Phosphoenolpyruvate carboxykinase-2 (PCK2) is a therapeutic target in triple-negative breast cancer.

PURPOSE: Metabolic rewiring in malignant transformation is often accompanied by altered expression of metabolic isozymes. Phosphoenolpyruvate carboxykinase-2 (PCK2) catalyzes the rate-limiting step of gluconeogenesis and is the dominant isoform in many cancers including triple-negative breast cancer (TNBC). Our goal was to identify small molecule inhibitors of PCK2 enzyme activity. METHODS: We assessed the impact of PCK2 down regulation with shRNA on TNBC cell growth in vitro and used AtomNet® deep convolutional neural network software to identify potential small molecule inhibitors of PCK2-based structure. We iteratively tested candidate compounds in an in vitro PCK-2 enzyme assay. The impact of the top hit on metabolic flux and cell viability was also assessed. RESULTS: PCK2 downregulation decreased growth of BT-549 and MDA-MB-231 cells and reduced metabolic flux through pyruvate carboxylase. The first AtomNet® in silico structural screen of 7 million compounds yielded 86 structures that were tested in PCK2 enzyme assay in vitro. The top hit (IC50 = 2.4 µM) was used to refine a second round of in silico screen that yielded 82 candidates to be tested in vitro, which resulted in 45 molecules with inhibition > 20%. In the second in vitro screen we also included 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate, previously suggested to be PCK2 inhibitor based on structure, which emerged as the top hit. The specificity of this compound was tested in PCK1 and PCK2 enzymatic assays and showed IC50 of 500 nM and 3.5-27 nM for PCK1 and PCK2, respectively. CONCLUSION: 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate is a high affinity PCK2 enzyme inhibitor that also has significant growth inhibitory activity in breast cell lines in vitro and represents a potential therapeutic lead compound.

Peripheral blood immune parameters, response, and adverse events after neoadjuvant chemotherapy plus durvalumab in early-stage triple-negative breast cancer.

PURPOSE: We evaluated T- and B-cell receptor (TCR and BCR) repertoire diversity and 38 serum cytokines in pre- and post-treatment peripheral blood of 66 patients with triple-negative breast cancer (TNBC) who received neoadjuvant chemotherapy plus durvalumab and assessed associations with pathologic response and immune-related adverse events (irAEs) during treatment. METHODS: Genomic DNA was isolated from buffy coat for TCR and BCR clonotype profiling using the Immunoseq platform and diversity was quantified with Pielou's evenness index. MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel was used to measure serum cytokine levels, which were compared between groups using moderated t-statistic with Benjamini-Hochberg correction for multiple testing. RESULTS: TCR and BCR diversity was high (Pielou's index > 0.75) in all samples. Baseline receptor diversities and change in diversity pre- and post-treatment were not associated with pathologic response or irAE status, except for BCR diversity that was significantly lower post-treatment in patients who developed irAE (unadjusted p = 0.0321). Five cytokines increased after treatment in patients with pathologic complete response (pCR) but decreased in patients with RD, most prominently IL-8. IFNγ, IL-7, and GM-CSF levels were higher in pre-treatment than in post-treatment samples of patients who developed irAEs but were lower in those without irAEs. CONCLUSION: Baseline peripheral blood cytokine levels may predict irAEs in patients treated with immune checkpoint inhibitors and chemotherapy, and increased post-treatment B-cell clonal expansion might mediate irAEs.

miR-122 and miR-21 are Stable Components of miRNA Signatures of Early Lung Cancer after Validation in Three Independent Cohorts.

Several panels of circulating miRNAs have been reported as potential biomarkers of early lung cancer, yet the overlap of components between different panels is limited, and the universality of proposed biomarkers has been minimal across proposed panels. To assess the stability of the diagnostic potential of plasma miRNA signature of early lung cancer among different cohorts, a panel of 24 miRNAs tested in the frame of one lung cancer screening study (MOLTEST-2013, Poland) was validated with material collected in the frame of two other screening studies (MOLTEST-BIS, Poland; and SMAC, Italy) using the same standardized analytical platform (the miRCURY LNA miRNA PCR assay). On analysis of selected miRNAs, two associated with lung cancer development, miR-122 and miR-21, repetitively differentiated healthy participants from individuals with lung cancer. Additionally, miR-144 differentiated controls from cases specifically in subcohorts with adenocarcinoma. Other tested miRNAs did not overlap in the three cohorts. Classification models based on neither a single miRNA nor multicomponent miRNA panels (24-mer and 7-mer) showed classification performance sufficient for a standalone diagnostic biomarker (AUC, 75%, 71%, and 53% in MOLTEST-2013, SMAC, and MOLTEST-BIS, respectively, in the 7-mer model). The performance of classification in the MOLTEST-BIS cohort with the lowest contribution of adenocarcinomas was increased when only this cancer type was considered (AUC, 60% in 7-mer model).

Evaluation of zero counts to better understand the discrepancies between bulk and single-cell RNA-Seq platforms.

Recent advances in sample preparation and sequencing technology have made it possible to profile the transcriptomes of individual cells using single-cell RNA sequencing (scRNA-Seq). Compared to bulk RNA-Seq data, single-cell data often contain a higher percentage of zero reads, mainly due to lower sequencing depth per cell, which affects mostly measurements of low-expression genes. However, discrepancies between platforms are observed regardless of expression level. Using four paired datasets with multiple samples each, we investigated technical and biological factors that can contribute to this expression shift. Using two separate machine learning models we found that, in addition to expression level, RNA integrity, gene or UTR3 length, and the number of transcripts potentially also influence the occurrence of zeros. These findings could enable the development of novel analytical methods for cross-platform expression shift correction. We also identified genes and biological pathways in our diverse datasets that consistently showed differences when assessed at the single cell versus bulk level to assist in interpreting analysis across transcriptomic platforms. At the gene level, 25 genes (0.12%) were found in all datasets as discordant, but at the pathway level, 7 pathways (2.02%) showed shared enrichment in discordant genes.

Pre-analytical effects on whole transcriptome and targeted RNA sequencing analysis in cytology: The effects of prolonged time in storage of effusion specimens prior to preservation.

OBJECTIVES: To investigate the pre-analytics of the molecular testing of cytology specimens, we studied the effects of time in refrigerator storage (4°C) of malignant effusions on RNA sequencing (RNAseq) results. METHODS: Ten effusion specimens were stored in a refrigerator (4°C) for different durations (day 0, 1, 4, and 7). All specimens were prepared as cytospins fixed in either Carnoy's solution or 95% ethanol (EtOH) and in an RNA preservative for a fresh frozen (FF) high-quality reference. Whole transcriptome (wt) and targeted (t)RNAseq of two multigene expression signatures were performed. We then compared transcript expression levels (including mutant allele fraction) according to pre-analytical variables using a concordance correlation coefficient (CCC) and a mixed effect model. RESULTS: Sequencing results were mostly stable over increasing time in storage. Cytospins fixed in Carnoy's solution were more concordant with FF samples than cytospins fixed in 95% EtOH at all timepoints. This finding was consistent for both wtRNAseq (averages: day 0 CCC = 0.98 vs 0.91; day 7 CCC = 0.88 vs 0.78) and tRNAseq methods (averages: day 0 CCC = 0.98 vs 0.81; day 7 CCC = 0.98 vs 0.90). Cytospins fixed in Carnoy's solution did not show significant changes in expression over timepoints or between expression signatures, whereas 95% EtOH did. CONCLUSION: RNAseq can be accurately performed on effusion specimens after prolonged refrigerator storage. RNA extracted from scraped cytospin slides fixed in Carnoy's solution was marginally superior to 95% EtOH fixation, but either method had comparable analytic performance to high-quality FF RNA samples.

Pathological changes or technical artefacts? The problem of the heterogenous databases in COVID-19 CXR image analysis.

BACKGROUND: When the COVID-19 pandemic commenced in 2020, scientists assisted medical specialists with diagnostic algorithm development. One scientific research area related to COVID-19 diagnosis was medical imaging and its potential to support molecular tests. Unfortunately, several systems reported high accuracy in development but did not fare well in clinical application. The reason was poor generalization, a long-standing issue in AI development. Researchers found many causes of this issue and decided to refer to them as confounders, meaning a set of artefacts and methodological errors associated with the method. We aim to contribute to this steed by highlighting an undiscussed confounder related to image resolution. METHODS: 20 216 chest X-ray images (CXR) from worldwide centres were analyzed. The CXRs were bijectively projected into the 2D domain by performing Uniform Manifold Approximation and Projection (UMAP) embedding on the radiomic features (rUMAP) or CNN-based neural features (nUMAP) from the pre-last layer of the pre-trained classification neural network. Additional 44 339 thorax CXRs were used for validation. The comprehensive analysis of the multimodality of the density distribution in rUMAP/nUMAP domains and its relation to the original image properties was used to identify the main confounders. RESULTS: nUMAP revealed a hidden bias of neural networks towards the image resolution, which the regular up-sampling procedure cannot compensate for. The issue appears regardless of the network architecture and is not observed in a high-resolution dataset. The impact of the resolution heterogeneity can be partially diminished by applying advanced deep-learning-based super-resolution networks. CONCLUSIONS: rUMAP and nUMAP are great tools for image homogeneity analysis and bias discovery, as demonstrated by applying them to COVID-19 image data. Nonetheless, nUMAP could be applied to any type of data for which a deep neural network could be constructed. Advanced image super-resolution solutions are needed to reduce the impact of the resolution diversity on the classification network decision.

POLCOVID: a multicenter multiclass chest X-ray database (Poland, 2020-2021).

The outbreak of the SARS-CoV-2 pandemic has put healthcare systems worldwide to their limits, resulting in increased waiting time for diagnosis and required medical assistance. With chest radiographs (CXR) being one of the most common COVID-19 diagnosis methods, many artificial intelligence tools for image-based COVID-19 detection have been developed, often trained on a small number of images from COVID-19-positive patients. Thus, the need for high-quality and well-annotated CXR image databases increased. This paper introduces POLCOVID dataset, containing chest X-ray (CXR) images of patients with COVID-19 or other-type pneumonia, and healthy individuals gathered from 15 Polish hospitals. The original radiographs are accompanied by the preprocessed images limited to the lung area and the corresponding lung masks obtained with the segmentation model. Moreover, the manually created lung masks are provided for a part of POLCOVID dataset and the other four publicly available CXR image collections. POLCOVID dataset can help in pneumonia or COVID-19 diagnosis, while the set of matched images and lung masks may serve for the development of lung segmentation solutions.

Assessment of stained direct cytology smears of breast cancer for whole transcriptome and targeted messenger RNA sequencing.

BACKGROUND: Rather than surgical resection, cytologic specimens are often used as first-line clinical diagnostic procedures due to higher safety, speed, and cost-effectiveness. Archival diagnostic cytology slides containing cancer can be equivalent to tissue biopsies for DNA mutation testing, but the accuracy of transcriptomic profiling by RNA sequencing (RNA-seq) is less understood. METHODS: This study compares the results from whole transcriptome RNA-seq and a targeted RNA-seq assay of stained cytology smears (CS) versus matched tumor tissue samples preserved fresh-frozen (FF) and processed as formalin-fixed paraffin-embedded (FFPE) sections. Cellular cytology scrapes from all 11 breast cancers were fixed and stained using three common protocols: Carnoy's (CS_C) or 95% ethanol (CS_E) fixation and then Papanicolaou stain or air-dried then methanol fixation and DiffQuik stain (CS_DQ). Agreement between samples was assessed using Lin's concordance correlation coefficient. RESULTS: Library yield for CS_DQ was too low, therefore it was not sequenced. The distributions of concordance correlation coefficient of gene expression levels in comparison to FF were comparable between CS_C and CS_E, but expression of genes enriched in stroma was lower in cytosmear samples than in FF or FFPE. Six signatures showed similar concordance to FF for all methods and two were slightly worse in CS_C and CS_E. Genomic signatures were highly concordant using targeted RNA-seq. The allele fraction of selected mutations calculated on cytosmear specimens was highly correlated with FF tissues using both RNA-seq methods. CONCLUSION: RNA can be reliably extracted from cytology smears and is suitable for transcriptome profiling or mutation detection, except for signatures of tumor stroma.

Molecular testing opportunities on cytology effusion specimens: the pre-analytic effects of various body fluid cytology preparation methods on RNA extraction quality and targeted sequencing.

INTRODUCTION: RNA sequencing (RNAseq) analysis is emerging as a clinical research or diagnostic approach for cytologic samples, but there is need for formal comparison of different sample preparation methods in the cytology laboratory to identify which pre-analytic methods could provide alternatives to formalin-fixed paraffin-embedded (FFPE) sections. MATERIALS AND METHODS: We prepared 13 malignant effusions (metastatic estrogen receptor-positive breast cancer) in the cytology laboratory using 6 routine cytologic methods: FFPE cell block, Carnoy's solution, 95% ethanol (EtOH), air-dried and Diff-Quik, ThinPrep, and SurePath preparations. Measurements of RNA quality, expression of 2 multigene expression signatures, molecular subtype, and 4 common activating mutation sites in each preparation were compared with fresh frozen (FF) cell pellet in RNA preservative using distribution of fragment length and concordance correlation coefficient (CCC). RESULTS: The fraction of RNA fragments measuring 200 bases or more (DV200) were 24% higher from cytospins fixed in Carnoy's solution or 95% EtOH than DV200 from FFPE cell blocks. SurePath samples failed RNAseq quality control. There was high concordance of gene expression measurements with FF samples using cytospins fixed in Carnoy's solution, 95% EtOH, Diff-Quik (CCC = 0.829, 0.812, 0.760, respectively), or ThinPrep (CCC = 0.736), but lower using FFPE cell block (CCC = 0.564). The proportion of mutant transcripts was concordant between FF and any cytologic preparation methods. CONCLUSIONS: Cytospin preparations fixed with Carnoy's or 95% ETOH then Papanicolaou stained produced RNAseq results that were equivalent to FF samples and superior to FFPE cell block sections.

Short- and long-term effects of radiation exposure at low dose and low dose rate in normal human VH10 fibroblasts.

Introduction: Experimental studies complement epidemiological data on the biological effects of low doses and dose rates of ionizing radiation and help in determining the dose and dose rate effectiveness factor. Methods: Human VH10 skin fibroblasts exposed to 25, 50, and 100 mGy of 137Cs gamma radiation at 1.6, 8, 12 mGy/h, and at a high dose rate of 23.4 Gy/h, were analyzed for radiation-induced short- and long-term effects. Two sample cohorts, i.e., discovery (n = 30) and validation (n = 12), were subjected to RNA sequencing. The pool of the results from those six experiments with shared conditions (1.6 mGy/h; 24 h), together with an earlier time point (0 h), constituted a third cohort (n = 12). Results: The 100 mGy-exposed cells at all abovementioned dose rates, harvested at 0/24 h and 21 days after exposure, showed no strong gene expression changes. DMXL2, involved in the regulation of the NOTCH signaling pathway, presented a consistent upregulation among both the discovery and validation cohorts, and was validated by qPCR. Gene set enrichment analysis revealed that the NOTCH pathway was upregulated in the pooled cohort (p = 0.76, normalized enrichment score (NES) = 0.86). Apart from upregulated apical junction and downregulated DNA repair, few pathways were consistently changed across exposed cohorts. Concurringly, cell viability assays, performed 1, 3, and 6 days post irradiation, and colony forming assay, seeded just after exposure, did not reveal any statistically significant early effects on cell growth or survival patterns. Tendencies of increased viability (day 6) and reduced colony size (day 21) were observed at 12 mGy/h and 23.4 Gy/min. Furthermore, no long-term changes were observed in cell growth curves generated up to 70 days after exposure. Discussion: In conclusion, low doses of gamma radiation given at low dose rates had no strong cytotoxic effects on radioresistant VH10 cells.

Combining Low-Dose Computer-Tomography-Based Radiomics and Serum Metabolomics for Diagnosis of Malignant Nodules in Participants of Lung Cancer Screening Studies.

Radiomics is an emerging approach to support the diagnosis of pulmonary nodules detected via low-dose computed tomography lung cancer screening. Serum metabolome is a promising source of auxiliary biomarkers that could help enhance the precision of lung cancer diagnosis in CT-based screening. Thus, we aimed to verify whether the combination of these two techniques, which provides local/morphological and systemic/molecular features of disease at the same time, increases the performance of lung cancer classification models. The collected cohort consists of 1086 patients with radiomic and 246 patients with serum metabolomic evaluations. Different machine learning techniques, i.e., random forest and logistic regression were applied for each omics. Next, model predictions were combined with various integration methods to create a final model. The best single omics models were characterized by an AUC of 83% in radiomics and 60% in serum metabolomics. The model integration only slightly increased the performance of the combined model (AUC equal to 85%), which was not statistically significant. We concluded that radiomics itself has a good ability to discriminate lung cancer from benign lesions. However, additional research is needed to test whether its combination with other molecular assessments would further improve the diagnosis of screening-detected lung nodules.

Molecular differences between younger versus older ER-positive and HER2-negative breast cancers.

The RxPONDER and TAILORx trials demonstrated benefit from adjuvant chemotherapy in patients age ≤ 50 with node-positive breast cancer and Recurrence Score (RS) 0-26, and in node-negative disease with RS 16-25, respectively, but no benefit in older women with the same clinical features. We analyzed transcriptomic and genomic data of ER+/HER2- breast cancers with in silico RS < 26 from TCGA (n = 530), two microarray cohorts (A: n = 865; B: n = 609), the METABRIC (n = 867), and the SCAN-B (n = 1636) datasets. There was no difference in proliferation-related gene expression between age groups. Older patients had higher mutation burden and more frequent ESR1 copy number gain, but lower frequency of GATA3 mutations. Younger patients had higher rate of ESR1 copy number loss. In all datasets, younger patients had significantly lower mRNA expression of ESR1 and ER-associated genes, and higher expression of immune-related genes. The ER- and immune-related gene signatures showed negative correlation and defined three subpopulations in younger women: immune-high/ER-low, immune-intermediate/ER-intermediate, and immune-low/ER-intermediate. We hypothesize that in immune-high cancers, the cytotoxic effect of chemotherapy may drive the benefit, whereas in immune-low/ER-intermediate cancers chemotherapy induced ovarian suppression may play important role.

Influence of single-cell RNA sequencing data integration on the performance of differential gene expression analysis.

Large-scale comprehensive single-cell experiments are often resource-intensive and require the involvement of many laboratories and/or taking measurements at various times. This inevitably leads to batch effects, and systematic variations in the data that might occur due to different technology platforms, reagent lots, or handling personnel. Such technical differences confound biological variations of interest and need to be corrected during the data integration process. Data integration is a challenging task due to the overlapping of biological and technical factors, which makes it difficult to distinguish their individual contribution to the overall observed effect. Moreover, the choice of integration method may impact the downstream analyses, including searching for differentially expressed genes. From the existing data integration methods, we selected only those that return the full expression matrix. We evaluated six methods in terms of their influence on the performance of differential gene expression analysis in two single-cell datasets with the same biological study design that differ only in the way the measurement was done: one dataset manifests strong batch effects due to the measurements of each sample at a different time. Integrated data were visualized using the UMAP method. The evaluation was done both on individual gene level using parametric and non-parametric approaches for finding differentially expressed genes and on gene set level using gene set enrichment analysis. As an evaluation metric, we used two correlation coefficients, Pearson and Spearman, of the obtained test statistics between reference, test, and corrected studies. Visual comparison of UMAP plots highlighted ComBat-seq, limma, and MNN, which reduced batch effects and preserved differences between biological conditions. Most of the tested methods changed the data distribution after integration, which negatively impacts the use of parametric methods for the analysis. Two algorithms, MNN and Scanorama, gave very poor results in terms of differential analysis on gene and gene set levels. Finally, we highlight ComBat-seq as it led to the highest correlation of test statistics between reference and corrected dataset among others. Moreover, it does not distort the original distribution of gene expression data, so it can be used in all types of downstream analyses.

Quantifying Spatial Heterogeneity of Tumor-Infiltrating Lymphocytes to Predict Survival of Individual Cancer Patients.

Tumor-infiltrating lymphocytes (TILs), identified on HE-stained histopathological images in the cancer area, are indicators of the adaptive immune response against cancers and play a major role in personalized cancer immunotherapy. Recent works indicate that the spatial organization of TILs may be prognostic of disease-specific survival and recurrence. However, there are a limited number of methods that were proposed and tested in analyses of the spatial structure of TILs. In this work, we evaluated 14 different spatial measures, including the one developed for other omics data, on 10,532 TIL maps from 23 cancer types in terms of reproducibility, uniqueness, and impact on patient survival. For each spatial measure, 16 different scenarios for the definition of prognostic factor were tested. We found no difference in survival prediction when TIL maps were stored as binary images or continuous TIL probability scores. When spatial measures were discretized into a low and high category, a higher correlation with survival was observed. Three measures with the highest cancer prognosis capability were spatial autocorrelation, GLCM M1, and closeness centrality. Most of the tested measures could be further tuned to increase prediction performance.

Tumor immune microenvironment of self-identified African American and non-African American triple negative breast cancer.

Differences in the tumor immune microenvironment may result in differences in prognosis and response to treatment in cancer patients. We hypothesized that differences in the tumor immune microenvironment may exist between African American (AA) and NonAA patients, due to ancestry-related or socioeconomic factors, that may partially explain differences in clinical outcomes. We analyzed clinically matched triple-negative breast cancer (TNBC) tissues from self-identified AA and NonAA patients and found that stromal TILs, PD-L1 IHC-positivity, mRNA expression of immune-related pathways, and immunotherapy response predictive signatures were significantly higher in AA samples (p < 0.05; Fisher's Exact Test, Mann-Whitney Test, Permutation Test). Cancer biology and metabolism pathways, TAM-M2, and Immune Exclusion were significantly higher in NonAA samples (p < 0.05; Permutation Test, Mann-Whitney Test). There were no differences in somatic tumor mutation burden. Overall, there is greater immune infiltration and inflammation in AA TNBC and these differences may impact response to immune checkpoint inhibitors and other therapeutic agents that modulate the immune microenvironment.

Predictive Markers of Response to Neoadjuvant Durvalumab with Nab-Paclitaxel and Dose-Dense Doxorubicin/Cyclophosphamide in Basal-Like Triple-Negative Breast Cancer.

PURPOSE: We examined gene expression, germline variant, and somatic mutation features associated with pathologic response to neoadjuvant durvalumab plus chemotherapy in basal-like triple-negative breast cancer (bTNBC). EXPERIMENTAL DESIGN: Germline and somatic whole-exome DNA and RNA sequencing, programmed death ligand 1 (PD-L1) IHC, and stromal tumor-infiltrating lymphocyte scoring were performed on 57 patients. We validated our results using 162 patients from the GeparNuevo randomized trial. RESULTS: Gene set enrichment analysis showed that pathways involved in immunity (adaptive, humoral, innate), JAK-STAT signaling, cancer drivers, cell cycle, apoptosis, and DNA repair were enriched in cases with pathologic complete response (pCR), whereas epithelial-mesenchymal transition, extracellular matrix, and TGFß pathways were enriched in cases with residual disease (RD). Immune-rich bTNBC with RD was enriched in CCL-3, -4, -5, -8, -23, CXCL-1, -3, -6, -10, and IL1, -23, -27, -34, and had higher expression of macrophage markers compared with immune-rich cancers with pCR that were enriched in IFNγ, IL2, -12, -21, chemokines CXCL-9, -13, CXCR5, and activated T- and B-cell markers (GZMB, CD79A). In the validation cohort, an immune-rich five-gene signature showed higher expression in pCR cases in the durvalumab arm (P = 0.040) but not in the placebo arm (P = 0.923) or in immune-poor cancers. Independent of immune markers, tumor mutation burden was higher, and PI3K, DNA damage repair, MAPK, and WNT/ß-catenin signaling pathways were enriched in germline and somatic mutations in cases with pCR. CONCLUSIONS: The TGFß pathway is associated with immune-poor phenotype and RD in bTNBC. Among immune-rich bTNBC RD, macrophage/neutrophil chemoattractants dominate the cytokine milieu, and IFNγ and activated B cells and T cells dominate immune-rich cancers with pCR.

Cancer Relevance of Human Genes.

BACKGROUND: We hypothesize that genes that directly or indirectly interact with core cancer genes (CCGs) in a comprehensive gene-gene interaction network may have functional importance in cancer. METHODS: We categorized 12 767 human genes into CCGs (n = 468), 1 (n = 5467), 2 (n = 5573), 3 (n = 915), and more than 3 steps (n = 416) removed from the nearest CCG in the Search Tool for the Retrieval of Interacting Genes/Proteins network. We estimated cancer-relevant functional importance in these neighborhood categories using 1) gene dependency score, which reflects the effect of a gene on cell viability after knockdown; 2) somatic mutation frequency in The Cancer Genome Atlas; 3) effect size that estimates to what extent a mutation in a gene enhances cell survival; and 4) negative selection pressure of germline protein-truncating variants in healthy populations. RESULTS: Cancer biology-related functional importance of genes decreases as their distance from the CCGs increases. Genes closer to cancer genes show greater connectedness in the network, have greater importance in maintaining cancer cell viability, are under greater negative germline selection pressure, and have higher somatic mutation frequency in cancer. Based on these 4 metrics, we provide cancer relevance annotation to known human genes. CONCLUSIONS: A large number of human genes are connected to CCGs and could influence cancer biology to various extent when dysregulated; any given mutation may be functionally important in one but not in another individual depending on genomic context.

Comprehensive Analysis of Metabolic Isozyme Targets in Cancer.

Metabolic reprogramming is a hallmark of malignant transformation, and loss of isozyme diversity (LID) contributes to this process. Isozymes are distinct proteins that catalyze the same enzymatic reaction but can have different kinetic characteristics, subcellular localization, and tissue specificity. Cancer-dominant isozymes that catalyze rate-limiting reactions in critical metabolic processes represent potential therapeutic targets. Here, we examined the isozyme expression patterns of 1,319 enzymatic reactions in 14 cancer types and their matching normal tissues using The Cancer Genome Atlas mRNA expression data to identify isozymes that become cancer-dominant. Of the reactions analyzed, 357 demonstrated LID in at least one cancer type. Assessment of the expression patterns in over 600 cell lines in the Cancer Cell Line Encyclopedia showed that these reactions reflect cellular changes instead of differences in tissue composition; 50% of the LID-affected isozymes showed cancer-dominant expression in the corresponding cell lines. The functional importance of the cancer-dominant isozymes was assessed in genome-wide CRISPR and RNAi loss-of-function screens: 17% were critical for cell proliferation, indicating their potential as therapeutic targets. Lists of prioritized novel metabolic targets were developed for 14 cancer types; the most broadly shared and functionally validated target was acetyl-CoA carboxylase 1 (ACC1). Small molecule inhibition of ACC reduced breast cancer viability in vitro and suppressed tumor growth in cell line- and patient-derived xenografts in vivo. Evaluation of the effects of drug treatment revealed significant metabolic and transcriptional perturbations. Overall, this systematic analysis of isozyme expression patterns elucidates an important aspect of cancer metabolic plasticity and reveals putative metabolic vulnerabilities. SIGNIFICANCE: This study exploits the loss of metabolic isozyme diversity common in cancer and reveals a rich pool of potential therapeutic targets that will allow the repurposing of existing inhibitors for anticancer therapy. See related commentary by Kehinde and Parker, p. 1695.

Importance of SNP Dependency Correction and Association Integration for Gene Set Analysis in Genome-Wide Association Studies.

A typical genome-wide association study (GWAS) analyzes millions of single-nucleotide polymorphisms (SNPs), several of which are in a region of the same gene. To conduct gene set analysis (GSA), information from SNPs needs to be unified at the gene level. A widely used practice is to use only the most relevant SNP per gene; however, there are other methods of integration that could be applied here. Also, the problem of nonrandom association of alleles at two or more loci is often neglected. Here, we tested the impact of incorporation of different integrations and linkage disequilibrium (LD) correction on the performance of several GSA methods. Matched normal and breast cancer samples from The Cancer Genome Atlas database were used to evaluate the performance of six GSA algorithms: Coincident Extreme Ranks in Numerical Observations (CERNO), Gene Set Enrichment Analysis (GSEA), GSEA-SNP, improved GSEA for GWAS (i-GSEA4GWAS), Meta-Analysis Gene-set Enrichment of variaNT Associations (MAGENTA), and Over-Representation Analysis (ORA). Association of SNPs to phenotype was calculated using modified McNemar's test. Results for SNPs mapped to the same gene were integrated using Fisher and Stouffer methods and compared with the minimum p-value method. Four common measures were used to quantify the performance of all combinations of methods. Results of GSA analysis on GWAS were compared to the one performed on gene expression data. Comparing all evaluation metrics across different GSA algorithms, integrations, and LD correction, we highlighted CERNO, and MAGENTA with Stouffer as the most efficient. Applying LD correction increased prioritization and specificity of enrichment outcomes for all tested algorithms. When Fisher or Stouffer were used with LD, sensitivity and reproducibility were also better. Using any integration method was beneficial in comparison with a minimum p-value method in specific combinations. The correlation between GSA results from genomic and transcriptomic level was the highest when Stouffer integration was combined with LD correction. We thoroughly evaluated different approaches to GSA in GWAS in terms of performance to guide others to select the most effective combinations. We showed that LD correction and Stouffer integration could increase the performance of enrichment analysis and encourage the usage of these techniques.