Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice.

In Duchenne muscular dystrophy (DMD), dystrophin mutation leads to progressive lethal skeletal muscle degeneration. For unknown reasons, dystrophin deficiency does not recapitulate DMD in mice (mdx), which have mild skeletal muscle defects and potent regenerative capacity. We postulated that human DMD progression is a consequence of loss of functional muscle stem cells (MuSC), and the mild mouse mdx phenotype results from greater MuSC reserve fueled by longer telomeres. We report that mdx mice lacking the RNA component of telomerase (mdx/mTR) have shortened telomeres in muscle cells and severe muscular dystrophy that progressively worsens with age. Muscle wasting severity parallels a decline in MuSC regenerative capacity and is ameliorated histologically by transplantation of wild-type MuSC. These data show that DMD progression results, in part, from a cell-autonomous failure of MuSC to maintain the damage-repair cycle initiated by dystrophin deficiency. The essential role of MuSC function has therapeutic implications for DMD.

DNA damage signaling mediates the functional antagonism between replicative senescence and terminal muscle differentiation.

The molecular determinants of muscle progenitor impairment to regenerate aged muscles are currently unclear. We show that, in a mouse model of replicative senescence, decline in muscle satellite cell-mediated regeneration coincides with activation of DNA damage response (DDR) and impaired ability to differentiate into myotubes. Inhibition of DDR restored satellite cell differentiation ability. Moreover, in replicative human senescent fibroblasts, DDR precluded MYOD-mediated activation of the myogenic program. A DDR-resistant MYOD mutant could overcome this barrier by resuming cell cycle progression. Likewise, DDR inhibition could also restore MYOD's ability to activate the myogenic program in human senescent fibroblasts. Of note, we found that cell cycle progression is necessary for the DDR-resistant MYOD mutant to reverse senescence-mediated inhibition of the myogenic program. These data provide the first evidence of DDR-mediated functional antagonism between senescence and MYOD-activated gene expression and indicate a previously unrecognized requirement of cell cycle progression for the activation of the myogenic program.

Id genes are essential for early heart formation.

Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1-4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.

Gray matter volume of the feline cerebral cortex and structural plasticity following perinatal deafness.

In response to sensory deprivation, the brain adapts according to contemporary demands to efficiently navigate a modified perceptual environment. This reorganization may result in improved processing of the remaining senses-a phenomenon referred to as compensatory crossmodal plasticity. One approach to explore this neuroplasticity is to consider the macrostructural changes in neural tissue that mirror this functional optimization. The current study is the first of its kind to measure MRI-derived gray matter (GM) volumes of control felines (n=30), while additionally identifying volumetric differences in response to perinatal deafness (30 ototoxically-deafened cats). To accomplish this purpose, regional and morphometric methods were performed in parallel. The regional analysis evaluated volumetric alterations of global GM, as well as the volumes of 146 regions of interest (ROIs) and 12 functional subgroupings of these ROIs. Results revealed whole-brain GM preservation; however, somatosensory and visual cortices exhibited an overall increase in volume. On a smaller scale, this analysis uncovered two auditory ROIs (second auditory cortex, A2, and ventral auditory field, VAF) that decreased in volume alongside two visual regions (anteromedial lateral suprasylvian area, AMLS and splenial visual area, SVA) that increased-all localized within the right hemisphere. Comparatively, the findings of tensor-based morphometry (TBM) generally aligned with those of the ROI-based method, as this voxel-wise approach demonstrated clusters of expansion coincident with visual- and somatosensory-related loci; although, it failed to detect any GM reductions following deafness. As distinct differences were identified in each analysis, the current study highlights the importance of employing multiple methods when exploring MRI volumetry. Overall, this study proposes that volumetric alterations within sensory loci allude to a redistribution of cortical space arising from modified perceptual demands following auditory deprivation.

Automated registration-based skull stripping procedure for feline neuroimaging.

Skull stripping is a fundamental preprocessing step in modern neuroimaging analyses that consists of removing non-brain voxels from structural images. When performed entirely manually, this laborious step can be rate-limiting for analyses, with the potential to influence the population size chosen. This emphasizes the need for a fully- or semi-automated masking procedure to decrease man-hours without an associated decline in accuracy. These algorithms are plentiful in human neuroimaging but are relatively lacking for the plethora of animal species used in research. Unfortunately, software designed for humans cannot be easily transformed for animal use due to the high amount of tailoring required to accurately account for the considerable degree of variation within the highly folded human cortex. As most animals have a relatively less complex cerebral morphology, intersubject variability is consequently decreased, presenting the possibility to simply warp the brain mask of a template image into subject space for the purpose of skull stripping. This study presents the use of the Cat Automated Registration-based Skull Stripper (CARSS) tool on feline structural images. Validation metrics revealed that this method was able to perform on par with manual raters on >90 % of scans tested, and that its consistency across multiple runs was superior to that of masking performed by two independent raters. Additionally, CARSS outperformed three well-known skull stripping programs on the validation dataset. Despite a handful of manual interventions required, the presented tool reduced the man-hours required to skull strip 60 feline images over tenfold when compared to a fully manual approach, proving to be invaluable for feline neuroimaging studies, particularly those with large population sizes.

Connectome alterations following perinatal deafness in the cat.

Following sensory deprivation, areas and networks in the brain may adapt and reorganize to compensate for the loss of input. These adaptations are manifestations of compensatory crossmodal plasticity, which has been documented in both human and animal models of deafness-including the domestic cat. Although there are abundant examples of structural plasticity in deaf felines from retrograde tracer-based studies, there is a lack of diffusion-based knowledge involving this model compared to the current breadth of human research. The purpose of this study was to explore white matter structural adaptations in the perinatally-deafened cat via tractography, increasing the methodological overlap between species. Plasticity was examined by identifying unique group connections and assessing altered connectional strength throughout the entirety of the brain. Results revealed a largely preserved connectome containing a limited number of group-specific or altered connections focused within and between sensory networks, which is generally corroborated by deaf feline anatomical tracer literature. Furthermore, five hubs of cortical plasticity and altered communication following perinatal deafness were observed. The limited differences found in the present study suggest that deafness-induced crossmodal plasticity is largely built upon intrinsic structural connections, with limited remodeling of underlying white matter.

Oligo-metastatic neoPlasms from the gastro-intestinal tract: iDentIfiCaTIon of cliNical and molecular drivers: the PREDICTION study.

BACKGROUND: Metastatic disease in tumors originating from the gastrointestinal tract can exhibit varying degrees of tumor burden at presentation. Some patients follow a less aggressive disease course, characterized by a limited number of metastatic sites, referred to as "oligo-metastatic disease" (OMD). The precise biological characteristics that define the oligometastatic behavior remain uncertain. In this study, we present a protocol designed to prospectively identify OMD, with the aim of proposing novel therapeutic approaches and monitoring strategies. METHODS: The PREDICTION study is a monocentric, prospective, observational investigation. Enrolled patients will receive standard treatment, while translational activities will involve analysis of the tumor microenvironment and genomic profiling using immunohistochemistry and next-generation sequencing, respectively. The first primary objective (descriptive) is to determine the prevalence of biological characteristics in OMD derived from gastrointestinal tract neoplasms, including high genetic concordance between primary tumors and metastases, a significant infiltration of T lymphocytes, and the absence of clonal evolution favoring specific driver genes (KRAS and PIK3CA). The second co-primary objective (analytic) is to identify a prognostic score for true OMD, with a primary focus on metastatic colorectal cancer. The score will comprise genetic concordance (> 80%), high T-lymphocyte infiltration, and the absence of clonal evolution favoring driver genes. It is hypothesized that patients with true OMD (score 3+) will have a lower rate of progression/recurrence within one year (20%) compared to those with false OMD (80%). The endpoint of the co-primary objective is the rate of recurrence/progression at one year. Considering a reasonable probability (60%) of the three factors occurring simultaneously in true OMD (score 3+), using a significance level of α = 0.05 and a test power of 90%, the study requires a minimum enrollment of 32 patients. DISCUSSION: Few studies have explored the precise genetic and biological features of OMD thus far. In clinical settings, the diagnosis of OMD is typically made retrospectively, as some patients who undergo intensive treatment for oligometastases develop polymetastatic diseases within a year, while others do not experience disease progression (true OMD). In the coming years, the identification of true OMD will allow us to employ more personalized and comprehensive strategies in cancer treatment. TRIAL REGISTRATION: ClinicalTrials.gov ID NCT05806151.

The MITO CERV-2 trial: A randomized phase II study of cetuximab plus carboplatin and paclitaxel, in advanced or recurrent cervical cancer.

BACKGROUND: Cervical cancer cells often express Epidermal Growth Factor Receptor (EGFR). Cetuximab (CET), an anti-EGFR antibody, can be safely combined with carboplatin (C) and paclitaxel (P), a standard treatment for advanced/recurrent cervical cancer (ARCC) patients. PATIENTS AND METHODS: ARCC patients, ECOG PS ≤ 1, were randomized to CP for 6 cycles with or without CET (400 mg/m2 one week before starting CP, then 250 mg/m2 weekly) until disease progression or unacceptable toxicity. Event-free survival (EFS) was the primary endpoint. With a 4.5 months expected median EFS and a 6.4 months predicted EFS (HR 0.70), 0.20 one-tailed α and 80% power, 89 events were required for the final intent-to-treat analysis. RESULTS: 108 patients were assigned to CP (n = 53) or CP-CET (n = 55). Median age was 50, 69% were PS0, 76% had recurrent disease, 91% had distant metastasis and 57% had received previous chemotherapy. After a median follow-up of 23 months, 102 patients had an event, 97 progressed and 61 died. Median EFS was 4.7 and 6.0 months (one-tail P = 0.43), median PFS was 5.2 and 7.6 months (one-tail P = 0.20) and median OS was 17.7 and 17 months (one-tail P = 0.27), with CP and CP-CET, respectively. There was no difference in the occurrence of severe adverse events, except for skin toxicity. Biomarker analysis, in a small subgroup of patients, suggests that PIK3CA mutation might be predictive of CET resistance. CONCLUSION: CP-CET was not more active than CP alone in unselected ARCC patients.

Early forming label-retaining muscle stem cells require p27kip1 for maintenance of the primitive state.

Across different niches, subsets of highly functional stem cells are maintained in a relatively dormant rather than proliferative state. Our understanding of proliferative dynamics in tissue-specific stem cells during conditions of increased tissue turnover remains limited. Using a TetO-H2B-GFP reporter of proliferative history, we identify skeletal muscle stem cell, or satellite cells, that retain (LRC) or lose (nonLRC) the H2B-GFP label. We show in mice that LRCs and nonLRCs are formed at birth and persist during postnatal growth and adult muscle repair. Functionally, LRCs and nonLRCs are born equivalent and transition during postnatal maturation into distinct and hierarchically organized subsets. Adult LRCs give rise to LRCs and nonLRCs; the former are able to self-renew, whereas the latter are restricted to differentiation. Expression analysis revealed the CIP/KIP family members p21(cip1) (Cdkn1a) and p27(kip1) (Cdkn1b) to be expressed at higher levels in LRCs. In accordance with a crucial role in LRC fate, loss of p27(kip1) promoted proliferation and differentiation of LRCs in vitro and impaired satellite cell self-renewal after muscle injury. By contrast, loss of p21(cip1) only affected nonLRCs, in which myogenic commitment was inhibited. Our results provide evidence that restriction of self-renewal potential to LRCs is established early in life and is maintained during increased tissue turnover through the cell cycle inhibitor p27(kip1). They also reveal the differential role of CIP/KIP family members at discrete steps within the stem cell hierarchy.

Signal transducer and activator of transcription 3 signaling as a potential target to treat muscle wasting diseases.

PURPOSE OF REVIEW: The review summarizes our current knowledge of the role of signal transducer and activator of transcription 3 (STAT3) signaling in skeletal muscle regeneration and the maintenance of muscle mass. RECENT FINDINGS: STAT3 signaling plays a pivotal role in regulating the function of multiple cell types in skeletal muscle. This includes muscle stem cells, myofibers, and macrophages. It regulates muscle stem cell function by antagonizing self-renewal. STAT3 also functions in myofibers to regulate skeletal muscle mass. This is highly relevant under pathological conditions where STAT3 activation promotes protein degradation and muscle atrophy. Transient pharmacological inhibition of STAT3 partially prevents muscle wasting. However, the mechanisms responsible for the improvement of muscle condition are not currently well understood. This is because of the complexity of the system, as STAT3 has a critical role in regulating the function of several cell types residing in skeletal muscle. SUMMARY: Muscle wasting is associated with several human diseases such as muscle dystrophies or cancer cachexia. However, currently there are no effective treatments for this condition, and there is a critical need to identify new potential targets for the development of efficient therapeutic approaches.

Innervation of dystrophic muscle after muscle stem cell therapy.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is caused by loss of the structural protein, dystrophin, resulting in muscle fragility. Muscle stem cell (MuSC) transplantation is a potential therapy for DMD. It is unknown whether donor-derived muscle fibers are structurally innervated. METHODS: Green fluorescent protein (GFP)-expressing MuSCs were transplanted into the tibials anterior of adult dystrophic mdx/mTR mice. Three weeks later the neuromuscular junction was labeled by immunohistochemistry. RESULTS: The percent overlap between pre- and postsynaptic immunolabeling was greater in donor-derived GFP(+) myofibers, and fewer GFP(+) myofibers were identified as denervated compared with control GFP(-) fibers (P = 0.001 and 0.03). GFP(+) fibers also demonstrated acetylcholine receptor fragmentation and expanded endplate area, indicators of muscle reinnervation (P = 0.008 and 0.033). CONCLUSION: It is unclear whether GFP(+) fibers are a result of de novo synthesis or fusion with damaged endogenous fibers. Either way, donor-derived fibers demonstrate clear histological innervation. Muscle Nerve 54: 763-768, 2016.

Reprogramming towards pluripotency requires AID-dependent DNA demethylation.

Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.

EGFR mutations in lung cancer: from tissue testing to liquid biopsy.

ABSTRACT The presence of EGFR mutations predicts the sensitivity to EGF receptor (EGFR)-tyrosine kinase inhibitors in a molecularly defined subset of non-small-cell lung carcinoma (NSCLC) patients. For this reason, EGFR testing of NSCLC is required to provide personalized treatment options and better outcomes for NSCLC patients. As surgery specimens are not available in the majority of NSCLC, other currently available DNA sources are small biopsies and cytological samples, providing however limited and low-quality material. In order to address this issue, the use of surrogate sources of DNA, such as blood, serum and plasma samples, which often contains circulating free tumor DNA or circulating tumor cells, is emerging as a new strategy for tumor genotyping.

MicroRNA programs in normal and aberrant stem and progenitor cells.

Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ∼22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.

Self-renewal and expansion of single transplanted muscle stem cells.

Adult muscle satellite cells have a principal role in postnatal skeletal muscle growth and regeneration. Satellite cells reside as quiescent cells underneath the basal lamina that surrounds muscle fibres and respond to damage by giving rise to transient amplifying cells (progenitors) and myoblasts that fuse with myofibres. Recent experiments showed that, in contrast to cultured myoblasts, satellite cells freshly isolated or satellite cells derived from the transplantation of one intact myofibre contribute robustly to muscle repair. However, because satellite cells are known to be heterogeneous, clonal analysis is required to demonstrate stem cell function. Here we show that when a single luciferase-expressing muscle stem cell is transplanted into the muscle of mice it is capable of extensive proliferation, contributes to muscle fibres, and Pax7(+)luciferase(+) mononucleated cells can be readily re-isolated, providing evidence of muscle stem cell self-renewal. In addition, we show using in vivo bioluminescence imaging that the dynamics of muscle stem cell behaviour during muscle repair can be followed in a manner not possible using traditional retrospective histological analyses. By imaging luciferase activity, real-time quantitative and kinetic analyses show that donor-derived muscle stem cells proliferate and engraft rapidly after injection until homeostasis is reached. On injury, donor-derived mononucleated cells generate massive waves of cell proliferation. Together, these results show that the progeny of a single luciferase-expressing muscle stem cell can both self-renew and differentiate after transplantation in mice, providing new evidence at the clonal level that self-renewal is an autonomous property of a single adult muscle stem cell.

Harmonization of tumor mutation burden testing with comprehensive genomic profiling assays: an IQN Path initiative.

BACKGROUND: Although conflicting results emerged from different studies, the tumor mutational burden (TMB) appears as one of most reliable biomarkers of sensitivity to immune checkpoint inhibitors. Several laboratories are reporting TMB values when performing comprehensive genomic profiling (CGP) without providing a clinical interpretation, due to the lack of validated cut-off values. The International Quality Network for Pathology launched an initiative to harmonize TMB testing with CGP assay and favor the clinical implementation of this biomarker. METHODS: TMB evaluation was performed with three commercially available CGP panels, TruSight Oncology 500 (TSO500), Oncomine Comprehensive Plus Assay (OCA) and QIAseq Multimodal Panel (QIA), versus the reference assay FoundationOne CDx (F1CDx). Archived clinical samples derived from 60 patients with non-small cell lung cancer were used for TMB assessment. Adjusted cut-off values for each panel were calculated. RESULTS: Testing was successful for 91.7%, 100%, 96.7% and 100% of cases using F1CDx, TSO500, OCA and QIA, respectively. The matrix comparison analysis, between the F1CDx and CGP assays, showed a linear correlation for all three panels, with a higher correlation between F1CDx and TSO500 (rho=0.88) than in the other two comparisons (rho=0.77 for QIA; 0.72 for OCA). The TSO500 showed the best area under the curve (AUC, value 0.96), with a statistically significant difference when compared with the AUC of OCA (0.83, p value=0.01) and QIA (0.88, p value=0.028). The Youden Index calculation allowed us to extrapolate TMB cut-offs of the different panels corresponding to the 10 mutations/megabase (muts/Mb) cut-off of F1CDx: 10.19, 10.4 and 12.37 muts/Mb for TSO500, OCA and QIA, respectively. Using these values, we calculated the relative accuracy measures for the three panels. TSO500 showed 86% specificity and 96% sensitivity, while OCA and QIA had lower yet similar values of specificity and sensitivity (73% and 88%, respectively). CONCLUSION: This study estimated TMB cut-off values for commercially available CGP panels. The results showed a good performance of all panels on clinical samples and the calculated cut-offs support better accuracy measures for TSO500. The validated cut-off values can drive clinical interpretation of TMB testing in clinical research and clinical practice.

STAT3 inhibition recovers regeneration of aged muscles by restoring autophagy in muscle stem cells.

Age-related reduction in muscle stem cell (MuSC) regenerative capacity is associated with cell-autonomous and non-cell-autonomous changes caused by alterations in systemic and skeletal muscle environments, ultimately leading to a decline in MuSC number and function. Previous studies demonstrated that STAT3 plays a key role in driving MuSC expansion and differentiation after injury-activated regeneration, by regulating autophagy in activated MuSCs. However, autophagy gradually declines in MuSCs during lifespan and contributes to the impairment of MuSC-mediated regeneration of aged muscles. Here, we show that STAT3 inhibition restores the autophagic process in aged MuSCs, thereby recovering MuSC ability to promote muscle regeneration in geriatric mice. We show that STAT3 inhibition could activate autophagy at the nuclear level, by promoting transcription of autophagy-related genes, and at the cytoplasmic level, by targeting STAT3/PKR phosphorylation of eIF2α. These results point to STAT3 inhibition as a potential intervention to reverse the age-related autophagic block that impairs MuSC ability to regenerate aged muscles. They also reveal that STAT3 regulates MuSC function by both transcription-dependent and transcription-independent regulation of autophagy.

Harmonization of homologous recombination deficiency testing in ovarian cancer: Results from the MITO16A/MaNGO-OV2 trial.

BACKGROUND: Homologous Recombination Deficiency (HRD) status predicts response to treatment with poly(ADP-ribose) polymerase inhibitors in Ovarian Cancer (OC) patients. The Myriad myChoiceCDx Assay is approved by Food and Drug Agency for the HRD assessment. Here we compared the HRD status obtained by three commercial panels with the results from Myriad reference test. METHODS: The HRD analysis was performed on DNA from formalin-fixed and paraffin-embedded tumor samples of 100 untreated OC patients for which Myriad assay results were available, using TruSight Oncology 500 HRD assay (Illumina), Oncomine Comprehensive Assay Plus (Thermo Fisher Scientific) and SOPHiA DDM HRD solution panel (SOPHiA Genetics). RESULTS: A good overall concordance with the reference method was demonstrated at three different levels: BRCA mutational status (from 94.4 % to 97.7 %), the genomic instability value (from 88.2 % to 95.3 %) and for the HRD status (from 90.4 % to 97.6 %). Moreover, a trend in favour of HRD positive patients for response rate, progression-free survival and overall survival similar to Myriad was observed for all three tests. DISCUSSION: Our data suggest the feasibility of commercial testing for assessing HRD status, with a good concordance with the reference method and association with clinical outcome.

External quality assessment (EQA) for tumor mutational burden: results of an international IQN path feasibility pilot scheme.

Tumor mutational burden (TMB) has recently been approved as an agnostic biomarker for immune checkpoint inhibitors. However, methods for TMB testing have not yet been standardized. The International Quality Network for Pathology (IQNPath) organized a pilot external quality assessment (EQA) scheme for TMB testing. The aim of this program was the validation of the materials and the procedures for the EQA of this complex biomarker. Five formalin-fixed paraffin-embedded (FFPE) cell lines were selected to mimic the various TMB values observed in clinical practice. The FFPE samples were tested with the FoundationOne CDx (F1CDx) assay as the reference test and three commercially available targeted sequencing panels. Following this internal validation, the five cell lines were sent to 29 laboratories selected on the basis of a previous survey. Nineteen of the 23 laboratories that submitted results (82.6%) used targeted sequencing for TMB estimation. Only two laboratories performed whole exome sequencing (WES) and two assessed TMB by clinical exome. A high variability in the reported TMB values was observed. The variability was higher for samples with the highest TMB value according to the F1CDx test. However, good reproducibility of the TMB score was shown by laboratories using the same panel. The majority of laboratories did not indicate a TMB cut-off value for clinical interpretation. In conclusion, this pilot EQA scheme suggests that it is feasible to run such an EQA program for TMB assessment. However, the results of our pilot highlight the numerous challenges for the standardization of this test.

Conserved transcription factors promote cell fate stability and restrict reprogramming potential in differentiated cells.

Defining the mechanisms safeguarding cell fate identity in differentiated cells is crucial to improve 1) - our understanding of how differentiation is maintained in healthy tissues or altered in a disease state, and 2) - our ability to use cell fate reprogramming for regenerative purposes. Here, using a genome-wide transcription factor screen followed by validation steps in a variety of reprogramming assays (cardiac, neural and iPSC in fibroblasts and endothelial cells), we identified a set of four transcription factors (ATF7IP, JUNB, SP7, and ZNF207 [AJSZ]) that robustly opposes cell fate reprogramming in both lineage and cell type independent manners. Mechanistically, our integrated multi-omics approach (ChIP, ATAC and RNA-seq) revealed that AJSZ oppose cell fate reprogramming by 1) - maintaining chromatin enriched for reprogramming TF motifs in a closed state and 2) - downregulating genes required for reprogramming. Finally, KD of AJSZ in combination with MGT overexpression, significantly reduced scar size and improved heart function by 50%, as compared to MGT alone post-myocardial infarction. Collectively, our study suggests that inhibition of barrier to reprogramming mechanisms represents a promising therapeutic avenue to improve adult organ function post-injury.