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Quantification of organotypic invasion assay screening.

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In squamous cell carcinoma (SCC), tissue invasion by collectively invading cells requires physical forces applied by tumor cells on their surrounding extracellular matrix (ECM). Cancer-related ECM is composed of thick collagen bundles organized by carcinoma-associated fibroblasts (CAF) within the tumor stroma. Here, we show that SCC cell collective invasion is driven by the matrix-dependent mechano-sensitization of EGF signaling in cancer cells. Calcium (Ca2+) was a potent intracellular second messenger that drove actomyosin contractility. Tumor-derived matrix stiffness and EGFR signaling triggered increased intracellular Ca2+ through CaV1.1 expression in SCC cells. Blocking L-type calcium channel expression or activity using Ca2+ channel blockers verapamil and diltiazem reduced SCC cell collective invasion both in vitro and in vivo. These results identify verapamil and diltiazem, two drugs long used in medical care, as novel therapeutic strategies to block the tumor-promoting activity of the tumor niche.Significance: This work demonstrates that calcium channels blockers verapamil and diltiazem inhibit mechano-sensitization of EGF-dependent cancer cell collective invasion, introducing potential clinical strategies against stromal-dependent collective invasion.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/18/5229/F1.large.jpg. Cancer Res; 78(18); 5229–42. ©2018 AACR.

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Degradation of signaling proteins is one of the most powerful tumor-suppressive mechanisms by which a cell can control its own growth. Here, we identify RHOA as the molecular target by which autophagy maintains genomic stability. Specifically, inhibition of autophagosome degradation by the loss of the v-ATPase a3 (TCIRG1) subunit is sufficient to induce aneuploidy. Underlying this phenotype, active RHOA is sequestered via p62 (SQSTM1) within autolysosomes and fails to localize to the plasma membrane or to the spindle midbody. Conversely, inhibition of autophagosome formation by ATG5 shRNA dramatically increases localization of active RHOA at the midbody, followed by diffusion to the flanking zones. As a result, all of the approaches we examined that compromise autophagy (irrespective of the defect: autophagosome formation, sequestration, or degradation) drive cytokinesis failure, multinucleation, and aneuploidy, processes that directly have an impact upon cancer progression. Consistently, we report a positive correlation between autophagy defects and the higher expression of RHOA in human lung carcinoma. We therefore propose that autophagy may act, in part, as a safeguard mechanism that degrades and thereby maintains the appropriate level of active RHOA at the midbody for faithful completion of cytokinesis and genome inheritance. Cancer Res; 73(14); 4311–22. ©2013 AACR.

AVI file - 5901K, Video 5 (related to Fig. 2G) is a time-lapse recording of a mononucleate a3-/- cell that formed a tetranucleate cell after two rounds of abortive mitosis.

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AVI file - 2112K, Video 4 (related to Fig. 2F) illustrates the failed abscission of a mononucleated a3-/- cell that exited mitosis as a binucleated cell after 18 attempts at cleavage furrow formation. Note that this cell developed ectopic furrows, which led to the formation of anuclear fragments that fused back to the cell.

Supplementary Materials and Methods, Figure Legends

AVI file - 645K, Video 6 (related to Fig. 6A) is a time-lapse recording showing that the ATG5-depleted A549 cells formed numerous ectopic furrows in addition to a wider-than-normal equatorial cleavage furrow.

AVI file - 2696K, Video 3 (related to Fig. 2E) is a time-lapse recording of four pairs of a3-/- cells that entered mitosis synchronously, likely bridged sister cells. Pairs of cells that entered mitosis synchronously are outlined in color in the first frame, and then marked with dots in the next frame to help visualize their progression in mitosis.

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Colitis-associated cancer (CAC) is a complication of inflammatory bowel disease (IBD). Binding of extracellular ATP to the purinergic receptor P2RX7 has emerged as a critical event in controlling intestinal inflammation, acting to limit elevation of proinflammatory mast cells and cytokines and promote survival of regulatory T cells (Treg) and enteric neurons. In this study, we investigated the effect of P2RX7 blockade in an established mouse model of CAC. Using genetic and pharmacologic tools, we found unexpectedly that while P2RX7 mediated inflammatory responses, it also acted at an early time to suppress CAC development. P2RX7 blockade enhanced proliferation of intestinal epithelial cells and protected them from apoptosis. The proliferative effects of P2RX7 blockade were associated with an increased production of TGFβ1 that was sufficient to stimulate the proliferation of intestinal epithelial cells. Finally, P2RX7 blockade also altered immune cell infiltration and promoted Treg accumulation within lesions of the digestive system. Taken together, our findings reveal an unexpected role for P2RX7 in preventing CAC, suggesting cautions in the use of P2RX7 inhibitors to treat IBD given the possibility of increasing risks CAC as a result. Cancer Res; 75(5); 835–45. ©2015 AACR.

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AVI file - 875K, Video 2 (related to Fig. 2D) shows the delayed abscission of an a3-/- cell that remained connected by an intracellular bridge for up to 8 h 45 min before separating.

Supplementary Figures S1-S7. Figure S1: Characterization of heterozygous P2rx7 +/- mice Figure S2: The A740003 pharmacological inhibitor of P2RX7 dampens colitis Figure S3: P2rx7-dependent colonic epithelial cell proliferation Figure S4: Scoring of pre-cancerous dysplasic lesions Figure S5: Body weight curves of WT, P2rx7-/- and A438079-treated mice on a CAC regimen Figure S6: Histological examination of cancerous colonic lesions of WT and P2rx7-/- mice Figure S7: The cancerous microenvironment of P2rx7-/- mice is enriched in neutrophils

Background Mucosal antibodies can prevent virus entry and replication in mucosal epithelial cells and therefore virus shedding. Parenteral booster injection of a vaccine against a mucosal pathogen promotes stronger mucosal immune responses following prior mucosal infection compared with injections of a parenteral vaccine in a mucosally naive subject. We investigated whether this was also the case for the BNT162b2 coronavirus disease 2019 (COVID-19) messenger RNA vaccine. Methods Twenty recovered COVID-19 subjects (RCSs) and 23 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–naive subjects were vaccinated with, respectively, 1 and 2 doses of the BNT162b2 COVID-19 vaccine. Nasal epithelial lining fluid (NELF) and plasma were collected before and after vaccination and assessed for immunoglobulin G (IgG) and IgA antibody levels to Spike and for their ability to neutralize binding of Spike to angiotensin-converting enzyme-2 receptor. Blood was analyzed 1 week after vaccination for the number of Spike-specific antibody-secreting cells (ASCs) with a mucosal tropism. Results All RCSs had both nasal and blood SARS-CoV-2–specific antibodies at least 90 days after initial diagnosis. In RCSs, a single dose of vaccine amplified preexisting Spike-specific IgG and IgA antibody responses in both NELF and blood against both vaccine homologous and variant strains, including Delta. These responses were associated with Spike-specific IgG and IgA ASCs with a mucosal tropism in blood. Nasal IgA and IgG antibody responses were lower in magnitude in SARS-CoV-2–naive subjects after 2 vaccine doses compared with RCSs after 1 dose. Conclusions Mucosal immune response to the SARS-CoV-2 Spike protein is higher in RCSs after a single vaccine dose compared with SARS-CoV-2–naive subjects after 2 doses.

International audience; Given the size of digitized Whole Slide Images (WSIs), it is generally laborious and time-consuming for pathologists to exhaustively delineate objects within them, especially with datasets containing hundreds of slides to annotate. Most of the time, only slide-level labels are available, giving rise to the development of weakly-supervised models. However, it is often difficult to obtain from such models accurate object localization, e.g., patches with tumor cells in a tumor detection task, as they are mainly designed for slide-level classification. Using the attention-based deep Multiple Instance Learning (MIL) model as our base weakly-supervised model, we propose to use mixed supervision-i.e., the use of both slide-level and patch-level labels-to improve both the classification and the localization performances of the original model, using only a limited amount of patch-level labeled slides. In addition, we propose an attention loss term to regularize the attention between key instances, and a paired batch method to create balanced batches for the model. First, we show that the changes made to the model already improve its performance and interpretability in the weakly-supervised setting. Furthermore, when using only between 12 and 62% of the total available patch-level annotations, we can reach performance close to fully-supervised models on the tumor classification datasets DigestPath2019 and Camelyon16.

Introduction International guidelines recommend BRAF mutational status assessment in treatment-naive advanced non-squamous non-small cell lung carcinoma (NSCLC) patients since the presence of a BRAFV600 mutation enables specific BRAF inhibitor treatment. For this purpose, the mutational status needs to be obtained in 10 working days. Herein, we prospectively evaluated the feasibility of systematic assessment of the BRAF status using immunohistochemistry (IHC) in a single institution (LPCE, Nice) at baseline for NSCLC diagnosed. Methods 1317 NSCLC were evaluated using BRAF IHC from 2011 to 2019. Initially the BRAF status was prospectively assessed using NGS and/or pyrosequencing in 618 consecutively diagnosed NSCLC patients from 2012 to 2016; BRAFV600E and BRAF nonV600E mutated tumors detected in this cohort were retrospectively evaluated using BRAF IHC. Secondarily, 699 biopsies of NSCLC were prospectively analyzed between 2017 and 2019 using BRAF IHC. BRAF IHC positive tumors were tested using a rapid BRAF specific PCR based assay. Results Initially, 21/618 (3%) of tumors (15 early and 6 late stage tumors) were BRAFV600E mutated according to the results of NGS and/or pyrosequencing. BRAF IHC was positive in 21/21 of these cases and negative in 51/51 (100 %) BRAF non V600E mutated cases. In the prospective BRAF IHC tested cohort of patients, 24/699 (3%) tumors (13 early and 11 late stage tumors) were positive with VE1 IHC. The BRAF PCR assay was positive in 20/24 (83 %) of these cases. Conclusion BRAFV600E IHC screening of treatment-naive NSCLC patients is a rapid, specific and very sensitive method which can lead in advanced stage positive NSCLC tumors to a BRAF inhibitor treatment. This test can be routinely integrated into mandatory predictive biomarker ‘testing of NSCLC. According to the organization of patient care and the physician’s request, this practice can be proposed as an alternative to NGS-based tissue biopsy made at baseline.

Resume L’evaluation des images histologiques peut etre parfois subjective et assez peu reproductible selon les cas. Par consequent, l’analyse computationnelle de l’imagerie histologique via des approches dites d’intelligence artificielle (IA) a recemment recu une attention considerable afin d’ameliorer cette precision diagnostique. Ainsi, l’analyse computationnelle de lames virtuelles a recemment ete evaluee pour l’optimisation du diagnostic histologique ou cytologique, la prediction du pronostic ou du profil genomique des patients atteints de cancer. L’utilisation de l’IA progresse de plus en plus dans le domaine de la recherche translationnelle en anatomopathologie, mais n’a pas encore ete mise en œuvre de la maniere la plus pratique pour le transfert en routine clinique. Malgre des resultats prometteurs, il subsiste plusieurs defis ou enjeux majeurs a relever afin de reussir le transfert reel de cette nouvelle approche en routine clinique. Il s'agit ici de faire le point sur les applications recentes de l’IA en pathologie cancereuse, mais aussi d’apporter des clarifications sur les avantages et les limites de cette approche, ainsi que les perspectives a mettre en œuvre pour un transfert potentiel dans la pratique quotidienne des pathologistes.

The oxygen-limiting (hypoxic) microenvironment of tumors induces metabolic reprogramming and cell survival, but the underlying mechanisms involving mitochondria remain poorly understood. We previously demonstrated that hypoxia-inducible factor 1 mediates the hyperfusion of mitochondria by inducing Bcl-2/adenovirus E1B 19-kDa interacting protein 3 and posttranslational truncation of the mitochondrial ATP transporter outer membrane voltage-dependent anion channel 1 in hypoxic cells. In addition, we showed that truncation is associated with increased resistance to drug-induced apoptosis and is indicative of increased patient chemoresistance. We now show that silencing of the tumor suppressor TP53 decreases truncation and increases drug-induced apoptosis. We also show that TP53 regulates truncation through induction of the mitochondrial protein Mieap. While we found that truncation was independent of mitophagy, we observed local microfusion between mitochondria and endolysosomes in hypoxic cells in culture and in patients' tumor tissues. Since we found that the endolysosomal asparagine endopeptidase was responsible for truncation, we propose that it is a readout of mitochondrial-endolysosomal microfusion in hypoxia. These novel findings provide the framework for a better understanding of hypoxic cell metabolism and cell survival through mitochondrial-endolysosomal microfusion regulated by hypoxia-inducible factor 1 and TP53.

Due to increased demand for testing, as well as restricted supply chain resources, testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to face many hurdles. Pooling several samples has been proposed as an alternative approach to address these issues. We investigated the feasibility of pooling nasopharyngeal swab (NPS) or saliva samples for SARS-CoV-2 testing with a commercial assay (Idylla SARS-CoV-2 test; Biocartis). We evaluated the 10-pool and 20-pool approaches for 149 subjects, with 30 positive samples and 119 negative samples. The 10-pool approach had sensitivity of 78.95% (95% confidence interval [CI], 54.43% to 93.95%) and specificity of 100% (95% CI, 71.51% to 100%), whereas the 20-pool approach had sensitivity of 55.56% (95% CI, 21.20% to 86.30%) and specificity of 100% (95% CI, 25% to 100%). No significant difference was observed between the results obtained with pooled NPS and saliva samples. Given the rapidity, full automation, and practical advantages of the Idylla SARS-CoV-2 assay, pooling of 10 samples has the potential to significantly increase testing capacity for both NPS and saliva samples, with good sensitivity. IMPORTANCE To control outbreaks of coronavirus disease 2019 (COVID-19) and to avoid reagent shortages, testing strategies must be adapted and maintained for the foreseeable future. We analyzed the feasibility of pooling NPS and saliva samples for SARS-CoV-2 testing with the Idylla SARS-CoV-2 test, and we found that sensitivity was dependent on the pool size. The SARS-CoV-2 testing capacity with both NPS and saliva samples could be significantly expanded by pooling 10 samples; however, pooling 20 samples resulted in lower sensitivity.

BackgroundThe current diagnostic standard for coronavirus 2019 disease (COVID-19) is reverse transcriptase-polymerase chain reaction (RT-PCR) testing with naso-pharyngeal (NP) swabs. The invasiveness and need for trained personnel make the NP technique unsuited for repeated community-based mass screening. We developed a technique to collect saliva in a simple and easy way with the sponges that are usually used for tamponade of epistaxis. This study was carried out to validate the clinical performance of oral sponge (OS) sampling for SARS-CoV-2 testing.MethodsOver a period of 22 weeks, we collected prospectively 409 paired NP and OS samples from consecutive subjects presenting to a public community-based free screening center. Subjects were referred by their attending physician because of recent COVID-19 symptoms (n=147) or by the contact tracing staff of the French public health insurance since they were considered as close contacts of a laboratory-confirmed COVID-19 case (n=262).ResultsIn symptomatic subjects, RT-PCR SARS-CoV-2 testing with OS showed a 96.5% (95%CI: 89.6-94.8) concordance with NP testing, and, a 93.3% [95%CI: 89.1-97.3] sensitivity. In close contacts the NP-OS concordance (93.8% [95%CI: 90.9-96.7]) and OS sensitivity (71.9% [95%CI: 66.5-77.3]) were slightly lower.ConclusionThese results strongly suggest that OS testing is a straightforward, low-cost and high-throughput sampling method that can be used for frequent RT-PCR testing of COVID-19 patients and mass screening of populations.Summary of the “take home” messageOS sampling for SARS-CoV2 RT-PCR is an easy to perform, straightforward self-administered sampling technique, which has a sensitivity of up to 93.3% in symptomatic patients and 71% in close contact subjects.

Background Recent advances obtained with immune checkpoint inhibitors (ICIs) targeting the programmed cell death-1 (PD-1) protein have significantly improved the outcome of patients with metastatic melanoma. The PD-L1 expression in tumour cells as detected by immunohistochemistry is a predictive biomarker in some solid tumours, but appears insufficient as prognostic or predictive factor of response to ICIs in metastatic melanomas. Objectives We investigated whether the presence and the features of pretreatment CD8+ tumour-infiltrating T lymphocytes (TILs) could be a complementary prognostic or predictive biomarker in patients with metastatic melanoma. Methods In this retrospective study, we evaluated the association of PD-L1 expression ≥5% of tumour cells combined with TIL features (CD8, CD28, Ki67) with the overall survival (OS) among 51 patients treated with ICIs and 54 patients treated with other treatment options (non-ICIs). Results PD-L1 positivity was observed in 33% and 39% of primary melanomas and matched metastases, respectively, with, however, poor concordance between the primary and the matched metastatic site (κ = 0.283). No significant association was noted between PD-L1 expression and CD8+ TIL profile analysed as single markers and OS or response to immunotherapy. Instead, their combined analysis in primary melanoma samples showed that the PD-L1-/CD8+ status was significantly associated with prolonged OS in the whole population (P = 0.04) and in the subgroup treated with non-ICIs (P = 0.009). Conversely, the PD-L1+/CD8+ status was a good prognostic factor in patients treated with ICIs (P = 0.022), whereas was significantly associated with poor prognosis in patients treated with non-ICIs (P = 0.014). While the expression of CD28 was not related to outcome, the Ki67 expression was significantly associated with poor OS in the subgroup CD8+ TIL+/PD-L1- (P = 0.02). Conclusions The pretreatment combination of PD-L1 expression with the level of CD8+ TILs could better assess OS and predict therapeutic response of patients with metastatic melanoma treated by either immunotherapy or other treatment regimens.

In non-small cell lung cancer (NSCLC), response to immune checkpoint blockade (ICB) is associated with programmed cell death ligand 1 expression that is induced by interferon-γ-produced by tumor-infiltrating CD8+ T cells. However, not all tumors with a PD-L1 expression and/or CD8+ T cell infiltrate respond to ICB, and some tumors without any PD-L1 expression respond to ICB. Moreover, little is known about all the mechanisms governing ICB resistance in NSCLC. The objective of the study was to investigate a circulating immunological signature (cytokines, chemokines and immune checkpoints) which could be predictive of resistance to ICB in patients with advanced NSCLC. We performed a multiplexed analysis on 23 TruCulture® (in vitro T cells activation system) and 41 plasma samples using the Luminex® platform (Bio-Techne, MN USA). We investigated the relationship between the levels at baseline of 30 circulating analytes and the response to ICB of advanced NSCLC patients. Through the TruCulture® samples analysis, we identified two types of responders depending on T cell functionality. The responders with a functional T cell activation had lower levels of neutrophil associated analytes (CXCL5/6; p-value Citation Format: Wassila Khatir, Olivier Humbert, Jaap Neels, Léa Berland, Jonathan Benzaquen, Fabian Andrés Gallardo Rivera, Maryline Allegra, Myriam Salah, Virginie Tanga, Olivier Bordone, Julien Fayada, Virginie Lespinet-Fabre, Elodie Long-Mira, Sandra Lassalle, Patrick Brest, Valérie Vouret, Charlotte Maniel, Jacques Boutros, Simon Heeke, Véronique Hofman, Charles-Hugo Marquette, Paul Hofman, Marius Ilie. Identification of a predictive circulating immunological signature of response to immune checkpoint inhibitors in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1265.

With the growing standardization of Whole Slide Images (WSIs), deep learning algorithms have shown promising results for the automated classification and localization of tumors. Yet, it is often difficult to train such algorithms, as they usually require careful detailed annotations from expert pathologists, which are tedious to produce. This is why in general only slide-level labels are accessible while annotations of small regions (or tiles) are limited. With only slide-level information, it is difficult to obtain accurate predictions of the localization of pathological tissues inside a slide, despite reaching good slide-level classification. Besides, existing algorithms show limited consistency between slide- and tile-level predictions, leading to difficult interpretation in case of healthy tissue. Using the attention-based multiple instance learning framework, we propose to combine slide-level labels on all slides with tile-level labels on a small fraction (e.g. 20%) of slides within a histology dataset to improve both classification and localization performances. With this mixed supervision of slides, we aim to enforce a better consistency between slide- and tile-level labels. To this end, we introduce an attention-based loss function to further guide the model’s attention on discriminative regions inside tumorous slides, and display an equal attention among all tiles of normal slides. On the Camelyon16 dataset, we reached precision and recall scores as high as 0.99 and 0.85 respectively with an AUC of 0.93 on the competition test set, using only 50% of the slides with tile-level annotations in the training set. Experiments using various proportions of fully annotated slides in the training set show promising results for an improved localization of tumors and classification of slides. In this work, we showed that using a limited amount of fully annotated slides we can improve both the classification and localization performances of an attention-based deep learning model. This increased consistency and performances should help pathologists to better interpret the algorithm output and to focus on suspicious regions in probable tumorous slides. Citation Format: Paul Tourniaire, Marius Ilie, Paul Hofman, Nicholas Ayache, Hervé Delingette. Mixed supervision to improve the classification and localization: Coherence of tumors in histological slides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 461.

The outcome of patients with cutaneous melanoma has been strongly modified by recent advances obtained with Immune Checkpoint Inhibitors (ICIs). However, despite this breakthrough, durable response to ICIs is limited to a subset of patients. We investigated whether the expression of TRF2, which preserves telomere integrity, and have an effect on tumor immunosurveillance notably by directly recruiting and activating myeloid-derived suppressor cells (MDSCs), could be a prognostic biomarker in patients with relapsed or metastatic melanoma based on different treatment regimens. We evaluated retrospectively the association of TRF2 expressed in melanoma cells in combination with intratumoral CD33+ CD15+ CD14- MDSCs, as detected by immunohistochemistry and quantified by digital analysis, to clinicopathological features and overall survival (OS) among 48 patients treated with ICIs and 77 patients treated with other treatment options. The densities/mm2 of TRF2+ cells (P=.003) and CD33+ cells (P=.004) were individually significantly related to poor OS. In addition, only the combined expression of CD33+/CD15+/CD14- cells/mm2 was significantly correlated to poor OS (P=.017) in the whole study population as well as in patients treated by ICIs (P=.023). There was no significant difference in OS when analyzing the other markers individually or in combination according to the treatment regimen. The pre-treatment assessment of TRF2 expression and CD33+ cells/mm2 along with the density of CD33+/CD15+/CD14- cells/mm2 could assess OS and better predict clinical response of patients with melanoma treated by ICIs.