Multi-Institutional Evaluation of Pathologists' Assessment Compared to Immunoscore.

BACKGROUND The Immunoscore (IS) is a quantitative digital pathology assay that evaluates the immune response in cancer patients. This study reports on the reproducibility of pathologists' visual assessment of CD3+- and CD8+-stained colon tumors, compared to IS quantification. METHODS An international group of expert pathologists evaluated 540 images from 270 randomly selected colon cancer (CC) cases. Concordance between pathologists' T-score, corresponding hematoxylin-eosin (H&E) slides, and the digital IS was evaluated for two- and three-category IS. RESULTS Non-concordant T-scores were reported in more than 92% of cases. Disagreement between semi-quantitative visual assessment of T-score and the reference IS was observed in 91% and 96% of cases before and after training, respectively. Statistical analyses showed that the concordance index between pathologists and the digital IS was weak in two- and three-category IS, respectively. After training, 42% of cases had a change in T-score, but no improvement was observed with a Kappa of 0.465 and 0.374. For the 20% of patients around the cut points, no concordance was observed between pathologists and digital pathology analysis in both two- and three-category IS, before or after training (all Kappa < 0.12). CONCLUSIONS The standardized IS assay outperformed expert pathologists' T-score evaluation in the clinical setting. This study demonstrates that digital pathology, in particular digital IS, represents a novel generation of immune pathology tools for reproducible and quantitative assessment of tumor-infiltrated immune cell subtypes

Analyse de la composante immunitaire des cancers colorectaux et de son impact potentiel sur les stratégies thérapeutiques

In this work, I analyzed the quality of the immune infiltrate of locally advanced rectal cancer (LARC) and showed that an adaptation of the Immunoscore test to diagnostic biopsies (ISB: tumor infiltration in T cells CD3+ and CD8+) was feasible. In two cohorts (n1=131, n2=118) of LARC patients treated with neoadjuvant radiochemotherapy (nRCT) before surgery, I observed that the quality of the immune infiltrate, assessed by ISB, had a prognostic value in terms of recurrence-free survival [HR= 0.21; 95%CI 0.06-0.78, P=0.009], and was predictive of the quality of the response to nRCT (P<0.001). Tumor transcriptomic and protein analyses showed heterogeneity in local immune response to nCRT. A significant increase in the expression level of genes involved in cytotoxicity (GZMA, GZMH, GZMK, PRF-1), Th1 response (TBX21, STAT4), activation (CD69) and inhibitory costimulation (CTLA-4, LAG3) was observed in responder patients. The post-nCRT immune activation status correlated with the initial ISB. ISB coupled with post-nRCT imaging improves the prediction of histological complete response to nRCT, and thus the selection of patients eligible for an organ preservation strategy (Watch&Wait, W&W). In 2 independent cohorts of W&W patients, a High ISB predicted a low risk of recurrence at 5 years (3%; CI95% 0-10%). Finally, we showed that nRCT responder patients could show evidence of adaptive T and B immune stimulation, highlighting the immune benefit of organ (and draining lymph node) preservation in the nRCT response setting.Dans ce travail de thèse, j’ai analysé la qualité de l’infiltrat immunitaire des cancers du rectum localement avancés (LARC) et montré qu’une adaptation du test Immunoscore aux biopsies diagnostiques (ISB : infiltration tumorale en lymphocytes T CD3+ et CD8+) était réalisable. Sur deux cohortes (n1 = 131, n2=118) de patients LARC traités par radiochimiothérapie néoadjuvante (nRCT) avant chirurgie, j’ai observé que la qualité de l’infiltrat immunitaire, évalué par ISB, avait une valeur pronostique en termes de survie sans récidive [HR= 0.21 ; 95%CI 0.06-0.78, P=0.009], et était prédictive de la qualité de la réponse à la nRCT (P<0.001). Les analyses transcriptomiques et protéiques tumorales ont montré une hétérogénéité de réponse immunitaire locale à la nCRT. Une augmentation significative du niveau d’expression de gènes impliqués dans la cytotoxicité (GZMA, GZMH, GZMK, PRF-1), la réponse Th1 (TBX21, STAT4), l’activation (CD69) et la co-stimulation inhibitrice (CTLA-4, LAG3) était observée chez les patients répondeurs. Le statut d’activation immunitaire post-nCRT, était corrélé à l’ISB initial. L’ISB couplé aux examens d’imagerie post-nRCT améliore la prédiction de réponse complète histologique à la nRCT, et donc la sélection de patients éligibles à une stratégie de conservation d’organe (Watch&Wait, W&W). Sur 2 cohortes indépendantes de patients W&W, un ISB High prédisait un faible risque de récidive à 5 ans (3% ; CI95% 0-10%). Enfin, nous avons montré que les patients répondeurs à la nRCT pourraient présenter des signes de stimulation immunitaire adaptative T et B, soulignant le bénéfice immunitaire d’une préservation d’organe (et des ganglions drainants) en situation de réponse à la nCRT

Analyse de la composante immunitaire des cancers colorectaux et de son impact potentiel sur les stratégies thérapeutiques

In this work, I analyzed the quality of the immune infiltrate of locally advanced rectal cancer (LARC) and showed that an adaptation of the Immunoscore test to diagnostic biopsies (ISB: tumor infiltration in T cells CD3+ and CD8+) was feasible. In two cohorts (n1=131, n2=118) of LARC patients treated with neoadjuvant radiochemotherapy (nRCT) before surgery, I observed that the quality of the immune infiltrate, assessed by ISB, had a prognostic value in terms of recurrence-free survival [HR= 0.21; 95%CI 0.06-0.78, P=0.009], and was predictive of the quality of the response to nRCT (P<0.001). Tumor transcriptomic and protein analyses showed heterogeneity in local immune response to nCRT. A significant increase in the expression level of genes involved in cytotoxicity (GZMA, GZMH, GZMK, PRF-1), Th1 response (TBX21, STAT4), activation (CD69) and inhibitory costimulation (CTLA-4, LAG3) was observed in responder patients. The post-nCRT immune activation status correlated with the initial ISB. ISB coupled with post-nRCT imaging improves the prediction of histological complete response to nRCT, and thus the selection of patients eligible for an organ preservation strategy (Watch&Wait, W&W). In 2 independent cohorts of W&W patients, a High ISB predicted a low risk of recurrence at 5 years (3%; CI95% 0-10%). Finally, we showed that nRCT responder patients could show evidence of adaptive T and B immune stimulation, highlighting the immune benefit of organ (and draining lymph node) preservation in the nRCT response setting.Dans ce travail de thèse, j’ai analysé la qualité de l’infiltrat immunitaire des cancers du rectum localement avancés (LARC) et montré qu’une adaptation du test Immunoscore aux biopsies diagnostiques (ISB : infiltration tumorale en lymphocytes T CD3+ et CD8+) était réalisable. Sur deux cohortes (n1 = 131, n2=118) de patients LARC traités par radiochimiothérapie néoadjuvante (nRCT) avant chirurgie, j’ai observé que la qualité de l’infiltrat immunitaire, évalué par ISB, avait une valeur pronostique en termes de survie sans récidive [HR= 0.21 ; 95%CI 0.06-0.78, P=0.009], et était prédictive de la qualité de la réponse à la nRCT (P<0.001). Les analyses transcriptomiques et protéiques tumorales ont montré une hétérogénéité de réponse immunitaire locale à la nCRT. Une augmentation significative du niveau d’expression de gènes impliqués dans la cytotoxicité (GZMA, GZMH, GZMK, PRF-1), la réponse Th1 (TBX21, STAT4), l’activation (CD69) et la co-stimulation inhibitrice (CTLA-4, LAG3) était observée chez les patients répondeurs. Le statut d’activation immunitaire post-nCRT, était corrélé à l’ISB initial. L’ISB couplé aux examens d’imagerie post-nRCT améliore la prédiction de réponse complète histologique à la nRCT, et donc la sélection de patients éligibles à une stratégie de conservation d’organe (Watch&Wait, W&W). Sur 2 cohortes indépendantes de patients W&W, un ISB High prédisait un faible risque de récidive à 5 ans (3% ; CI95% 0-10%). Enfin, nous avons montré que les patients répondeurs à la nRCT pourraient présenter des signes de stimulation immunitaire adaptative T et B, soulignant le bénéfice immunitaire d’une préservation d’organe (et des ganglions drainants) en situation de réponse à la nCRT

Analysis of the immune component of colorectal cancers and its potential impact on therapeutic strategies

Dans ce travail de thèse, j’ai analysé la qualité de l’infiltrat immunitaire des cancers du rectum localement avancés (LARC) et montré qu’une adaptation du test Immunoscore aux biopsies diagnostiques (ISB : infiltration tumorale en lymphocytes T CD3+ et CD8+) était réalisable. Sur deux cohortes (n1 = 131, n2=118) de patients LARC traités par radiochimiothérapie néoadjuvante (nRCT) avant chirurgie, j’ai observé que la qualité de l’infiltrat immunitaire, évalué par ISB, avait une valeur pronostique en termes de survie sans récidive [HR= 0.21 ; 95%CI 0.06-0.78, P=0.009], et était prédictive de la qualité de la réponse à la nRCT (P<0.001). Les analyses transcriptomiques et protéiques tumorales ont montré une hétérogénéité de réponse immunitaire locale à la nCRT. Une augmentation significative du niveau d’expression de gènes impliqués dans la cytotoxicité (GZMA, GZMH, GZMK, PRF-1), la réponse Th1 (TBX21, STAT4), l’activation (CD69) et la co-stimulation inhibitrice (CTLA-4, LAG3) était observée chez les patients répondeurs. Le statut d’activation immunitaire post-nCRT, était corrélé à l’ISB initial. L’ISB couplé aux examens d’imagerie post-nRCT améliore la prédiction de réponse complète histologique à la nRCT, et donc la sélection de patients éligibles à une stratégie de conservation d’organe (Watch&Wait, W&W). Sur 2 cohortes indépendantes de patients W&W, un ISB High prédisait un faible risque de récidive à 5 ans (3% ; CI95% 0-10%). Enfin, nous avons montré que les patients répondeurs à la nRCT pourraient présenter des signes de stimulation immunitaire adaptative T et B, soulignant le bénéfice immunitaire d’une préservation d’organe (et des ganglions drainants) en situation de réponse à la nCRT.In this work, I analyzed the quality of the immune infiltrate of locally advanced rectal cancer (LARC) and showed that an adaptation of the Immunoscore test to diagnostic biopsies (ISB: tumor infiltration in T cells CD3+ and CD8+) was feasible. In two cohorts (n1=131, n2=118) of LARC patients treated with neoadjuvant radiochemotherapy (nRCT) before surgery, I observed that the quality of the immune infiltrate, assessed by ISB, had a prognostic value in terms of recurrence-free survival [HR= 0.21; 95%CI 0.06-0.78, P=0.009], and was predictive of the quality of the response to nRCT (P<0.001). Tumor transcriptomic and protein analyses showed heterogeneity in local immune response to nCRT. A significant increase in the expression level of genes involved in cytotoxicity (GZMA, GZMH, GZMK, PRF-1), Th1 response (TBX21, STAT4), activation (CD69) and inhibitory costimulation (CTLA-4, LAG3) was observed in responder patients. The post-nCRT immune activation status correlated with the initial ISB. ISB coupled with post-nRCT imaging improves the prediction of histological complete response to nRCT, and thus the selection of patients eligible for an organ preservation strategy (Watch&Wait, W&W). In 2 independent cohorts of W&W patients, a High ISB predicted a low risk of recurrence at 5 years (3%; CI95% 0-10%). Finally, we showed that nRCT responder patients could show evidence of adaptive T and B immune stimulation, highlighting the immune benefit of organ (and draining lymph node) preservation in the nRCT response setting

Analyse de l’infiltration immunitaire et conséquences thérapeutiques potentielles dans les cancers du rectum traités par radiochimiothérapie néoadjuvante

Les cancers du rectum sont traités par radio chimiothérapie (RCT) néoadjuvante avant l’exérèse chirurgicale. La réponse à la RCT est variable et de nouvelles options thérapeutiques sont actuellement en cours d’étude. Dans le cancer colorectal, la qualité et la densité de l’infiltrat immunitaire (CD3 et CD8) sont des facteurs pronostiques majeurs. L’intégration de cette composante immunitaire est en cours de translation clinique. Dans les cancers du rectum traités par RCT, la compréhension du rôle de l’infiltrat immunitaire nécessite l’analyse des biopsies diagnostiques prélevées avant tout traitement. L’analyse de la densité en CD3 et en CD8 par immunohistochimie sur ces biopsies nous a permis de montrer une corrélation entre la densité de l’infiltration immunitaire et la réponse à la RCT. Une bonne infiltration immunitaire était également corrélée à un meilleur taux de survie sans récidive. Les analyses transcriptomique et protéique sur des cancers du rectum traités ou non par RCT, nous ont permis de montrer l’induction d’une réponse Th1 cytotoxique ainsi qu’une surexpression de molécules de co-stimulation inhibitrices dans les cancers traités par RCT. L’association du critère immunitaire au critère de réponse clinique à la RCT permet d’individualiser les patients bons répondeurs à la RCT qui pourraient bénéficier d’une stratégie de conservation d’organe (watch and wait)

Therapeutic Implications of the Immunoscore in Patients with Colorectal Cancer

International audienceFour decades were needed to progress from the first demonstration of the independent prognostic value of lymphocytes infiltration in rectal cancers to the first recommendation from the international guidelines for the use of a standardized immune assay, namely the “Immunoscore” (IS), to accurately prognosticate colon cancers beyond the TNM-system. The standardization process included not only the IS conceptualization, development, fine-tuning, and validation by a large international consortium, but also a demonstration of the robustness and reproducibility across the world and testing of international norms and their effects on the IS. This is the first step of a major change of paradigm that now perceives cancer as the result of contradicting driving forces, i.e., the tumor expansion and the immune response, interacting dynamically and influencing the prognosis and the response to therapies. This prompted us to evaluate and evidence the capacity of the tumor immune status, as reflected by the IS, to accurately predict chemotherapy responses in an international, randomized cohort study of colon cancer. Moreover, we developed a derived IS performed on initial diagnostic biopsies (ISB) to assess response levels to neoadjuvant therapies. In rectal cancer, ISB was positively correlated with the degree of histologic response to neoadjuvant chemoradiotherapy and identified - alone and even more accurately if combined with clinical data- patients eligible for a noninvasive strategy. Based on these results, we are currently setting up an international cohort for confirmation. The potential role of IS with immunotherapies must be anticipate

Delayed Generalized Necrotic Purpuric Rash in a C6-deficient 12-year-old Girl Treated for Group W Meningococcal Disease.

International audienceWe report an unusual case of generalized necrotic purpuric rash that started 48 hours after the initiation of effective third-generation cephalosporin therapy to treat Neisseria meningitidis W infection in a 12-year-old girl. The course was favorable with no shock, and she recovered completely without sequelae. This infection revealed C6 deficiency in our patient

Acquired Flucytosine Resistance during Combination Therapy with Caspofungin and Flucytosine for Candida glabrata Cystitis

International audienceTreatment of Candida glabrata cystitis remains a therapeutic challenge, and an antifungal combination using flucytosine is one option. We describe two patients with refractory C. glabrata cystitis who failed flucytosine combined with caspofungin with early-acquired high-level resistance to flucytosine due to nonsense mutations in the FUR1 gene. Rapidly acquired flucytosine resistance with microbiological failure should discourage combination of caspofungin and flucytosine during urinary candidiasis

Strains Responsible for Invasive Meningococcal Disease in Patients With Terminal Complement Pathway Deficiencies.

International audienceBackground:Patients with terminal complement pathway deficiency (TPD) are susceptible to recurrent invasive meningococcal disease (IMD). Neisseria meningitidis (Nm) strains infecting these patients are poorly documented in the literature.Methods:We identified patients with TPD and available Nm strains isolated during IMD. We investigated the genetic basis of the different TPDs and the characteristics of the Nm strains.Results:We included 56 patients with C5 (n = 8), C6 (n = 20), C7 (n = 18), C8 (n = 9), or C9 (n = 1) deficiency. Genetic study was performed in 47 patients and 30 pathogenic variants were identified in the genes coding for C5 (n = 4), C6 (n = 5), C7 (n = 12), C8 (n = 7), and C9 (n = 2). We characterized 61 Nm strains responsible for IMD in the 56 patients with TPD. The most frequent strains belonged to groups Y (n = 27 [44%]), B (n = 18 [30%]), and W (n = 8 [13%]). Hyperinvasive clonal complexes (CC11, CC32, CC41/44, and CC269) were responsible for 21% of IMD cases. The CC23 predominates and represented 26% of all invasive isolates. Eleven of the 15 clonal complexes identified fit to 12 different clonal complexes belonging to carriage strains.Conclusions:Unusual meningococcal strains with low level of virulence similar to carriage strains are most frequently responsible for IMD in patients with TPD