Single-cell multiomics profiling reveals heterogeneous transcriptional programs and microenvironment in DSRCTs.

Desmoplastic small round cell tumor (DSRCT) is a rare, aggressive sarcoma driven by the EWSR1::WT1 chimeric transcription factor. Despite this unique oncogenic driver, DSRCT displays a polyphenotypic differentiation of unknown causality. Using single-cell multi-omics on 12 samples from five patients, we find that DSRCT tumor cells cluster into consistent subpopulations with partially overlapping lineage- and metabolism-related transcriptional programs. In vitro modeling shows that high EWSR1::WT1 DNA-binding activity associates with most lineage-related states, in contrast to glycolytic and profibrotic states. Single-cell chromatin accessibility analysis suggests that EWSR1::WT1 binding site variability may drive distinct lineage-related transcriptional programs, supporting some level of cell-intrinsic plasticity. Spatial transcriptomics reveals that glycolytic and profibrotic states specifically localize within hypoxic niches at the periphery of tumor cell islets, suggesting an additional role of tumor cell-extrinsic microenvironmental cues. We finally identify a single-cell transcriptomics-derived epithelial signature associated with improved patient survival, highlighting the clinical relevance of our findings.

Spatial Profiling of Ovarian Carcinoma and Tumor Microenvironment Evolution under Neoadjuvant Chemotherapy.

PURPOSE: This study investigates changes in CD8+ cells, CD8+/Foxp3 ratio, HLA I expression, and immune coregulator density at diagnosis and upon neoadjuvant chemotherapy (NACT), correlating changes with clinical outcomes. EXPERIMENTAL DESIGN: Multiplexed immune profiling and cell clustering analysis were performed on paired matched ovarian cancer samples to characterize the immune tumor microenvironment (iTME) at diagnosis and under NACT in patients enrolled in the CHIVA trial (NCT01583322). RESULTS: Several immune cell (IC) subsets and immune coregulators were quantified pre/post-NACT. At diagnosis, patients with higher CD8+ T cells and HLA I+-enriched tumors were associated with a better outcome. The CD8+/Foxp3+ ratio increased significantly post-NACT in favor of increased immune surveillance, and the influx of CD8+ T cells predicted better outcomes. Clustering analysis stratified pre-NACT tumors into four subsets: high Binf, enriched in B clusters; high Tinf and low Tinf, according to their CD8+ density; and desert clusters. At baseline, these clusters were not correlated with patient outcomes. Under NACT, tumors were segregated into three clusters: high BinfTinf, low Tinf, and desert. The high BinfTinf, more diverse in IC composition encompassing T, B, and NK cells, correlated with improved survival. PDL1 was rarely expressed, whereas TIM3, LAG3, and IDO1 were more prevalent. CONCLUSIONS: Several iTMEs exist during tumor evolution, and the NACT impact on iTME is heterogeneous. Clustering analysis of patients unravels several IC subsets within ovarian cancer and can guide future personalized approaches. Targeting different checkpoints such as TIM3, LAG3, and IDO1, more prevalent than PDL1, could more effectively harness antitumor immunity in this anti-PDL1-resistant malignancy.

Molecular profiling of biliary tract cancers reveals distinct genomic landscapes between circulating and tissue tumor DNA.

Biliary tract cancers (BTCs) are heterogeneous malignancies with dismal prognosis due to tumor aggressiveness and poor response to limited current therapeutic options. Tumor exome profiling has allowed to successfully establish targeted therapeutic strategies in the clinical management of cholangiocarcinoma (CCA). Still, whether liquid biopsy profiling could inform on BTC biology and patient management is unknown. In order to test this and generate novel insight into BTC biology, we analyzed the molecular landscape of 128 CCA patients, using a 394-gene NGS panel (Foundation Medicine). Among them, 32 patients had matched circulating tumor (ct) DNA and tumor DNA samples, where both samples were profiled. In both tumor and liquid biopsies, we identified an increased frequency of alterations in genes involved in genome integrity or chromatin remodeling, including ARID1A (15%), PBRM1 (9%), and BAP1 (14%), which were validated using an in-house-developed immunohistochemistry panel. ctDNA and tumor DNA showed variable concordance, with a significant correlation in the total number of detected variants, but some heterogeneity in the detection of actionable mutations. FGFR2 mutations were more frequently identified in liquid biopsies, whereas KRAS alterations were mostly found in tumors. All IDH1 mutations detected in tumor DNA were also identified in liquid biopsies. These findings provide novel insights in the concordance between the tumor and liquid biopsies genomic landscape in a large cohort of patients with BTC and highlight the complementarity of both analyses when guiding therapeutic prescription.

Trastuzumab deruxtecan in metastatic breast cancer with variable HER2 expression: the phase 2 DAISY trial.

The mechanisms of action of and resistance to trastuzumab deruxtecan (T-DXd), an anti-HER2-drug conjugate for breast cancer treatment, remain unclear. The phase 2 DAISY trial evaluated the efficacy of T-DXd in patients with HER2-overexpressing (n = 72, cohort 1), HER2-low (n = 74, cohort 2) and HER2 non-expressing (n = 40, cohort 3) metastatic breast cancer. In the full analysis set population (n = 177), the confirmed objective response rate (primary endpoint) was 70.6% (95% confidence interval (CI) 58.3-81) in cohort 1, 37.5% (95% CI 26.4-49.7) in cohort 2 and 29.7% (95% CI 15.9-47) in cohort 3. The primary endpoint was met in cohorts 1 and 2. Secondary endpoints included safety. No new safety signals were observed. During treatment, HER2-expressing tumors (n = 4) presented strong T-DXd staining. Conversely, HER2 immunohistochemistry 0 samples (n = 3) presented no or very few T-DXd staining (Pearson correlation coefficient r = 0.75, P = 0.053). Among patients with HER2 immunohistochemistry 0 metastatic breast cancer, 5 of 14 (35.7%, 95% CI 12.8-64.9) with ERBB2 expression below the median presented a confirmed objective response as compared to 3 of 10 (30%, 95% CI 6.7-65.2) with ERBB2 expression above the median. Although HER2 expression is a determinant of T-DXd efficacy, our study suggests that additional mechanisms may also be involved. (ClinicalTrials.gov identifier NCT04132960 .).

Deleting Snord115 genes in mice remodels monoaminergic systems activity in the brain toward cortico-subcortical imbalances.

The neuronal-specific SNORD115 has gathered interest because its deficiency may contribute to the pathophysiology of Prader-Willi syndrome (PWS), possibly by altering post-transcriptional regulation of the gene encoding the serotonin (HTR2C) receptor. Yet, Snord115-KO mice do not resume the main symptoms of PWS, and only subtle-altered A-to-I RNA editing of Htr2c mRNAs was uncovered. Because HTR2C signaling fine-tunes the activity of monoaminergic neurons, we addressed the hypothesis that lack of Snord115 alters monoaminergic systems. We first showed that Snord115 was expressed in both monoaminergic and non-monoaminergic cells of the ventral tegmental area (VTA) and the dorsal raphe nucleus (DRN) harboring cell bodies of dopaminergic and serotonergic neurons, respectively. Measuring the tissue level of monoamines and metabolites, we found very few differences except that the content of homovanillic acid-a metabolite of dopamine-was decreased in the orbitofrontal and prefrontal cortex of Snord115-KO mice. The latter effects were, however, associated with a few changes in monoamine tissue content connectivity across the 12 sampled brain regions. Using in vivo single-cell extracellular recordings, we reported that the firing rate of VTA dopaminergic neurons and DRN serotonergic neurons was significantly increased in Snord115-KO mice. These neural circuit dysfunctions were not, however, associated with apparent defects in binge eating, conditioned place preference to cocaine, cocaine-induced hyperlocomotion or compulsive behavior. Altogether, our multiscale study shows that the absence of Snord115 impacts central monoaminergic circuits to an extent that does not elicit gross behavioral abnormalities.

Escherichia coli-Specific CXCL13-Producing TFH Are Associated with Clinical Efficacy of Neoadjuvant PD-1 Blockade against Muscle-Invasive Bladder Cancer.

Biomarkers guiding the neoadjuvant use of immune-checkpoint blockers (ICB) are needed for patients with localized muscle-invasive bladder cancers (MIBC). Profiling tumor and blood samples, we found that follicular helper CD4+ T cells (TFH) are among the best therapeutic targets of pembrolizumab correlating with progression-free survival. TFH were associated with tumoral CD8 and PD-L1 expression at baseline and the induction of tertiary lymphoid structures after pembrolizumab. Blood central memory TFH accumulated in tumors where they produce CXCL13, a chemokine found in the plasma of responders only. IgG4+CD38+ TFH residing in bladder tissues correlated with clinical benefit. Finally, TFH and IgG directed against urothelium-invasive Escherichia coli dictated clinical responses to pembrolizumab in three independent cohorts. The links between tumor infection and success of ICB immunomodulation should be prospectively assessed at a larger scale. SIGNIFICANCE: In patients with bladder cancer treated with neoadjuvant pembrolizumab, E. coli-specific CXCL13 producing TFH and IgG constitute biomarkers that predict clinical benefit. Beyond its role as a biomarker, such immune responses against E. coli might be harnessed for future therapeutic strategies. This article is highlighted in the In This Issue feature, p. 2221.

Targeting genome integrity dysfunctions impedes metastatic potency in non-small cell lung cancer circulating tumor cell-derived explants.

DNA damage and genomic instability contribute to non-small cell lung cancer (NSCLC) etiology and progression. However, their therapeutic exploitation is disappointing. CTC-derived explants (CDX) offer systems for mechanistic investigation of CTC metastatic potency and may provide rationale for biology-driven therapeutics. Four CDX models and 3 CDX-derived cell lines were established from NSCLC CTCs and recapitulated patient tumor histology and response to platinum-based chemotherapy. CDX (GR-CDXL1, GR-CDXL2, GR-CDXL3, GR-CDXL4) demonstrated considerable mutational landscape similarity with patient tumor biopsy and/or single CTCs. Truncal alterations in key DNA damage response (DDR) and genome integrity-related genes were prevalent across models and assessed as therapeutic targets in vitro, in ovo, and in vivo. GR-CDXL1 presented homologous recombination deficiency linked to biallelic BRCA2 mutation and FANCA deletion, unrepaired DNA lesions after mitosis, and olaparib sensitivity, despite resistance to chemotherapy. SLFN11 overexpression in GR-CDXL4 led to olaparib sensitivity and was in coherence with neuroendocrine marker expression in patient tumor biopsy, suggesting a predictive value of SLFN11 in NSCLC histological transformation into small cell lung cancer (SCLC). Centrosome clustering promoted targetable chromosomal instability in GR-CDXL3 cells. These CDX unravel DDR and genome integrity-related defects as a central mechanism underpinning metastatic potency of CTCs and provide rationale for their therapeutic targeting in metastatic NSCLC.

A loss-of-function polymorphism in ATG16L1 compromises therapeutic outcome in head and neck carcinoma patients.

The anticancer immune response is shaped by immunogenic cell stress and death pathways. Thus, cancer cells can release danger-associated molecular patterns that act on pattern recognition receptors expressed by dendritic cells and their precursors to elicit an antitumor immune response. Here, we investigated the impact of single nucleotide polymorphisms (SNPs) in genes affecting this cancer-immunity dialogue in the context of head and neck squamous cell carcinoma (HNSCC). We observed that homozygosity for a loss-of-function SNP (rs2241880, leading to the substitution of a threonine residue in position 300 by an alanine) affecting autophagy related 16 like 1 (ATG16L1) is coupled to poor progression-free survival in platinum-treated HNSCC patients. This result was obtained on a cohort of patients enrolled at the Gustave Roussy Cancer Campus and was validated on an independent cohort of The Cancer Genome Atlas (TCGA). Homozygosity in rs2241880 is well known to predispose to Crohn's disease, and epidemiological associations between Crohn's disease and HNSCC have been reported at the levels of cancer incidence and prognosis. We speculate that rs2241880 might be partially responsible for this association.

Expression of the Immune Checkpoint Regulators LAG-3 and TIM-3 in Classical Hodgkin Lymphoma.

INTRODUCTION: The role of the programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) axis is well established in classical Hodgkin lymphoma (HL), where PD-1 blockade demonstrated spectacular efficacy in relapsed/refractory disease. However, little is known about the frequency and cellular distribution of other immune checkpoints in HL samples. PATIENTS AND METHODS: Using immunohistochemistry, we investigated, along with PD-L1 and PD-1, the expression of lymphocyte-activation gene 3 (LAG-3) and T-cell immunoglobulin and mucin-domain containing 3 (TIM-3) in 57 biopsy samples of patients with classical HL. RESULTS: Hodgkin and Reed/Sternberg (HRS) cells were strongly positive for PD-L1 in nearly all cases. HRS cells were TIM-3 positive in 36% of samples, whereas LAG-3 was rarely expressed (5.2%). In the microenvironment, PD-1, LAG-3, and TIM-3 were expressed by ≥ 5% of cells in 65%, 98%, and 96% of cases, respectively. T-cell rosettes surrounding HRS cells consisted of CD4+ FoxP3- helper T cells expressing both PD-1 and LAG-3, with a variable expression of TIM-3. CONCLUSION: This study demonstrates for the first time that LAG-3 and TIM-3 are nearly always expressed in the microenvironment of classical HL. This may constitute the basis for targeting LAG-3 or TIM-3 in combination with anti-PD-1 antibodies in the treatment of relapsed/refractory HL.

Reassessment of the involvement of Snord115 in the serotonin 2c receptor pathway in a genetically relevant mouse model.

SNORD115 has been proposed to promote the activity of serotonin (HTR2C) receptor via its ability to base pair with its pre-mRNA and regulate alternative RNA splicing and/or A-to-I RNA editing. Because SNORD115 genes are deleted in most patients with the Prader-Willi syndrome (PWS), diminished HTR2C receptor activity could contribute to the impaired emotional response and/or compulsive overeating characteristic of this disease. In order to test this appealing but never demonstrated hypothesis in vivo, we created a CRISPR/Cas9-mediated Snord115 knockout mouse. Surprisingly, we uncovered only modest region-specific alterations in Htr2c RNA editing profiles, while Htr2c alternative RNA splicing was unchanged. These subtle changes, whose functional relevance remains uncertain, were not accompanied by any discernible defects in anxio-depressive-like phenotypes. Energy balance and eating behavior were also normal, even after exposure to high-fat diet. Our study raises questions concerning the physiological role of SNORD115, notably its involvement in behavioural disturbance associated with PWS.

DNA FISH Diagnostic Assay on Cytological Samples of Thyroid Follicular Neoplasms.

Although fine-needle aspiration cytology (FNAC) is helpful in determining whether thyroid nodules are benign or malignant, this distinction remains a cytological challenge in follicular neoplasms. Identification of genomic alterations in cytological specimens with direct and routine techniques would therefore have great clinical value. A series of 153 cases consisting of 72 and 81 histopathologically confirmed classic follicular adenomas (cFAs) and classic follicular thyroid carcinomas (cFTCs), respectively, was studied by means of different molecular techniques in three different cohorts of patients (pts). In the first cohort (training set) of 66 pts, three specific alterations characterized by array comparative genomic hybridization (aCGH) were exclusively found in half of cFTCs. These structural abnormalities corresponded to losses of 1p36.33-35.1 and 22q13.2-13.31, and gain of whole chromosome X. The second independent cohort (validation set) of 60 pts confirmed these data on touch preparations of frozen follicular neoplasms by triple DNA fluorescent in situ hybridization using selected commercially available probes. The third cohort, consisting of 27 archived cytological samples from an equal number of pts that had been obtained for preoperative FNAC and morphologically classified as and histologically verified to be follicular neoplasms, confirmed our previous findings and showed the feasibility of the DNA FISH (DNA fluorescent in situ hybridization) assay. All together, these data suggest that our triple DNA FISH diagnostic assay may detect 50% of cFTCs with a specificity higher than 98% and be useful as a low-cost adjunct to cytomorphology to help further classify follicular neoplasms on already routinely stained cytological specimens.

Genetic characterization of a unique neuroendocrine transdifferentiation prostate circulating tumor cell-derived eXplant model.

Transformation of castration-resistant prostate cancer (CRPC) into an aggressive neuroendocrine disease (CRPC-NE) represents a major clinical challenge and experimental models are lacking. A CTC-derived eXplant (CDX) and a CDX-derived cell line are established using circulating tumor cells (CTCs) obtained by diagnostic leukapheresis from a CRPC patient resistant to enzalutamide. The CDX and the derived-cell line conserve 16% of primary tumor (PT) and 56% of CTC mutations, as well as 83% of PT copy-number aberrations including clonal TMPRSS2-ERG fusion and NKX3.1 loss. Both harbor an androgen receptor-null neuroendocrine phenotype, TP53, PTEN and RB1 loss. While PTEN and RB1 loss are acquired in CTCs, evolutionary analysis suggest that a PT subclone harboring TP53 loss is the driver of the metastatic event leading to the CDX. This CDX model provides insights on the sequential acquisition of key drivers of neuroendocrine transdifferentiation and offers a unique tool for effective drug screening in CRPC-NE management.

Developmental changes of rRNA ribose methylations in the mouse.

A sequencing-based profiling method (RiboMeth-seq) for ribose methylations was used to study methylation patterns in mouse adult tissues and during development. In contrast to previous reports based on studies of human cancer cell lines, almost all methylation sites were close to fully methylated in adult tissues. A subset of sites was differentially modified in developing tissues compared to their adult counterparts and showed clear developmental dynamics. This provides the first evidence for ribosome heterogeneity at the level of rRNA modifications during mouse development. In a prominent example, the expression levels of SNORD78 during development appeared to be regulated by alternative splicing of the Gas5 host-gene and to correlate with the methylation level of its target site at LSU-G4593. The results are discussed in the context of the specialized ribosome hypothesis.

Does smoking alter the mutation profile of human papillomavirus-driven head and neck cancers?

BACKGROUND: Human papillomavirus (HPV)-driven oropharyngeal cancer (OPC) patients are characterised by a better prognosis than their HPV-negative counterparts. However, this significant survival advantage is not homogeneous and among HPV-positive patients those with a smoking history have a significantly increased risk of oncologic failure. The reason why tobacco consumption impacts negatively the prognosis is still elusive. Tobacco might induce additional genetic alterations leading to a more aggressive phenotype. The purpose of this study was to characterise the mutational profile of HPV-positive OPCs by smoking status. We hypothesise a higher frequency of mutations affecting smokers. METHODS: Targeted next-generation sequencing of 39 genes that are recurrently mutated in head and neck cancers (HNCs) caused by tobacco/alcohol consumption was performed in 62 HPV-driven OPC cases including smokers and non-smokers. RESULTS: The study population included 37 (60%) non-smokers and 25 (40%) smokers. Twenty (32%) patients had no mutation, 14 (23%) had 1 mutation and 28 (45%) had 2 or more mutations. The most commonly mutated genes regardless of tobacco consumption were PIK3CA (19%), MLL2 (19%), TP53 (8%), FAT 1 (15%), FBXW7 (16%), NOTCH1 (10%) and FGFR3 (10%). Mutation rate was not significantly different in smokers compared with non-smokers even when analyses focused on heavy smokers (>20 pack-years vs. <20 pack-years). Similarly, there was no significant difference in mutations patterns according to tobacco consumption. CONCLUSION: In HPV-positive patients, smoking does not increase the mutation rate of genes that are recurrently mutated in traditional HNC. Additional studies are warranted to further describe the molecular landscape of HPV-driven OPC according to tobacco consumption.

Small-RNA sequencing identifies dynamic microRNA deregulation during skeletal muscle lineage progression.

Skeletal muscle satellite cells are quiescent adult resident stem cells that activate, proliferate and differentiate to generate myofibres following injury. They harbour a robust proliferation potential and self-renewing capacity enabling lifelong muscle regeneration. Although several classes of microRNAs were shown to regulate adult myogenesis, systematic examination of stage-specific microRNAs during lineage progression from the quiescent state is lacking. Here we provide a genome-wide assessment of the expression of small RNAs during the quiescence/activation transition and differentiation by RNA-sequencing. We show that the majority of small RNAs present in quiescent, activated and differentiated muscle cells belong to the microRNA class. Furthermore, by comparing expression in distinct cell states, we report a massive and dynamic regulation of microRNAs, both in numbers and amplitude, highlighting their pivotal role in regulation of quiescence, activation and differentiation. We also identify a number of microRNAs with reliable and specific expression in quiescence including several maternally-expressed miRNAs generated at the imprinted Dlk1-Dio3 locus. Unexpectedly, the majority of class-switching miRNAs are associated with the quiescence/activation transition suggesting a poised program that is actively repressed. These data constitute a key resource for functional analyses of miRNAs in skeletal myogenesis, and more broadly, in the regulation of stem cell self-renewal and tissue homeostasis.

Assessment of programmed death-ligand 1 expression and tumor-associated immune cells in pediatric cancer tissues.

BACKGROUND: Programmed death 1 (PD-1) signaling in the tumor microenvironment dampens immune responses to cancer, and blocking this axis induces antitumor effects in several malignancies. Clinical studies of PD-1 blockade are only now being initiated in pediatric patients, and little is known regarding programmed death-ligand 1 (PD-L1) expression in common childhood cancers. The authors characterized PD-L1 expression and tumor-associated immune cells (TAICs) (lymphocytes and macrophages) in common pediatric cancers. METHODS: Whole slide sections and tissue microarrays were evaluated by immunohistochemistry for PD-L1 expression and for the presence of TAICs. TAICs were also screened for PD-L1 expression. RESULTS: Thirty-nine of 451 evaluable tumors (9%) expressed PD-L1 in at least 1% of tumor cells. The highest frequency histotypes comprised Burkitt lymphoma (80%; 8 of 10 tumors), glioblastoma multiforme (36%; 5 of 14 tumors), and neuroblastoma (14%; 17 of 118 tumors). PD-L1 staining was associated with inferior survival among patients with neuroblastoma (P = .004). Seventy-four percent of tumors contained lymphocytes and/or macrophages. Macrophages were significantly more likely to be identified in PD-L1-positive versus PD-L1-negative tumors (P < .001). CONCLUSIONS: A subset of diagnostic pediatric cancers exhibit PD-L1 expression, whereas a much larger fraction demonstrates infiltration with tumor-associated lymphocytes. PD-L1 expression may be a biomarker for poor outcome in neuroblastoma. Further preclinical and clinical investigation will define the predictive nature of PD-L1 expression in childhood cancers both at diagnosis and after exposure to chemoradiotherapy. Cancer 2017;123:3807-3815. © 2017 American Cancer Society.

Transcriptional response to hypoxic stress in melanoma and prognostic potential of GBE1 and BNIP3.

Gradients of hypoxia occur in most solid tumors and cells found in hypoxic regions are associated with the most aggressive and therapy-resistant fractions of the tumor. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia in melanoma. Using microarray technology, whole genome gene expression profiling was first performed on established melanoma cell lines. From gene set enrichment analyses, we derived a robust 35 probes signature (hypomel for HYPOxia MELanoma) associated with hypoxia-response pathways, including 26 genes up regulated, and 9 genes down regulated. The microarray data were validated by RT-qPCR for the 35 transcripts. We then validated the signature in hypoxic zones from 8 patient specimens using laser microdissection or macrodissection of Formalin fixed-paraffin-embedded (FFPE) material, followed with RT-qPCR. Moreover, a similar hypoxia-associated gene expression profile was observed using NanoString technology to analyze RNAs from FFPE melanoma tissues of a cohort of 19 patients treated with anti-PD1. Analysis of NanoString data from validation sets using Non-Negative Matrix Factorization (NMF) analysis (26 genes up regulated in hypoxia) and dual clustering (samples and genes) further revealed that the increased level of BNIP3 (Bcl-2 adenovirus E1B 19 kDa-interacting protein 3)/GBE1 (glycogen branching enzyme1) differential pair correlates with the lack of response of melanoma patients to anti-PD1 (pembrolizumab) immunotherapy. These studies suggest that through elevated glycogenic flux and induction of autophagy, hypoxia is a critical molecular program that could be considered as a prognostic factor for melanoma.

Phenotypic and genetic heterogeneity of tumor tissue and circulating tumor cells in patients with metastatic castration-resistant prostate cancer: A report from the PETRUS prospective study.

Molecular characterization of cancer samples is hampered by tumor tissue availability in metastatic castration-resistant prostate cancer (mCRPC) patients. We reported the results of prospective PETRUS study of biomarker assessment in paired primary prostatic tumors, metastatic biopsies and circulating tumor cells (CTCs). Among 54 mCRPC patients enrolled, 38 (70%) had biopsies containing more than 50% tumour cells. 28 (52%) patients were analyzed for both tissue samples and CTCs. FISH for AR-amplification and TMPRSS2-ERG translocation were successful in 54% and 32% in metastatic biopsies and primary tumors, respectively. By comparing CellSearch and filtration (ISET)-enrichment combined to four color immunofluorescent staining, we showed that CellSearch and ISET isolated distinct subpopulations of CTCs: CTCs undergoing epithelial-to-mesenchymal transition, CTC clusters and large CTCs with cytomorphological characteristics but no detectable markers were isolated using ISET. Epithelial CTCs detected by the CellSearch were mostly lost during the ISET-filtration. AR-amplification was detected in CellSearch-captured CTCs, but not in ISET-enriched CTCs which harbor exclusively AR gain of copies. Eighty-eight percent concordance for ERG-rearrangement was observed between metastatic biopsies and CTCs even if additional ERG-alteration patterns were detected in ISET-enriched CTCs indicating a higher heterogeneity in CTCs.Molecular screening of metastatic biopsies is achievable in a multicenter context. Our data indicate that CTCs detected by the CellSearch and the ISET-filtration systems are not only phenotypically but also genetically different. Close attention must be paid to CTC characterization since neither approach tested here fully reflects the tremendous phenotypic and genetic heterogeneity present in CTCs from mCRPC patients.

Deletion of the miR-379/miR-410 gene cluster at the imprinted Dlk1-Dio3 locus enhances anxiety-related behaviour.

The brain-specific miR-379/miR-410 gene cluster at the imprinted Dlk1-Dio3 domain is implicated in several aspects of brain development and function, particularly in fine-tuning the dendritic outgrowth and spine remodelling of hippocampal neurons. Whether it might influence behaviour and memory-related processes has not yet been explored at the whole organism level. We previously reported that constitutive deletion of the miR-379/miR-410 gene cluster affects metabolic adaptation in neonatal mice. Here, we examined the role of this cluster in adult brain functions by subjecting mice with the constitutive deletion to a battery of behavioural and cognitive tests. We found that the lack of miR-379/miR-410 expression is associated with abnormal emotional responses, as demonstrated by increased anxiety-related behaviour in unfamiliar environments. In contrast, spontaneous exploration, general locomotion, mood levels and sociability remained unaltered. Surprisingly, miR-379/miR-410-deficient mice also showed normal learning and spatial (or contextual) memory abilities in hippocampus-dependent tasks involving neuronal plasticity. Taken together, the imprinted miR-379/miR-410 gene cluster thus emerges as a novel regulator of the two main post-natal physiological processes previously associated with imprinted, protein-coding genes: behaviour and energy homeostasis.

Mutation-Independent Activation of the Anaplastic Lymphoma Kinase in Neuroblastoma.

Activating mutations of anaplastic lymphoma kinase (ALK) have been identified as important players in neuroblastoma development. Our goal was to evaluate the significance of overall ALK activation in neuroblastoma. Expression of phosphorylated ALK, ALK, and its putative ligands, pleiotrophin and midkine, was screened in 289 neuroblastomas and 56 paired normal tissues. ALK was expressed in 99% of tumors and phosphorylated in 48% of cases. Pleiotrophin and midkine were expressed in 58% and 79% of tumors, respectively. ALK activation was significantly higher in tumors than in paired normal tissues, together with ALK and midkine expression. ALK activation was largely independent of mutations and correlated with midkine expression in tumors. ALK activation in tumors was associated with favorable features, including a younger age at diagnosis, hyperdiploidy, and detection by mass screening. Antitumor activity of the ALK inhibitor TAE684 was evaluated in wild-type or mutated ALK neuroblastoma cell lines and xenografts. TAE684 was cytotoxic in vitro in all cell lines, especially those harboring an ALK mutation. TAE684 efficiently inhibited ALK phosphorylation in vivo in both F1174I and R1275Q xenografts but demonstrated antitumor activity only against the R1275Q xenograft. In conclusion, ALK activation occurs frequently during neuroblastoma oncogenesis, mainly through mutation-independent mechanisms. However, ALK activation is not associated with a poor outcome and is not always a driver of cell proliferation and/or survival in neuroblastoma.