutation in a molecularly defined endometrial cancer cohort including 47 POLE-mutant tumors. We sought to confirm our results by analysis of RNAseq data from the TCGA endometrial cancer series and used the same series to examine whether differences in immune infiltration could be explained by an enrichment of immunogenic neoepitopes in POLE-mutant endometrial cancers. Results: Compared with other endometrial cancers, POLE mutants displayed an enhanced cytotoxic T-cell response, evidenced by increased numbers of CD8+ tumor-infiltrating lymphocytes and CD8A expression, enrichment for a tumor-infiltrating T-cell gene signature, and strong upregulation of the T-cell cytotoxic differentiation and effector markers T-bet, Eomes, IFNG, PRF, and granzyme B. This was accompanied by upregulation of T-cell exhaustion markers, consistent with chronic antigen exposure. In silico analysis confirmed that POLE-mutant cancers are predicted to display more antigenic neoepitopes than other endometrial cancers, providing a potential explanation for our findings. Conclusions: Ultramutated POLE proofreading-mutant endometrial cancers are characterized by a robust intratumoral T-cell response, which correlates with, and may be caused by an enrichment of antigenic neopeptides. Our study provides a plausible mechanism for the excellent prognosis of these cancers. Clin Cancer Res; 21(14); 3347–55. ©2015 AACR.

Refining prognosis and identifying targetable pathways for high-risk endometrial cancer; a TransPORTEC initiative

This study aimed to investigate whether molecular analysis can be used to refine risk assessment, direct adjuvant therapy, and identify actionable alterations in high-risk endometrial cancer. TransPORTEC, an international consortium related to the PORTEC3 trial, was established for translational research in high-risk endometrial cancer. In this explorative study, routine molecular analyses were used to detect prognostic subgroups: p53 immunohistochemistry, microsatellite instability and POLE proofreading mutation. Furthermore, DNA was analyzed for hotspot mutations in 13 additional genes (BRAF, CDKNA2, CTNNB1, FBXW7, FGFR2, FGFR3, FOXL2, HRAS, KRAS, NRAS, PIK3CA, PPP2R1A, and PTEN) and protein expression of ER, PR, PTEN, and ARID1a was analyzed. Rates of distant metastasis, recurrence-free, and overall survival were calculated using the Kaplan–Meier method and log-rank test. In total, samples of 116 high-risk endometrial cancer patients were included: 86 endometrioid; 12 serous; and 18 clear cell. For endometrioid, serous, and clear cell cancers, 5-year recurrence-free survival rates were 68%, 27%, and 50% (P=0.014) and distant metastasis rates 23%, 64%, and 50% (P=0.001), respectively. Four prognostic subgroups were identified: (1) a group of p53-mutant tumors; (2) microsatellite instable tumors; (3) POLE proofreading-mutant tumors; and (4) a group with no specific molecular profile (NSMP). In group 3 (POLE-mutant; n=14) and group 2 (microsatellite instable; n=19) patients, no distant metastasis occurred, compared with 50% distant metastasis rate in group 1 (p53-mutant; n=36) and 39% in group 4 (NSMP; P<0.001). Five-year recurrence-free survival was 93% and 95% for group 3 (POLE-mutant) and group 2 (microsatellite instable) vs 42% (group 1, p53-mutant) and 52% (group 4, NSMP; P<0.001). Targetable FBXW7 and FGFR2 mutations (6%), alterations in the PI3K-AKT pathway (60%) and hormone receptor positivity (45%) were frequently found. In conclusion, molecular analysis of high-risk endometrial cancer identifies four distinct prognostic subgroups, with potential therapeutic implications. High frequencies of targetable alterations were identified and may serve as targets for individualized treatment.

Loss of ARID1A expression and its relationship with PI3K-Akt pathway alterations, TP53 and microsatellite instability in endometrial cancer

The switch/sucrose non-fermentable (SWI/SNF) subunit ARID1A (AT-rich interactive domain 1A gene) has been recently postulated as a novel tumor suppressor of gynecologic cancer and one of the driver genes in endometrial carcinogenesis. However, specific relationships with established molecular alterations in endometrioid endometrial cancer (EEC) are currently unknown. We analyzed the expression of ARID1A in 146 endometrial cancers (130 EECs and 16 non-EECs) in relation to alterations in the PI3K-Akt pathway (PTEN expression/KRAS/PIK3CA mutations), TP53 status (TP53 immunohistochemistry) and microsatellite instability. To discriminate between microsatellite instability due to somatic MLH1 promoter hypermethylation or germline mutations in one of the mismatch repair genes (Lynch syndrome), we included a ‘Lynch syndrome set’. This set included 21 cases with confirmed germline mutations and 15 cases that were suspected to have a germline mutation. Loss of ARID1A expression was exclusively found in EECs in 31% (40/130) of the EEC cases. No loss of expression of the other subunits of the SWI/SNF complex, SMARCD3 and SMARCB1, was detected. Alterations in the PI3K-Akt pathway were more frequent when ARID1A expression was lost. Loss of ARID1A and mutant-like TP53 expression was nearly mutually exclusive (P=0.0004). In contrast to Lynch-associated tumors, a strong association between ARID1A loss and sporadic microsatellite instability was found. Only five cases (14%) of the ‘Lynch syndrome set’ as compared with 24 cases (75%, P<0.0001) of the sporadic microsatellite-unstable tumors showed loss of ARID1A. These observations suggest that ARID1A is a causative gene, instead of a target gene, of microsatellite instability by having a role in epigenetic silencing of the MLH1 gene in endometrial cancer.

Improved Risk Assessment by Integrating Molecular and Clinicopathological Factors in Early-stage Endometrial Cancer-Combined Analysis of the PORTEC Cohorts

Purpose: Recommendations for adjuvant treatment for women with early-stage endometrial carcinoma are based on clinicopathologic features. Comprehensive genomic characterization defined four subgroups: p53-mutant, microsatellite instability (MSI), POLE-mutant, and no specific molecular profile (NSMP). We aimed to confirm the prognostic capacity of these subgroups in large randomized trial populations, investigate potential other prognostic classifiers, and integrate these into an integrated molecular risk assessment guiding adjuvant therapy. Experimental Design: Analysis of MSI, hotspot mutations in 14 genes including POLE, protein expression of p53, ARID1a, β-catenin, L1CAM, PTEN, ER, and PR was undertaken on 947 available early-stage endometrioid endometrial carcinomas from the PORTEC-1 and -2 trials, mostly high-intermediate risk (n = 614). Prognostic value was determined using univariable and multivariable Cox proportional hazard models. AUCs of different risk stratification models were compared. Results: Molecular analyses were feasible in >96% of the patients and confirmed the four molecular subgroups: p53-mutant (9%), MSI (26%), POLE-mutant (6%), and NSMP (59%). Integration of prognostic molecular alterations with established clinicopathologic factors resulted in a stronger model with improved risk prognostication. Approximately 15% of high-intermediate risk patients had unfavorable features (substantial lymphovascular space invasion, p53-mutant, and/or >10% L1CAM), 50% favorable features (POLE-mutant, NSMP being microsatellite stable, and CTNNB1 wild-type), and 35% intermediate features (MSI or CTNNB1-mutant). Conclusions: Integrating clinicopathologic and molecular factors improves the risk assessment of patients with early-stage endometrial carcinoma. Assessment of this integrated risk profile is feasible in daily practice, and holds promise to reduce both overtreatment and undertreatment. Clin Cancer Res; 22(16); 4215–24. ©2016 AACR.

A Novel Tumor-Specific Agent for Intraoperative Near-Infrared Fluorescence Imaging: A Translational Study in Healthy Volunteers and Patients with Ovarian Cancer

Purpose: Completeness of cytoreductive surgery is a key prognostic factor for survival in patients with ovarian cancer. The ability to differentiate clearly between malignant and healthy tissue is essential for achieving complete cytoreduction. Using current approaches, this differentiation is often difficult and can lead to incomplete tumor removal. Near-infrared fluorescence imaging has the potential to improve the detection of malignant tissue during surgery, significantly improving outcome. Here, we report the use of OTL38, a near-infrared (796 nm) fluorescent agent, that binds folate receptor alpha, which is expressed in >90% of epithelial ovarian cancers. Experimental Design: We first performed a randomized, placebo-controlled study in 30 healthy volunteers. Four single increasing doses of OTL38 were delivered intravenously. At fixed times following drug delivery, tolerability and blood/skin pharmacokinetics were assessed. Next, using the results of the first study, three doses were selected and administered to 12 patients who had epithelial ovarian cancer and were scheduled for cytoreductive surgery. We measured tolerability and blood pharmacokinetics, as well as the ability to detect the tumor using intraoperative fluorescence imaging. Results: Intravenous infusion of OTL38 in 30 healthy volunteers yielded an optimal dosage range and time window for intraoperative imaging. In 12 patients with ovarian cancer, OTL38 accumulated in folate receptor alpha–positive tumors and metastases, enabling the surgeon to resect an additional 29% of malignant lesions that were not identified previously using inspection and/or palpation. Conclusions: This study demonstrates that performing real-time intraoperative near-infrared fluorescence imaging using a tumor-specific agent is feasible and potentially clinically beneficial. Clin Cancer Res; 22(12); 2929–38. ©2016 AACR.

L1 cell adhesion molecule is a strong predictor for distant recurrence and overall survival in early stage endometrial cancer: pooled PORTEC trial results.

BACKGROUND L1 cell adhesion molecule (L1CAM) expression has been implicated as risk factor for disease recurrence in endometrial cancer (EC), most likely due to its role in promoting tumour cell motility. We tested the performance of L1CAM expression in predicting the risk of recurrence in the randomised post operative radiation therapy in endometrial carcinoma (PORTEC)-1 and -2 trials. METHODS In the PORTEC trials, stage I EC patients were randomised to external beam radiotherapy (EBRT) versus no additional treatment (PORTEC-1, n=714), or to EBRT versus vaginal brachytherapy (PORTEC-2, n=427). Tumour samples of 865 (75.8%) patients were available for L1CAM expression analysis by immunohistochemistry. An established scoring system for EC was used, with >10% L1CAM staining defined as positive. RESULTS Positive L1CAM expression was significantly correlated with risk of distant recurrence, with a hazard ratio (HR) of 5.1 (95% confidence interval (CI) 3.1-8.7) but not with vaginal relapse, while a trend for pelvic nodal relapse was found. Tumours with the highest expression levels (>50% positive) had the strongest risk of distant recurrence (HR 5.3, CI 2.7-10.4). In multivariate Cox analysis with the risk factors age, depth of invasion, grade, lympho-vascular space invasion (LVSI) and treatment, L1CAM expression remained an independent prognostic factor for distant recurrence (HR 3.5, CI 1.92-6.30) and overall survival (HR 2.1, CI 1.41-2.98). CONCLUSION L1CAM expression is a strong independent predictor for distant recurrence and overall survival in stage I endometrial cancer. These results warrant prospective validation of L1CAM as marker for selecting patients who could benefit from more extensive diagnostic and/or therapeutic procedures.

Improved risk assessment of endometrial cancer by combined analysis of MSI, PI3K–AKT, Wnt/β-catenin and P53 pathway activation ☆

OBJECTIVE To investigate if analysis of genetic alterations in the main pathways involved in endometrioid type carcinogenesis (PI3K-AKT, Wnt/β-catenin, P53-activation and MSI) improves the current risk assessment based on clinicopathological factors. METHODS Formalin fixed paraffin embedded (FFPE) primary tumor samples of 65 patients with FIGO-stage I endometrioid type endometrial cancer (EEC) were selected from the randomized PORTEC-2 trial. Tumors were stained by immunohistochemistry for P53, PTEN and β-catenin. Tumor DNA was isolated for sequence analysis of TP53 (exons 4 to 8), hotspot mutation analysis of KRAS (exon 1) and PI3K (exon 9 and 20) and microsatellite-instability (MSI) analysis including MLH1 promotor-methylation status. Univariate and multivariate analyses for disease-free survival (DFS) using Cox regression models were performed. RESULTS P53 status (HR 6.7, 95%CI 1.75-26.0, p=0.006) and MSI were the strongest single genetic prognostic factors for decreased DFS, while high PI3K-AKT pathway activation showed a trend and β-catenin was not prognostic. The combination of multiple activated pathways was the most powerful prognostic factor for decreased DFS (HR 5.0; 95%CI 1.59-15.6 p=0.006). Multiple pathway activation, found in 8% of patients, was strongly associated with aggressive clinical course. In contrast, 40% of patients had no alterations in the investigated pathways and had a very low risk of disease progression. CONCLUSIONS Activation of multiple oncogenic pathways in EEC was the most powerful prognostic factor for decreased DFS, resulting in an individual risk assessment superior to the current approach based on clinicopathological factors.