The inhibitory receptor CD94/NKG2A on CD8+ tumor-infiltrating lymphocytes in colorectal cancer: a promising new druggable immune checkpoint in the context of HLAE/ß2m overexpression.

We previously demonstrated that HLA-E/ß2m overexpression by tumor cells in colorectal cancers is associated with an unfavorable prognosis. However, the expression of its specific receptor CD94/NKG2 by intraepithelial tumor-infiltrating lymphocytes, their exact phenotype and function, as well as the relation with the molecular status of colorectal cancer and prognosis remain unknown. Based on a retrospective cohort of 234 colorectal cancer patients, we assessed the expression of HLA-E, ß2m, CD94, CD8, and NKp46 by immunohistochemistry on tissue microarray. The expression profile of HLA-E/ß2m on tumor cells and the density of tumor-infiltrating lymphocytes were correlated to the clinicopathological and molecular features (Microsatellite status, BRAF and RAS mutations). Then, from the primary tumors of 27 prospective colorectal cancers, we characterized by multiparameter flow cytometry the nature (T and/or NK cells) and the co-expression of the inhibitory NKG2A or activating NKG2C chain of ex vivo isolated CD94+ tumor-infiltrating lymphocytes. Their biological function was determined using an in vitro redirected cytolytic activity assay. Our results showed that HLA-E/ß2m was preferentially overexpressed in microsatellite instable tumors compared with microsatellite stable ones (45% vs. 19%, respectively, p = 0.0001), irrespective of the RAS or BRAF mutational status. However, HLA-E/ß2m+ colorectal cancers were significantly enriched in CD94+ intraepithelial tumor-infiltrating lymphocytes in microsatellite instable as well as in microsatellite stable tumors. Those CD94+ tumor-infiltrating lymphocytes mostly corresponded to CD8+ αß T cells, and  to a lesser extent to NK cells, and mainly co-expressed a functional inhibitory NKG2A chain. Finally, a high number of CD94+ intraepithelial tumor-infiltrating lymphocytes in close contact with tumor cells was independently associated with a worse overall survival. In conclusion, these findings strongly suggest that HLA-E/ß2m-CD94/NKG2A represents a new druggable inhibitory immune checkpoint, preferentially expressed in microsatellite instable tumors, but also in a subgroup of microsatellite stable tumors, leading to a new opportunity in colorectal cancer immunotherapies.

[uPA/PAI-1, Oncotype DX™, MammaPrint(®). Prognosis and predictive values for clinical utility in breast cancer management]. / uPA/PAI-1, Oncotype DX™, Mammaprint(®). Valeurs pronostique et prédictive pour une utilité clinique dans la prise en charge du cancer du sein.

CONTEXT AND AIMS: Breast cancer prognosis and predictive biomarkers development would allow sparing some patients from chemotherapy or identifying patients for whom chemotherapy would be indicated. In this context, in 2009, the French National Cancer Institute, a National Health and Science Agency dedicated to cancer, in collaboration with the French society of senology and breast pathology (SFSPM) published a report on the assessment of the prognostic and the predictive clinical validity of tissular biomarkers, uPA/PAI-1, Oncotype DX™ and MammaPrint(®), in breast cancer management. They concluded that only the uPA/PAI-1 prognosis value reached the highest level of evidence (LOE I according to Hayes 1998 classification). In 2012, it was decided to update this report since new data have emerged and because information disparities among clinicians have been identified. This article aims to present the main conclusions together with the levels of evidence associated with those conclusions. METHODS: The updating process was based on literature published since 2009 appraisal and on multidisciplinary and independent experts' opinion. The levels of evidence (LOE) used are those of the classification defined by Simon in 2009 (updated Hayes 1998 classification): LOE IA and LOE IB: high level of evidence; LOE IIB and LOE IIC: intermediate level of evidence; LOE IIIC and LOE IV-VD: low level of evidence. CONCLUSIONS: Among patients without lymph-node involvement, uPA/PAI-1, invasion process biomarkers, reach the highest level of evidence for 10 years recurrence free survival prognosis (LOE IA according to Simon). The predictive value to anthracyclins chemotherapy remains to be confirmed. Oncotype DX™ and MammaPrint(®) prognosis and predictive value do not reach the LOE I level. This updating' process confirms the 2009 levels of evidence for all the three biomarkers prognosis value. Besides, concerning Oncotype DX™ and MammaPrint(®), new data do not allow to conclude neither to their complementary clinical information to other clinicopathological existing biomarkers nor to a favorable cost-efficiency ratio in therapeutic decision making and this because of the methodological weakness and uncertainty that are identified in the selected studies. Practically, beyond the prognosis and predictive biomarkers validity, the clinical utility of a new biomarker for chemotherapy indication depends on its clinical added information with regard to validated biomarkers (HR, HER2 and Ki67) and to clinicopathological parameters. Since they are the sole validated biomarkers of the invasion process, uPA/PAI-1 could complete clinical information of other clinicopathological factors and consequently could confer an added clinical value. However, data concerning the impact of this information on chemotherapy clinical indication are lacking.

The density of Tbet+ tumor-infiltrating T lymphocytes reflects an effective and druggable preexisting adaptive antitumor immune response in colorectal cancer, irrespective of the microsatellite status.

Purpose: The recent success of anti-PD1 antibody in metastatic colorectal cancer (CRC) patients with microsatellite instability (MSI), known to be associated with an upregulated Th1/Tc1 gene signature, provides new promising therapeutic strategies. However, the partial objective response highlights a crucial need for relevant, easily evaluable, predictive biomarkers. Here we explore whether in situ assessment of Tbet+ tumor infiltrating lymphocytes (TILs) reflects a pre-existing functional antitumor Th1/Tc1/IFNγ response, in relation with clinicopathological features, microsatellite status and expression of immunoregulatory molecules (PD1, PDL1, IDO-1). Methodology: In two independent cohorts of CRC (retrospective n = 80; prospective n = 27) we assessed TILs density (CD3, Tbet, PD1) and expression profile of PDL1 and IDO-1 by immunohistochemistry/image analysis. Furthermore, the prospective cohort allowed to perform ex vivo CRC explant cultures and measure by Elisa the IFNγ response, at baseline and upon anti-PD1 treatment. Results: The density of Tbet+ TILs was significantly higher in MSI CRC, especially in the medullary subtype but also in a subgroup of MSS (microsatellite stable), and positively correlated with PD1 and PDL1 expression, but not with IDO-1. Finally, a high number of Tbet+ TILs was associated with a favorable overall survival. These Tbet+ TILs were functional as their density positively correlated with basal IFNγ levels. In addition, the combined score of Tbet+ PD1+ TILs coupled with IDO-1 expression predicted the magnitude of the IFNγ response upon anti-PD1. Conclusion: Altogether, immunohistochemical quantification of Tbet+ TILs is a reliable and accurate tool to recapitulate a preexisting Th1/Tc1/IFNγ antitumor response that can be reinvigorated by anti-PD1 treatment.

Clinical, histological, and molecular risk factors for cancer recurrence in patients with stage II colon cancer.

INTRODUCTION: The assessment of risk factors of cancer recurrence in patients with stage II colon cancer (CC) is crucial. Our aim was to study the clinical, histological, and molecular features associated with 3-year disease-free survival in a series of consecutive patients with stage II CC treated in three regional digestive oncology centers. METHODS: Clinical and histological data of all patients after curative surgery for stage II CC, treated from 2001 until 2009, were collected retrospectively. Histological samples were obtained and tested prospectively for microsatellite instability using fluorescent PCR amplification. Cox proportional hazards regression models were used to calculate P values, hazard ratios (HRs), and 95% confidence intervals (CIs). RESULTS: Among 195 patients studied, 22 (11%) had disease recurrence during the 3-year period following diagnosis. On multivariate analysis, only low number of lymph nodes (HR=3.81, 95% CI: 1.19-12.19, P=0.02) and T4 status (HR=5.49, 95% CI: 1.06-28.43, P=0.04) were associated significantly with an increased risk of relapse. CONCLUSION: In this series of stage II CC patients, only T4 status and low number of lymph nodes were independent risk factors for poor 3-year disease-free survival, suggesting that patients with these features should be considered for adjuvant chemotherapy.