The practical utility of AI-assisted molecular profiling in the diagnosis and management of cancer of unknown primary: an updated review.

Cancer of unknown primary (CUP) presents a complex diagnostic challenge, characterized by metastatic tumors of unknown tissue origin and a dismal prognosis. This review delves into the emerging significance of artificial intelligence (AI) and machine learning (ML) in transforming the landscape of CUP diagnosis, classification, and treatment. ML approaches, trained on extensive molecular profiling data, have shown promise in accurately predicting tissue of origin. Genomic profiling, encompassing driver mutations and copy number variations, plays a pivotal role in CUP diagnosis by providing insights into tumor type-specific oncogenic alterations. Mutational signatures (MS), reflecting somatic mutation patterns, offer further insights into CUP diagnosis. Known MS with established etiology, such as ultraviolet (UV) light-induced DNA damage and tobacco exposure, have been identified in cases of dedifferentiated/transdifferentiated melanoma and carcinoma. Deep learning models that integrate gene expression data and DNA methylation patterns offer insights into tissue lineage and tumor classification. In digital pathology, machine learning algorithms analyze whole-slide images to aid in CUP classification. Finally, precision oncology, guided by molecular profiling, offers targeted therapies independent of primary tissue identification. Clinical trials assigning CUP patients to molecularly guided therapies, including targetable alterations and tumor mutation burden as an immunotherapy biomarker, have resulted in improved overall survival in a subset of patients. In conclusion, AI- and ML-driven approaches are revolutionizing CUP management by enhancing diagnostic accuracy. Precision oncology utilizing enhanced molecular profiling facilitates the identification of targeted therapies that transcend the need to identify the tissue of origin, ultimately improving patient outcomes.

Dysregulated cellular redox status during hyperammonemia causes mitochondrial dysfunction and senescence by inhibiting sirtuin-mediated deacetylation.

Perturbed metabolism of ammonia, an endogenous cytotoxin, causes mitochondrial dysfunction, reduced NAD+ /NADH (redox) ratio, and postmitotic senescence. Sirtuins are NAD+ -dependent deacetylases that delay senescence. In multiomics analyses, NAD metabolism and sirtuin pathways are enriched during hyperammonemia. Consistently, NAD+ -dependent Sirtuin3 (Sirt3) expression and deacetylase activity were decreased, and protein acetylation was increased in human and murine skeletal muscle/myotubes. Global acetylomics and subcellular fractions from myotubes showed hyperammonemia-induced hyperacetylation of cellular signaling and mitochondrial proteins. We dissected the mechanisms and consequences of hyperammonemia-induced NAD metabolism by complementary genetic and chemical approaches. Hyperammonemia inhibited electron transport chain components, specifically complex I that oxidizes NADH to NAD+ , that resulted in lower redox ratio. Ammonia also caused mitochondrial oxidative dysfunction, lower mitochondrial NAD+ -sensor Sirt3, protein hyperacetylation, and postmitotic senescence. Mitochondrial-targeted Lactobacillus brevis NADH oxidase (MitoLbNOX), but not NAD+ precursor nicotinamide riboside, reversed ammonia-induced oxidative dysfunction, electron transport chain supercomplex disassembly, lower ATP and NAD+ content, protein hyperacetylation, Sirt3 dysfunction and postmitotic senescence in myotubes. Even though Sirt3 overexpression reversed ammonia-induced hyperacetylation, lower redox status or mitochondrial oxidative dysfunction were not reversed. These data show that acetylation is a consequence of, but is not the mechanism of, lower redox status or oxidative dysfunction during hyperammonemia. Targeting NADH oxidation is a potential approach to reverse and potentially prevent ammonia-induced postmitotic senescence in skeletal muscle. Since dysregulated ammonia metabolism occurs with aging, and NAD+ biosynthesis is reduced in sarcopenia, our studies provide a biochemical basis for cellular senescence and have relevance in multiple tissues.

CSL112 Infusion Rapidly Increases APOA1 Exchange Rate via Specific Serum Amyloid-Poor HDL Subpopulations When Administered to Patients Post-Myocardial Infarction.

BACKGROUND: To characterize the effects of CSL112 (human APOA1 [apolipoprotein A1]) on the APOA1 exchange rate (AER) and the relationships with specific HDL (high-density lipoprotein) subpopulations when administered in the 90-day high-risk period post-acute myocardial infarction. METHODS: A subset of patients (n=50) from the AEGIS-I (ApoA-I Event Reducing in Ischemic Syndromes I) study received either placebo or CSL112 post-acute myocardial infarction. AER was measured in AEGIS-I plasma samples incubated with lipid-sensitive fluorescent APOA1 reporter. HDL particle size distribution was assessed by native gel electrophoresis followed by fluorescent imaging and detection of APOA1 and SAA (serum amyloid A) by immunoblotting. RESULTS: CSL112 infusion increased AER peaking at 2 hours and returning to baseline 24 hours post-infusion. AER correlated with cholesterol efflux capacity (r=0.49), HDL-cholesterol (r=0.30), APOA1 (r=0.48), and phospholipids (r=0.48; all P<0.001) over all time points. Mechanistically, changes in cholesterol efflux capacity and AER induced by CSL112 reflected HDL particle remodeling resulting in increased small HDL species that are highly active in mediating ABCA1 (ATP-binding cassette transporter 1)-dependent efflux, and large HDL species with high capacity for APOA1 exchange. The lipid-sensitive APOA1 reporter predominantly exchanged into SAA-poor HDL particles and weakly incorporated into SAA-enriched HDL species. CONCLUSIONS: Infusion of CSL112 enhances metrics of HDL functionality in patients with acute myocardial infarction. This study demonstrates that in post-acute myocardial infarction patients, HDL-APOA1 exchange involves specific SAA-poor HDL populations. Our data suggest that progressive enrichment of HDL with SAA may generate dysfunctional particles with impaired HDL-APOA1 exchange capacity, and that infusion of CSL112 improves the functional status of HDL with respect to HDL-APOA1 exchange. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT02108262.

HDL Is Not Dead Yet.

High-density lipoprotein cholesterol (HDL-C) levels are inversely correlated with coronary heart disease (CHD) in multiple epidemiological studies, but whether HDL is causal or merely associated with CHD is unclear. Recent trials for HDL-raising drugs were either not effective in reducing CHD events or, if beneficial in reducing CHD events, were not conclusive as the findings could be attributed to the drugs' LDL-reducing activity. Furthermore, the first large Mendelian randomization study did not causally relate HDL-C levels to decreased CHD. Thus, the hypothesis that HDL is protective against CHD has been rightfully challenged. However, subsequent Mendelian randomization studies found HDL characteristics that are causally related to decreased CHD. Many aspects of HDL structure and function, especially in reverse cholesterol transport, may be better indicators of HDL's protective activity than simply measuring HDL-C. Cholesterol efflux capacity is associated with lower levels of prevalent and incident CHD, even after adjustment for HDL-C and apolipoprotein A-1 levels. Also, subjects with very high levels of HDL-C, including those with rare mutations that disrupt hepatic HDL uptake and reverse cholesterol transport, may be at higher risk for CHD than those with moderate levels. We describe here several cell-based and cell-free in vitro assays of HDL structure and function that may be used in clinical studies to determine which of HDL's functions are best associated with protection against CHD. We conclude that the HDL hypothesis may need revision based on studies of HDL structure and function, but that the HDL hypothesis is not dead yet.

Quantitative trait locus mapping identifies the Gpnmb gene as a modifier of mouse macrophage lysosome function.

We have previously shown that the DBA/2J versus AKR/J mouse strain is associated with decreased autophagy-mediated lysosomal hydrolysis of cholesterol esters. Our objective was to determine differences in lysosome function in AKR/J and DBA/2J macrophages, and identify the responsible genes. Using a novel dual-labeled indicator of lysosome function, DBA/2J versus AKR/J bone marrow derived macrophages had significantly decreased lysosome function. We performed quantitative trait loci mapping of lysosome function in bone marrow macrophages from an AKR/J × DBA/2J strain intercross. Four distinct lysosome function loci were identified, which we named macrophage lysosome function modifier (Mlfm) Mlfm1 through Mlfm4. The strongest locus Mlfm1 harbors the Gpnmb gene, which has been shown to recruit autophagy protein light chain 3 to autophagosomes for lysosome fusion. The parental DBA/2J strain has a nonsense variant in Gpnmb. siRNA knockdown of Gpnmb in AKR/J macrophages decreased lysosome function, and Gpnmb deletion through CRISP/Cas9 editing in RAW 264.7 mouse macrophages also demonstrated a similar result. Furthermore, a DBA/2 substrain, called DBA/2J-Gpnmb+/SjJ, contains the wildtype Gpnmb gene, and macrophages from this Gpnmb-preserved DBA/2 substrain exhibited recovered lysosome function. In conclusion, we identified Gpnmb as a causal modifier gene of lysosome function in this strain pair.

A Novel Cell-Free Fluorescent Assay for HDL Function: Low Apolipoprotein A1 Exchange Rate Associated with Increased Incident Cardiovascular Events.

BACKGROUND: Cholesterol efflux capacity is a tissue culture assay for HDL function that is not amenable for high-throughput monitoring of risk assessment. METHODS: We devised a cell-free HDL function assay to measure the exchange rate of exogenous apoA1 into serum HDL using NBD/Alexa647 double-labeled apoA1, whose NBD/Alexa647 emission ratio increased upon exchange into HDL. ApoA1 exchange rate (AER) was assayed by incubating labeled apoA1 with human serum, and the rate of the increase of the NBD/Alexa647 ratio over time was calculated as AER. RESULTS: Fast protein liquid chromatography analysis of serum confirmed that the labeled apoA1 selectively exchanged into the HDL lipoprotein fraction. Characterization studies demonstrated that the AER assay had excellent intra- and inter-day reproducibility, was stable over 3 freeze-thaw cycles, and yielded similar results with serum or plasma. We quantified AER in serum from randomly selected stable subjects undergoing elective diagnostic coronary angiography (n = 997). AER was correlated with HDL-cholesterol (r = 0.58, P < 0.0001) and apoA1 levels (r = 0.56, P < 0.0001). Kaplan-Meier survival plot showed subjects in the lowest quartile of AER experienced a significantly higher rate of incident major adverse cardiovascular events (MACE = myocardial infarction, stroke, or death) (P < 0.0069 log rank). Moreover, compared to subjects in the lowest AER quartile, the remaining subjects showed significantly lower incident (3 year) risk for MACE, even after adjustment for traditional risk factors and apoA1 (HR 0.58; 95% CI 0.40-0.85; P = 0.005). CONCLUSIONS: In a prospective cohort of stable subjects undergoing elective diagnostic cardiac evaluations, low AER was associated with increased incident risk of MACE.

Uptake of high-density lipoprotein by scavenger receptor class B type 1 is associated with prostate cancer proliferation and tumor progression in mice.

High-density lipoprotein (HDL) metabolism is facilitated in part by scavenger receptor class B, type 1 (SR-B1) that mediates HDL uptake into cells. Higher levels of HDL have been associated with protection in other diseases, however, its role in prostate cancer is not definitive. SR-B1 is up-regulated in prostate cancer tissue, suggesting a possible role of this receptor in tumor progression. Here, we report that knockout (KO) of SR-B1 in both human and mouse prostate cancer cell lines through CRISPR/Cas9-mediated genome editing reduces HDL uptake into the prostate cancer cells and reduces their proliferation in response to HDL. In vivo studies using syngeneic SR-B1 WT (SR-B1+/+) and SR-B1 KO (SR-B1-/-) prostate cancer cells in WT and apolipoprotein-AI KO (apoA1-KO) C57BL/6J mice revealed that WT hosts, containing higher levels of total and HDL-cholesterol, grew larger tumors than apoA1-KO hosts with lower levels of total and HDL-cholesterol. Furthermore, SR-B1-/- prostate cancer cells formed smaller tumors in WT hosts than SR-B1+/+ cells in the same host model. Increased tumor volume was overall associated with reduced survival. We conclude that knocking out SR-B1 in prostate cancer tumors reduces HDL-associated increases in prostate cancer cell proliferation and disease progression.

Bariatric Surgery Improves HDL Function Examined by ApoA1 Exchange Rate and Cholesterol Efflux Capacity in Patients with Obesity and Type 2 Diabetes.

Bariatric surgery improves glycemic control better than medical therapy; however, the effect of bariatric surgery on HDL function is not well characterized. Serum samples were available at baseline, 1-, and 5-years post procedures, for 90 patients with obesity and type 2 diabetes who were randomized to intensive medical therapy (n = 20), Roux-en-Y gastric bypass (RYGB, n = 37), or sleeve gastrectomy (SG, n = 33) as part of the STAMPEDE clinical trial. We examined serum HDL function by two independent assays, apolipoprotein A-1 exchange rate (AER) and cholesterol efflux capacity (CEC). Compared with baseline, AER was significantly higher at 5 years for participants in all treatment groups, but only increased significantly at 1 year in the RYGB and SG groups. CEC was divided into ABCA1-dependent and independent fractions, and the later was correlated with AER. ABCA1-independent CEC increased significantly only at 5 years in both surgical groups, but did not significantly change in the medical therapy group. There was no significant change in ABCA1-dependent CEC in any group. The increase in AER, but not ABCA1-independent CEC, was correlated with the reduction in body mass index and glycated hemoglobin levels among all subjects at 5 years, indicating that AER as a measure of HDL function would be a better reflection of therapy versus CEC.

V-ATPase (Vacuolar ATPase) Activity Required for ABCA1 (ATP-Binding Cassette Protein A1)-Mediated Cholesterol Efflux.

Objective- We have shown that ABCA1 (ATP-binding cassette protein A1) mediates unfolding of the apoA1 (apolipoprotein A1) N-terminal helical hairpin during apoA1 lipidation. Others have shown that an acidic pH exposes the hydrophobic surface of apoA1. We postulated that the V-ATPase (vacuolar ATPase) proton pump facilitates apoA1 unfolding and promotes ABCA1-mediated cholesterol efflux. Approach and Results- We found that V-ATPase inhibitors dose-dependently decreased ABCA1-mediated cholesterol efflux to apoA1 in baby hamster kidney cells and RAW264.7 cells; and similarly, siRNA knockdown of ATP6V0C inhibited ABCA1-mediated cholesterol efflux to apoA1 in RAW264.7 cells. Although ABCA1 expression did not alter total cellular levels of V-ATPase, ABCA1 increased the cell surface levels of the V0A1 and V1E1 subunits of V-ATPase. We generated a fluorescein isothiocyanate/Alexa647 double-labeled fluorescent ratiometric apoA1 pH indicator whose fluorescein isothiocyanate/Alexa647 emission ratio decreased as the pH drops. We found that ABCA1 induction in baby hamster kidney cells led to acidification of the cell-associated apoA1 pH indicator, compared with control cells without ABCA1 expression. The V-ATPase inhibitor bafilomycin A1 dose-dependently inhibited the apoA1 pH shift in ABCA1-expressing cells, without affecting the levels of cell-associated apoA1. However, we were not able to detect ABCA1-mediated extracellular proton release. We showed that acidic pH facilitated apoA1 unfolding, apoA1 solubilization of phosphatidycholine:phosphatidyserine liposomes, and increased lipid fluidity of these liposomes. Conclusions- Our results support a model that ABCA1 recruits V-ATPase to the plasma membrane where V-ATPase mediates apoA1 acidification and membrane remodeling that promote apoA1 unfolding and ABCA1-mediated HDL (high-density lipoprotein) biogenesis and lipid efflux.