Multisite clinical cross-validation and variant interpretation of a next generation sequencing panel for lymphoid cancer prognostication.

AIMS: Genomic sequencing of lymphomas is under-represented in routine clinical testing despite having prognostic and predictive value. Clinical implementation is challenging due to a lack of consensus on reportable targets and a paucity of reference samples. We organised a cross-validation study of a lymphoma-tailored next-generation sequencing panel between two College of American Pathologists (CAP)-accredited clinical laboratories to mitigate these challenges. METHODS: A consensus for the genomic targets was discussed between the two institutes based on recurrence in diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukaemia and T-cell lymphomas. Using the same genomic targets, each laboratory ordered libraries independently and a cross-validation study was designed to exchange samples (8 cell lines and 22 clinical samples) and their FASTQ files. RESULTS: The sensitivity of the panel when comparing different library preparation and bioinformatic workflows was between 97% and 99% and specificity was 100% when a 5% limit of detection cut-off was applied. To evaluate how the current standards for variant classification of tumours apply to lymphomas, the Association for Molecular Pathology/American Society of Clinical Oncology/CAP and OncoKB classification systems were applied to the panel. The majority of variants were assigned a possibly actionable class or likely pathogenic due to more limited evidence in the literature. CONCLUSIONS: The cross-validation study highlights the benefits of sample and data exchange for clinical validation and provided a framework for reporting the findings in lymphoid malignancies.

Navigating nonalcoholic fatty liver disease (NAFLD): Exploring the roles of estrogens, pharmacological and medical interventions, and life style.

The pursuit of studying this subject is driven by the urgency to address the increasing global prevalence of Non-Alcoholic Fatty Liver Disease (NAFLD) and its profound health implications. NAFLD represents a significant public health concern due to its association with metabolic disorders, cardiovascular complications, and the potential progression to more severe conditions like non-alcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. Liver estrogen signaling is important for maintaining liver function, and loss of estrogens increases the likelihood of NAFLD in postmenopausal women. Understanding the multifaceted mechanisms underlying NAFLD pathogenesis, its varied treatment strategies, and their effectiveness is crucial for devising comprehensive and targeted interventions. By unraveling the intricate interplay between genetics, lifestyle, hormonal regulation, and gut microbiota, we can unlock insights into risk stratification, early detection, and personalized therapeutic approaches. Furthermore, investigating the emerging pharmaceutical interventions and dietary modifications offers the potential to revolutionize disease management. This review reinforces the role of collaboration in refining NAFLD comprehension, unveiling novel therapeutic pathways, and ultimately improving patient outcomes for this intricate hepatic condition.

Liver Metastatic Breast Cancer: Epidemiology, Dietary Interventions, and Related Metabolism.

The median overall survival of patients with metastatic breast cancer is only 2-3 years, and for patients with untreated liver metastasis, it is as short as 4-8 months. Improving the survival of women with breast cancer requires more effective anti-cancer strategies, especially for metastatic disease. Nutrients can influence tumor microenvironments, and cancer metabolism can be manipulated via a dietary modification to enhance anti-cancer strategies. Yet, there are no standard evidence-based recommendations for diet therapies before or during cancer treatment, and few studies provide definitive data that certain diets can mediate tumor progression or therapeutic effectiveness in human cancer. This review focuses on metastatic breast cancer, in particular liver metastatic forms, and recent studies on the impact of diets on disease progression and treatment.

Targeting Metabolic Adaptations in the Breast Cancer-Liver Metastatic Niche Using Dietary Approaches to Improve Endocrine Therapy Efficacy.

Estrogen receptor-positive (ER+) metastatic tumors contribute to nearly 70% of breast cancer-related deaths. Most patients with ER+ metastatic breast cancer (MBC) undergo treatment with the estrogen receptor antagonist fulvestrant as standard of care. Yet, among such patients, metastasis in liver is associated with reduced overall survival compared with other metastasis sites. The factors underlying the reduced responsiveness of liver metastases to ER-targeting agents remain unknown, impeding the development of more effective treatment approaches to improve outcomes for patients with ER+ liver metastases. We therefore evaluated site-specific changes in MBC cells and determined the mechanisms through which the liver metastatic niche specifically influences ER+ tumor metabolism and drug resistance. We characterized ER activity of MBC cells both in vitro, using a novel system of tissue-specific extracellular matrix hydrogels representing the stroma of ER+ tumor metastatic sites (liver, lung, and bone), and in vivo, in liver and lung metastasis mouse models. ER+ metastatic liver tumors and MBC cells grown in liver hydrogels displayed upregulated expression of glucose metabolism enzymes in response to fulvestrant. Furthermore, differential ERα activity, but not expression, was detected in liver hydrogels. In vivo, increased glucose metabolism led to increased glycogen deposition in liver metastatic tumors, while a fasting-mimicking diet increased efficacy of fulvestrant treatment to reduce the metastatic burden. Our findings identify a novel mechanism of endocrine resistance driven by the liver tumor microenvironment. IMPLICATIONS: These results may guide the development of dietary strategies to circumvent drug resistance in liver metastasis, with potential applicability in other metastatic diseases.

Gradient of Developmental and Injury Response transcriptional states defines functional vulnerabilities underpinning glioblastoma heterogeneity.

Glioblastomas harbor diverse cell populations, including rare glioblastoma stem cells (GSCs) that drive tumorigenesis. To characterize functional diversity within this population, we performed single-cell RNA sequencing on >69,000 GSCs cultured from the tumors of 26 patients. We observed a high degree of inter- and intra-GSC transcriptional heterogeneity that could not be fully explained by DNA somatic alterations. Instead, we found that GSCs mapped along a transcriptional gradient spanning two cellular states reminiscent of normal neural development and inflammatory wound response. Genome-wide CRISPR-Cas9 dropout screens independently recapitulated this observation, with each state characterized by unique essential genes. Further single-cell RNA sequencing of >56,000 malignant cells from primary tumors found that the majority organize along an orthogonal astrocyte maturation gradient yet retain expression of founder GSC transcriptional programs. We propose that glioblastomas grow out of a fundamental GSC-based neural wound response transcriptional program, which is a promising target for new therapy development.

Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt.

Glioblastoma multiforme (GBM) contains a subpopulation of cells, GBM stem cells (GSCs), that maintain the bulk tumor and represent a key therapeutic target. Norrin is a Wnt ligand that binds Frizzled class receptor 4 (FZD4) to activate canonical Wnt signaling. Although Norrin, encoded by NDP, has a well-described role in vascular development, its function in human tumorigenesis is largely unexplored. Here, we show that NDP expression is enriched in neurological cancers, including GBM, and its levels positively correlated with survival in a GBM subtype defined by low expression of ASCL1, a proneural factor. We investigated the function of Norrin and FZD4 in GSCs and found that it mediated opposing tumor-suppressive and -promoting effects on ASCL1lo and ASCL1hi GSCs. Consistent with a potential tumor-suppressive effect of Norrin suggested by the tumor outcome data, we found that Norrin signaling through FZD4 inhibited growth in ASCL1lo GSCs. In contrast, in ASCL1hi GSCs Norrin promoted Notch signaling, independently of WNT, to promote tumor progression. Forced ASCL1 expression reversed the tumor-suppressive effects of Norrin in ASCL1lo GSCs. Our results identify Norrin as a modulator of human brain cancer progression and reveal an unanticipated Notch-mediated function of Norrin in regulating cancer stem cell biology. This study identifies an unanticipated role of Norrin in human brain cancer progression. In addition, we provide preclinical evidence suggesting Norrin and canonical Wnt signaling as potential therapeutic targets for GBM subtype-restricted cancer stem cells.

Wnt and Notch signaling govern self-renewal and differentiation in a subset of human glioblastoma stem cells.

Developmental signal transduction pathways act diversely, with context-dependent roles across systems and disease types. Glioblastomas (GBMs), which are the poorest prognosis primary brain cancers, strongly resemble developmental systems, but these growth processes have not been exploited therapeutically, likely in part due to the extreme cellular and genetic heterogeneity observed in these tumors. The role of Wnt/ßcatenin signaling in GBM stem cell (GSC) renewal and fate decisions remains controversial. Here, we report context-specific actions of Wnt/ßcatenin signaling in directing cellular fate specification and renewal. A subset of primary GBM-derived stem cells requires Wnt proteins for self-renewal, and this subset specifically relies on Wnt/ßcatenin signaling for enhanced tumor burden in xenograft models. In an orthotopic Wnt reporter model, Wnthi GBM cells (which exhibit high levels of ßcatenin signaling) are a faster-cycling, highly self-renewing stem cell pool. In contrast, Wntlo cells (with low levels of signaling) are slower cycling and have decreased self-renewing potential. Dual inhibition of Wnt/ßcatenin and Notch signaling in GSCs that express high levels of the proneural transcription factor ASCL1 leads to robust neuronal differentiation and inhibits clonogenic potential. Our work identifies new contexts for Wnt modulation for targeting stem cell differentiation and self-renewal in GBM heterogeneity, which deserve further exploration therapeutically.

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy.

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

ASCL1 Reorganizes Chromatin to Direct Neuronal Fate and Suppress Tumorigenicity of Glioblastoma Stem Cells.

Glioblastomas exhibit a hierarchical cellular organization, suggesting that they are driven by neoplastic stem cells that retain partial yet abnormal differentiation potential. Here, we show that a large subset of patient-derived glioblastoma stem cells (GSCs) express high levels of Achaete-scute homolog 1 (ASCL1), a proneural transcription factor involved in normal neurogenesis. ASCL1hi GSCs exhibit a latent capacity for terminal neuronal differentiation in response to inhibition of Notch signaling, whereas ASCL1lo GSCs do not. Increasing ASCL1 levels in ASCL1lo GSCs restores neuronal lineage potential, promotes terminal differentiation, and attenuates tumorigenicity. ASCL1 mediates these effects by functioning as a pioneer factor at closed chromatin, opening new sites to activate a neurogenic gene expression program. Directing GSCs toward terminal differentiation may provide therapeutic applications for a subset of GBM patients and strongly supports efforts to restore differentiation potential in GBM and other cancers.

Inhibition of Dopamine Receptor D4 Impedes Autophagic Flux, Proliferation, and Survival of Glioblastoma Stem Cells.

Glioblastomas (GBM) grow in a rich neurochemical milieu, but the impact of neurochemicals on GBM growth is largely unexplored. We interrogated 680 neurochemical compounds in patient-derived GBM neural stem cells (GNS) to determine the effects on proliferation and survival. Compounds that modulate dopaminergic, serotonergic, and cholinergic signaling pathways selectively affected GNS growth. In particular, dopamine receptor D4 (DRD4) antagonists selectively inhibited GNS growth and promoted differentiation of normal neural stem cells. DRD4 antagonists inhibited the downstream effectors PDGFRß, ERK1/2, and mTOR and disrupted the autophagy-lysosomal pathway, leading to accumulation of autophagic vacuoles followed by G0/G1 arrest and apoptosis. These results demonstrate a role for neurochemical pathways in governing GBM stem cell proliferation and suggest therapeutic approaches for GBM.

MLL5 Orchestrates a Cancer Self-Renewal State by Repressing the Histone Variant H3.3 and Globally Reorganizing Chromatin.

Mutations in the histone 3 variant H3.3 have been identified in one-third of pediatric glioblastomas (GBMs), but not in adult tumors. Here we show that H3.3 is a dynamic determinant of functional properties in adult GBM. H3.3 is repressed by mixed lineage leukemia 5 (MLL5) in self-renewing GBM cells. MLL5 is a global epigenetic repressor that orchestrates reorganization of chromatin structure by punctuating chromosomes with foci of compacted chromatin, favoring tumorigenic and self-renewing properties. Conversely, H3.3 antagonizes self-renewal and promotes differentiation. We exploited these epigenetic states to rationally identify two small molecules that effectively curb cancer stem cell properties in a preclinical model. Our work uncovers a role for MLL5 and H3.3 in maintaining self-renewal hierarchies in adult GBM.

Structural and genic characterization of stable genomic regions in breast cancer: relevance to chemotherapy.

BACKGROUND: Cancer genomes accumulate frequent and diverse chromosomal abnormalities as well as gene mutations but must maintain the ability to survive in vivo. We hypothesize that genetic selection acts to maintain tumour survival by preserving copy number of specific genes and genomic regions. Genomic regions and genes that remain unaltered in copy number and expression, respectively, may be essential for maintaining tumour survival. METHODS: We analyzed copy number data of 243 previously reported breast tumours and computationally derived stable copy number regions. To identify genes in stable copy number regions with nominal changes in expression, datasets for tumour and normal samples were compared. Results were replicated by analysis of a series of independent copy number, expression and genomic sequencing studies. A subset of stable regions, including stable paralogous regions, were confirmed by quantitative PCR and fluorescence in situ hybridization (FISH) in 5 breast cancer cell lines. We deduced a comprehensive set of dually stable genes (i.e. maintaining nominal copy number and expression) which were categorized according to pathway and ontology assignments. The stability of genes encoding therapeutic drug targets was also assessed. RESULTS AND CONCLUSION: Tumour genome analysis revealed 766 unstable (amplified and/or deleted) and 812 stable contiguous genomic regions. Replication analysis of an independent set of 171 breast tumours confirmed copy number stability of 1.3 Gb of the genome. We found that 5804 of these genes were dually stable. The composition of this gene set remained essentially unchanged (<2% reduction) after accounting for commonly mutated breast cancer genes found by sequencing and differential expression. The stable breast cancer genome is enriched for cellular metabolism, regulation of gene expression, DNA packaging (chromatin and nucleosome assembly), and regulation of apoptosis functions. Stable genes participating in multiple essential pathways were consistently found to be targets of chemotherapies. Preservation of stable, essential genes may be related to the effectiveness of certain chemotherapeutic agents that act on multiple gene products in this set.

Effect of caffeine ingestion on muscular strength and endurance: a meta-analysis.

PURPOSE: Our objective was to perform a systematic review and meta-analysis of the research literature assessing the effect of caffeine ingestion on maximal voluntary contraction (MVC) strength and muscular endurance. METHODS: Thirty-four relevant studies between 1939 and 2008 were included in the meta-analyses of caffeine's effects on MVC strength (n = 27 studies) and muscular endurance (n = 23 studies). Effect sizes (ES) were calculated as the standardized mean difference and meta-analyses were completed using a random-effects model. RESULTS: Overall, caffeine ingestion was found to result in a small beneficial effect on MVC strength (overall ES = 0.19, P = 0.0003). However, caffeine appears to improve MVC strength primarily in the knee extensors (i.e., by approximately 7%, ES = 0.37) and not in other muscle groups such as the forearm or the knee flexors. In an attempt to offer a physiological mechanism behind caffeine's ability to improve MVC strength, a meta-analysis was run on ES from nine studies that measured percent muscle activation during MVC in trials comparing caffeine versus placebo; the overall ES (0.67) was highly significant (P = 0.00008) and of moderate to large size, thus implicating an effect of caffeine on the CNS. Caffeine ingestion was also found to exert a small beneficial effect on muscular endurance (overall ES = 0.28, P = 0.00005). However, it appears caffeine improves muscular endurance only when it is assessed using open (i.e., by approximately 18%, ES = 0.37) and not fixed end point tests. CONCLUSIONS: Overall, caffeine ingestion improves MVC strength and muscular endurance. The effect on strength appears exclusively in the knee extensors, and the effect on muscular endurance appears only detectable with open end point tests.

Caffeines enhancement of maximal voluntary strength and activation in uninjured but not injured muscle.

The study's objective was to determine whether orally ingested caffeine could help overcome excitation-contraction-coupling failure, which has been suggested to explain part of the strength loss associated with eccentric-contraction-induced muscle injury. A sample of 13 college students (4 men and 9 women) was used in a double-blind, repeated-measures experimental design. Each participant performed 2 experimental trials, 1 with each leg, with each trial lasting 4 consecutive days. On a given day, each participant was randomly assigned to ingest a capsule containing 6 mg/kg of either caffeine or flour (placebo). On the day of and the first 2 days after a bout of 50 injurious eccentric contractions done by the knee extensors, the interpolated-twitch technique was used to assess electrically evoked strength, maximal voluntary isometric contraction (MVIC) strength, and percent muscle activation during MVIC both before and after capsule ingestion. These variables were also measured before and after capsule ingestion the day before the eccentric-contraction bout--when the muscle was uninjured. In injured muscle, caffeine had no effect on any variable. In uninjured muscle, caffeine also had no effect on electrically evoked strength but increased MVIC strength by 10.4% compared with placebo (p = .00002), and this was attributed to an increase in muscle activation (6.2%; p = .01). In conclusion, the data provide no evidence that caffeine ingestion can help overcome excitation-contraction-coupling failure, if it exists, in injured human muscle. The data do indicate that caffeine ingestion can increase MVIC strength and activation in uninjured muscle but not in injured muscle.