Abstract Circulating tumor cells (CTCs) are the potential seeds of distant metastases; however, little is known about how they survive in the bloodstream. Using a large cohort of colorectal cancer (CRC) patients, we found that the pseudokinase receptor PTK7 is highly expressed in primary tumors and metastatic lesions. Consistent with previous reports, high PTK7 expression is associated with reduced disease-free survival and increased metastatic dissemination. Surprisingly, PTK7 is absent from most CTCs and undergoes a cell-autonomous ON tumor /OFF CTC /ON metastasis switch that can be recapitulated in a xenografted mouse model, in in vitro systems, and a fluidic platform. PTK7-negative cancer cells exhibit increased expression of YAP1-driven genes, senescence-like features, and enhanced resistance to hemodynamic stress following loss of cell-cell and cell-matrix adhesion. This adaptive phenotype depends on metalloproteases, notably ADAM17, whose cleavage activity remodels the CTCs surfaceome. Functionally, the PTK7 OFF CTC state confers enhanced metastatic potential in vivo , and can be pharmacologically suppressed using metalloprotease inhibitors. Collectively, our findings identify a reversible, cell-autonomous, protease-driven surfaceome remodeling program that enables metastatic adaptation during hematogenous dissemination. Highlights / statement of significance By investigating potential markers for circulating colorectal tumor cells with strong metastatic potential, we describe a reversible and cell-autonomous remodeling of the circulating tumor cell surfaceome in patients that confers resistance to anoikis and stress induced by entry into the bloodstream. One Sentence Summary The dynamic regulation of PTK7 serves as a surrogate marker for tumor cell plasticity, aggressiveness, survival in the bloodstream, and efficiency in forming metastases. Trial registration CTC colon Cohort: registered on https://ClinicalTrials.gov identifier NCT03256084 ; date of registration 2017-07-17 B-Org cohort: registered on https://ClinicalTrials.gov NCT05384184 ; date of registration 2019-06-06 Ethics statement for animal experiments Studies on animals were conducted in accordance with the current ethical standards of the European Community (Directive 2010/63/EU), the Ethics Committee for Animal Experimentation (CEEA#14) and the French Ministry of Higher Education and Research, which approved and authorized the entire procedure described in this paper (project number APAFIS #35294).

Background: Breast cancer (BC) is the most frequent and the most deadly cancer among women worldwide. Different classifications of disease (anatomoclinical, pathological, prognostic) are used for guiding the management of patients. Unfortunately, they fail to reflect the whole clinical heterogeneity of the disease. Because the phenotype of tumors is dependent on many genes, a large-scale and integrated molecular characterization of the genetic and epigenetic alterations should allow the identification of new molecular classes clinically relevant, as well as among the altered genes and/or pathways, the identification of new therapeutic targets. We focused on luminal B breast cancer molecular subtype whose clinical course is particularly pejorative and for which no targeted therapy exists.

Cancer stem cell (CSC) has paved the way to many fundamental and translational studies. Recent studies have highlighted differentiated breast cancer cells (non-CSCs) switching phenotype to CSCs in response to various stimuli, depicting the existence of cancer stem cell plasticity. Although strategies to reduce the phenotypic plasticity of non-CSCs into CSCs are likely to prevent treatment-resilient cancer cells driving recurrence, most phenotypic plasticity mechanisms involve Notch, Wnt or MAPK signaling pathways. In this study, breast cancer cells were irradiated to identify soluble reprogramming factors. Using conditioned medias, protein arrays analyses, flow cytometry and in cellulo/in vivo functional assays, we demonstrated, for the first time, that radiation-induced chemokine expression, especially CXCL1 and CCL5 and their receptors CXCR2, CCR1 and CCR5, stimulates reprogramming of breast non-CSCs into CSCs. Treatment of non-CSCs with recombinant CXCL1 and CCL5 is sufficient to induce cell reprogramming, while their inhibition can be used to prevent reprogramming and sensitize tumor to radiation. Moreover, analysis of gene expression profiles from 38 public merged databases demonstrated that combined over-expression of CXCL1/CXCR2, CCL5/CCR1 or CCL5/CCR5 has a poorer prognosis in patients treated with radiotherapy, suggesting a promising way for patient stratification, where individuals with elevated cytokine levels could benefit from radiotherapy in conjunction with cytokine inhibitors. Taken together, our findings provide a rationale to consider these axes as potential targets and predictive biomarkers in breast cancer patients.

Cytogenetic normal AML with NPM1 mutations forms a distinct AML entity, associated with an intermediate prognosis and a heterogeneous response to treatment. We previously described an epigenetic biomarker, defined by the level of H3K27me3 on 70kb of the HIST1 cluster in patient blast DNA. This epigenetic mark separates cytogenetically normal NPM1 mut AML into two groups of patients differing in their survival rate following chemotherapy. To better characterize the influence of the biomarker on disease progression, we performed transcriptomic and histone mark profiling on patient blasts according to the level of H3K27me3 HIST1 . Our integrated analysis revealed that the two groups of patients display differences in terms of transcriptomic, chromatin landscape and cell surface markers, which could explain the clinical difference. Our profiling revealed novel targets and therefore constitutes an (epi)transcriptomic resource for NPM1 AML. It also highlights the power of epigenetic profiling to dissect the heterogeneity of a single AML genetic entity.### Competing Interest StatementThe authors have declared no competing interest.

Neoadjuvant chemotherapy (NACT) became a standard treatment strategy for patients with inflammatory breast cancer (IBC) because of high disease aggressiveness. However, given the heterogeneity of IBC, no molecular feature reliably predicts the response to chemotherapy. Whole-exome sequencing (WES) of clinical tumor samples provides an opportunity to identify genomic alterations associated with chemosensitivity. We retrospectively applied WES to 44 untreated IBC primary tumor samples and matched normal DNA. The pathological response to NACT, assessed on operative specimen, distinguished the patients with versus without pathological complete response (pCR versus no-pCR respectively). We compared the mutational profiles, spectra and signatures, pathway mutations, copy number alterations (CNAs), HRD, and heterogeneity scores between pCR versus no-pCR patients. The TMB, HRD, and mutational spectra were not different between the complete (N = 13) versus non-complete (N = 31) responders. The two most frequently mutated genes were TP53 and PIK3CA. They were more frequently mutated in the complete responders, but the difference was not significant. Only two genes, NLRP3 and SLC9B1, were significantly more frequently mutated in the complete responders (23

Background Inflammatory breast cancer (IBC) is the most pro-metastatic form of BC. Better understanding of its enigmatic pathophysiology is crucial. We report here the largest whole-exome sequencing (WES) study of clinical IBC samples.Methods We retrospectively applied WES to 54 untreated IBC primary tumor samples and matched normal DNA. The comparator samples were 102 stage-matched non-IBC samples from TCGA. We compared the somatic mutational profiles, spectra and signatures, copy number alterations (CNAs), HRD and heterogeneity scores, and frequencies of actionable genomic alterations (AGAs) between IBCs and non-IBCs. The comparisons were adjusted for the molecular subtypes.Results The number of somatic mutations, TMB, and mutational spectra were not different between IBCs and non-IBCs, and no gene was differentially mutated or showed differential frequency of CNAs. Among the COSMIC signatures, only the age-related signature was more frequent in non-IBCs than in IBCs. We also identified in IBCs two new mutational signatures not associated with any environmental exposure, one of them having been previously related to HIF pathway activation. Overall, the HRD score was not different between both groups, but was higher in TN IBCs than TN non-IBCs. IBCs were less frequently classified as heterogeneous according to heterogeneity H-index than non-IBCs (21% vs 33%), and clonal mutations were more frequent and subclonal mutations less frequent in IBCs. More than 50% of patients with IBC harbored at least one high-level of evidence (LOE) AGA (OncoKB LOE 1-2, ESCAT LOE I-II), similarly to patients with non-IBC.Conclusions We provide the largest mutational landscape of IBC. Only a few subtle differences were identified with non-IBCs. The most clinically relevant one was the higher HRD score in TN IBCs than in TN non-IBCs, whereas the most intriguing one was the smaller intratumor heterogeneity of IBCs.

By identifying somatic mutations, whole-exome sequencing (WES) has become a technology of choice for the diagnosis and guiding treatment decisions in many cancers. Despite advances in the field of somatic variant detection and the emergence of sophisticated tools incorporating machine learning, accurately identifying somatic variants remains challenging. Each new somatic variant caller is often accompanied by claims of superior performance compared to predecessors. Furthermore, most comparative studies focus on a limited set of tools and reference datasets, leading to inconsistent results and making it difficult for laboratories to select the optimal solution. Our study comprehensively evaluated 20 somatic variant callers across four reference WES datasets. We subsequently assessed the performance of ensemble approaches by exploring all possible combinations of these callers, generating 8178 and 1013 combinations for single-nucleotide variants (SNVs) and indels, respectively, with varying voting thresholds. Our analysis identified five high-performing individual somatic variant callers: Muse, Mutect2, Dragen, TNScope, and NeuSomatic. For somatic SNVs, an ensemble combining LoFreq, Muse, Mutect2, SomaticSniper, Strelka, and Lancet outperformed the top-performing caller (Dragen) by >3.6% (mean F1 score = 0.927). Similarly, for somatic indels, an ensemble of Mutect2, Strelka, Varscan2, and Pindel outperformed the best individual caller (Neusomatic) by >3.5% (mean F1 score = 0.867). By considering the computational costs of each combination, we were able to identify an optimal solution involving four somatic variant callers, Muse, Mutect2, and Strelka for the SNVs and Mutect2, Strelka, and Varscan2 for the indels, enabling accurate and cost-effective somatic variant detection in whole exome.

Introduction. BH3 mimetics such as the BCL2 specific inhibitor (-inh) venetoclax (VEN) and the BCL2/BLCxL-inh navitoclax (NAV), are small molecules inducing intrinsic apoptotic cell death by inhibiting antiapoptotic proteins. VEN has changed the paradigm of treatment of acute myeloid leukemia (AML) unfit for intensive chemotherapy based on the VIALE-A study. Nevertheless, approximately 1/3 of AML is primary refractory to VEN (VEN-R), with no current treatment option. Monocytic differentiation, signaling mutations and MCL1 antiapoptotic dependency are among the most frequent and interconnected resistance mechanisms. Method. To find new therapeutics active in resistant AML, we collected clinical data and bone marrow or peripheral blasts at AML diagnosis in a prospective biobanking clinical trial named HEMATO-BIO-IPC 2013-015 (NCT02320656, PI: Prof. N Vey), between 2014 and 2019. We performed targeted DNA sequencing using a NGS panel of recurrently mutated AML genes and ex vivo drug sensitivity/resistance profiling (DSRP). Results. Taking the first 108 AML analyzed samples, we identified that 17 (15.7%) were resistant to NAV (NAV-R), taking a Z score >0.75 as a threshold. The patient median age was 62.5 yo, median leukocyte count, platelet count, percentage of blasts were 29.7 G/L (ranges, 1.1-86.3), 67 G/L (ranges, 18-163) and 72% (ranges, 21-93), respectively. Twelve AML were classified as FAB4 (n=6) and FAB5 (n=6). Cytogenetics was normal in 9 cases. NPM1 mutation was found in 6 and FLT3-ITD in 4 cases. Signaling mutations, including NRAS, KRAS and PTPN11 mutations, were found in 3, 2 and 2 cases, respectively. No samples were found with KIT mutations and only 2 samples had a TP53 mutation. We noticed a strong anticorrelation between NAV-R and sensitivity to most of the tested kinase inhibitors, including the Pi3K-inh BKM120 (Buparlisib) and Idelalisib, the JAK2-inh Ruxolitinib, the MEK-inh Trametinib, the mTOR-inh Temsirolimus, the FLT3-inh Quizartinib, and the BCR-ABL-inh Imatinib and Dasatinib (DASA). On the other hand, we did not observe any anticorrelation with the EGFR-inh erlotinib nor gefitinib. Amongst the most active kinase inhibitors, DASA had the lowest median IC50 (Z-score=0,01511). As DASA has already been used in clinical settings in AML, we chose DASA for further tests. We first confirmed the anticorrelation between DASA and the BCL2/BCLxL-inh ABT797 in the BEAT-AML cohort. As NAV and ABT797 are both known BCL2/BCLxL-inh, we hypothesized that blasts sensitive do DASA (DASA-S) were in fact dependent on MCL1. Indeed, western blot (WB) analysis showed higher MCL1 and proapoptotic BIM protein levels. To link a possible dependence of DASA-S blasts to MCL1, we performed BH3 profiling in 25 AML samples. We confirmed that DASA-S samples had a strong MCL1 dependency as shown by a dose-dependent mitochondrial membrane depolarization using MS1 peptide and a high MS1/BAD ratio. We next hypothesized that DASA was targeting MCL1 through an indirect manner. We performed a WB of MCL1 using AML cell line K562 and HL60, and observed a dose dependent decrease in MCL1 protein levels upon DASA treatment while BCL2 expression was not altered. Collectively, these results suggest that DASA could be a potential MCL1 indirect inhibitor that could be used in the clinical settings to treat VEN-R AML. To prove this hypothesis, we designed a phase II clinical trial named VEN-R DASA-IPC 2022 067 (EUCT 2023-505846-24-00) that is currently enrolling patients failing a minimum of two VEN-AZA cycles. Conclusion. DASA may be efficient in targeting VEN-R by inhibiting MCL1. Preclinical studies based on the AML collections HEMATIO-BIO allowed us to ask clinical questions that can be addressed in early-phase clinical trial.

Supplementary Figure 1A from Integrated Profiling of Basal and Luminal Breast Cancers

Abstract Most studies of genomic rearrangements in common cancers have focused on regional gains and losses, but some rearrangements may break within specific genes. We previously reported that five breast cancer cell lines have chromosome translocations that break in the NRG1 gene and that could cause abnormal NRG1 expression. NRG1 encodes the Neuregulins 1 (formerly the Heregulins), ligands for members of the ErbB/epidermal growth factor-receptor family, which includes ErbB2/HER2. We have now screened for breaks at NRG1 in paraffin sections of breast tumors. Tissue microarrays were screened by fluorescence in situ hybridization, with hybridization probes proximal and distal to the expected breakpoints. This screen detects breaks but does not distinguish between translocation or deletion breakpoints. The screen was validated with array-comparative genomic hybridization on a custom 8p12 high-density genomic array to detect a lower copy number of the sequences that were lost distal to the breaks. We also precisely mapped the breaks in five tumors with different hybridization probes. Breaks in NRG1 were detected in 6% (19 of 323) of breast cancers and in some lung and ovarian cancers. In an unselected series of 213 cases with follow-up, breast cancers where the break was detected tended to be high-grade (65% grade III compared with 28% of negative cases). They were, like breast tumors in general, mainly ErbB2 low (11 of 13 were low) and estrogen receptor positive (11 of 13 positive).

Supplementary Table 2 from Breast Cancer Cell Lines Contain Functional Cancer Stem Cells with Metastatic Capacity and a Distinct Molecular Signature

Supplementary Figures 1-8 from Distinct Roles of BARD1 Isoforms in Mitosis: Full-Length BARD1 Mediates Aurora B Degradation, Cancer-Associated BARD1β Scaffolds Aurora B and BRCA2

β-catenin activation in GIST is associated with survival

PDF file 43K, Phenotype of BCLs after abexinostat treatment

PDF file - 320K, Supplemental Data 1: Ligands and receptors respectively expressed by tumor and NK-cells. Natural killer (NK) cells are components of the innate immune system that play a central role against tumor cells. They have an innate ability to distinguish normal from "modified" cells, such as cancer cells, through a broad range of activating and inhibitory receptors. These receptors act like little sensors of the malignant self (Moretta et al, 2006); the integration of these opposite signals determines whether an NK-cell will eliminate a potential target or not. NK-cell activating receptors are the natural cytotoxicity receptors (NKp30, NKp46 and NKp44) and NKG2D. They recognize various ligands expressed by transformed cells and/or stressed or activated cells. To date, NKp30 has two described ligands, B7H6 and BAT3. NKG2D can bind MICA, MICB and the members of the ULBP family. The ligand(s) of NKp46 or NKp44 are unknown. The main NK-cell inhibitory receptors are the killer immunoglobulin receptors (KIRs) and NKG2A (Moretta et al., 1995 & 2001), which recognize members of the HLA-I family expressed by normal cells, respectively HLA-A, -B or -C for the KIRs and HLA-E for NKG2A. The signal is fine-tuned by co-activators such as DNAM-1, CD2, NKp80, 2B4, NTBA and co-inhibitors such as the CD85 family, B7-H3 or LAIR. Among co-activator receptors, DNAM-1 binds the heterodimer PVR/Nectin2 receptors, CD2 binds CD58, NTBA binds another NTBA molecule, and 2B4 binds CD48. The co-inhibitory receptors from the CD85 family bind HLA-I molecules, while ligands of the LAIR family and B7H3 are still unknown.

Table SVI: Correlation between histoclinical and molecular parameters and our BCSC-GES in 2609 patients from public datasets

Progastrin inhibition decreases colonosphere incidence, CSC frequency and ALDHhigh cell proportion in vitro.