Intra-patient Inter-metastatic Genetic Heterogeneity in Colorectal Cancer as a Key Determinant of Survival after Curative Liver Resection.

Chromosomal instability is a well-defined hallmark of tumor aggressiveness and metastatic progression in colorectal cancer. The magnitude of genetic heterogeneity among distinct liver metastases from the same patient at the copy number level, as well as its relationship with chemotherapy exposure and patient outcome, remains unknown. We performed high-resolution DNA copy number analyses of 134 liver metastatic deposits from 45 colorectal cancer patients to assess: (i) intra-patient inter-metastatic genetic heterogeneity using a heterogeneity score based on pair-wise genetic distances among tumor deposits; and (ii) genomic complexity, defined as the proportion of the genome harboring aberrant DNA copy numbers. Results were analyzed in relation to the patients' clinical course; previous chemotherapy exposure and outcome after surgical resection of liver metastases. We observed substantial variation in the level of intra-patient inter-metastatic heterogeneity. Heterogeneity was not associated with the number of metastatic lesions or their genomic complexity. In metachronous disease, heterogeneity was higher in patients previously exposed to chemotherapy. Importantly, intra-patient inter-metastatic heterogeneity was a strong prognostic determinant, stronger than known clinicopathological prognostic parameters. Patients with a low level of heterogeneity (below the median level) had a three-year progression-free and overall survival rate of 23% and 66% respectively, versus 5% and 18% for patients with a high level (hazard ratio0.4, 95% confidence interval 0.2-0.8, P = 0.01; and hazard ratio0.3,95% confidence interval 0.1-0.7, P = 0.007). A low patient-wise level of genomic complexity (below 25%) was also a favorable prognostic factor; however, the prognostic association of intra-patient heterogeneity was independent of genomic complexity in multivariable analyses. In conclusion, intra-patient inter-metastatic genetic heterogeneity is a pronounced feature of metastatic colorectal cancer, and the strong prognostic association reinforces its clinical relevance and places it as a key feature to be explored in future patient cohorts.

Can I trust my fake data - A comprehensive quality assessment framework for synthetic tabular data in healthcare.

BACKGROUND: Ensuring safe adoption of AI tools in healthcare hinges on access to sufficient data for training, testing and validation. Synthetic data has been suggested in response to privacy concerns and regulatory requirements and can be created by training a generator on real data to produce a dataset with similar statistical properties. Competing metrics with differing taxonomies for quality evaluation have been proposed, resulting in a complex landscape. Optimising quality entails balancing considerations that make the data fit for use, yet relevant dimensions are left out of existing frameworks. METHOD: We performed a comprehensive literature review on the use of quality evaluation metrics on synthetic data within the scope of synthetic tabular healthcare data using deep generative methods. Based on this and the collective team experiences, we developed a conceptual framework for quality assurance. The applicability was benchmarked against a practical case from the Dutch National Cancer Registry. CONCLUSION: We present a conceptual framework for quality assuranceof synthetic data for AI applications in healthcare that aligns diverging taxonomies, expands on common quality dimensions to include the dimensions of Fairness and Carbon footprint, and proposes stages necessary to support real-life applications. Building trust in synthetic data by increasing transparency and reducing the safety risk will accelerate the development and uptake of trustworthy AI tools for the benefit of patients. DISCUSSION: Despite the growing emphasis on algorithmic fairness and carbon footprint, these metrics were scarce in the literature review. The overwhelming focus was on statistical similarity using distance metrics while sequential logic detection was scarce. A consensus-backed framework that includes all relevant quality dimensions can provide assurance for safe and responsible real-life applications of synthetic data. As the choice of appropriate metrics are highly context dependent, further research is needed on validation studies to guide metric choices and support the development of technical standards.

Federated Networks for Distributed Analysis of Health Data.

Access to health data, important for population health planning, basic and clinical research and health industry utilization, remains problematic. Legislation intended to improve access to personal data across national borders has proven to be a double-edged sword, where complexity and implications from misinterpretations have paradoxically resulted in data becoming more siloed. As a result, the potential for development of health specific AI and clinical decision support tools built on real-world data have yet to be fully realized. In this perspective, we propose federated networks as a solution to enable access to diverse data sets and tackle known and emerging health problems. The perspective draws on experience from the World Economic Forum Breaking Barriers to Health Data project, the Personal Health Train and Vantage6 infrastructures, and industry insights. We first define the concept of federated networks in a healthcare context, present the value they can bring to multiple stakeholders, and discuss their establishment, operation and implementation. Challenges of federated networks in healthcare are highlighted, as well as the resulting need for and value of an independent orchestrator for their safe, sustainable and scalable implementation.

Genomic and prognostic heterogeneity among RAS/BRAFV600E /TP53 co-mutated resectable colorectal liver metastases.

Hepatic resection is potentially curative for patients with colorectal liver metastases, but the treatment benefit varies. KRAS/NRAS (RAS)/TP53 co-mutations are associated with a poor prognosis after resection, but there is large variation in patient outcome within the mutation groups, and genetic testing is currently not used to evaluate benefit from surgery. We have investigated the potential for improved prognostic stratification by combined biomarker analysis with DNA copy number aberrations (CNAs), and taking tumor heterogeneity into account. We determined the mutation status of RAS, BRAFV600 , and TP53 in 441 liver lesions from 171 patients treated by partial hepatectomy for metastatic colorectal cancer. CNAs were profiled in 232 tumors from 67 of the patients. Mutations and high-level amplifications of cancer-critical genes, the latter including ERBB2 and EGFR, were predominantly homogeneous within patients. RAS/BRAFV600E and TP53 co-mutations were associated with a poor patient outcome (hazard ratio, HR, 3.9, 95% confidence interval, CI, 1.3-11.1, P = 0.012) in multivariable analyses with clinicopathological variables. The genome-wide CNA burden and intrapatient intermetastatic CNA heterogeneity varied within the mutation groups, and the CNA burden had prognostic associations in univariable analysis. Combined prognostic analyses of RAS/BRAFV600E /TP53 mutations and CNAs, either as a high CNA burden or high intermetastatic CNA heterogeneity, identified patients with a particularly poor outcome (co-mutation/high CNA burden: HR 2.7, 95% CI 1.2-5.9, P = 0.013; co-mutation/high CNA heterogeneity: HR 2.5, 95% CI 1.1-5.6, P = 0.022). In conclusion, DNA copy number profiling identified genomic and prognostic heterogeneity among patients with resectable colorectal liver metastases with co-mutated RAS/BRAFV600E /TP53.

Frequent copy number gains of SLC2A3 and ETV1 in testicular embryonal carcinomas.

Testicular germ cell tumours (TGCTs) appear as different histological subtypes or mixtures of these. They show similar, multiple DNA copy number changes, where gain of 12p is pathognomonic. However, few high-resolution analyses have been performed and focal DNA copy number changes with corresponding candidate target genes remain poorly described for individual subtypes. We present the first high-resolution DNA copy number aberration (CNA) analysis on the subtype embryonal carcinomas (ECs), including 13 primary ECs and 5 EC cell lines. We identified recurrent gains and losses and allele-specific CNAs. Within these regions, we nominate 30 genes that may be of interest to the EC subtype. By in silico analysis of data from 150 TGCTs from The Cancer Genome Atlas (TCGA), we further investigated CNAs, RNA expression, somatic mutations and fusion transcripts of these genes. Among primary ECs, ploidy ranged between 2.3 and 5.0, and the most common aberrations were DNA copy number gains at chromosome (arm) 7, 8, 12p, and 17, losses at 4, 10, 11, and 18, replicating known TGCT genome characteristics. Gain of whole or parts of 12p was found in all samples, including a highly amplified 100 kbp segment at 12p13.31, containing SLC2A3. Gain at 7p21, encompassing ETV1, was the second most frequent aberration. In conclusion, we present novel CNAs and the genes located within these regions, where the copy number gain of SLC2A3 and ETV1 are of interest, and which copy number levels also correlate with expression in TGCTs.

Flavodoxin relaxes in microseconds upon excitation of the flavin chromophore: detection of a UV-visible silent intermediate by laser photocalorimetry.

A small contraction concomitant with the relaxation of the protein in ca. 3 mus is observed upon ns-laser excitation at 455 nm of the Cys69Ala (C69A) mutant of flavodoxin II from Azotobacter vinelandii. This constitutes another example of detection of a UV-vis silent transient species through a photocalorimetric technique. The contraction is attributed to reorganization of protein dihedral bonds and of water molecules at relatively long distances from the flavin chromophore, after the protein has received the heat shock from the relaxing photoproduced charge transfer state. This study constitutes a preliminary step towards the understanding of the origin of protein movements taking place upon electron transfer reactions, e.g. between a photoinduced electron donor (or acceptor) and an accepting (or donating) cofactor in a protein.

Gene expression profiles of CMS2-epithelial/canonical colorectal cancers are largely driven by DNA copy number gains.

About 80% of colorectal cancers (CRCs) have chromosomal instability, which is an integral part of aggressive malignancy development, but the importance of specific copy number aberrations (CNAs) in modulating gene expression, particularly within the framework of clinically relevant molecular subtypes, remains mostly elusive. We performed DNA copy number profiling of 257 stage I-IV primary CRCs and integrative gene expression analysis in 151 microsatellite stable (MSS) tumors, focusing on high-level amplifications and the effect of CNAs on the characteristics of the gene expression-based consensus molecular subtypes (CMS). The results were validated in 323 MSS tumors from TCGA. Novel recurrent high-level amplifications (≥15 additional copies) with a major impact on gene expression were found for TOX3 (16q) at 1.5% frequency, as well as for CCND2 (12p) and ANXA11 (10q) at 1% frequency, in addition to the well-known targets ERBB2 (17q) and MYC (8q). Focal amplifications with ≥15 or ≥5 additional copies of at least one of these regions were associated with a poor overall survival among patients with stage I-III MSS CRCs (multivariable hazard ratio ≥3.2, p ≤ 0.01). All high-level amplifications were focal and had a more consistent relationship with gene expression than lower amplitude and/or broad-range amplifications, suggesting specific targeting during carcinogenesis. Genome-wide, copy number driven gene expression was enriched for pathways characteristic of the CMS2-epithelial/canonical subtype, including DNA repair and cell cycle progression. Furthermore, 50% of upregulated genes in CMS2-epithelial/canonical MSS CRCs were driven by CNAs, an enrichment compared with the other CMS groups, and associated with the stronger correspondence between CNAs and gene expression in malignant epithelial cells than in the cells of the tumor microenvironment (fibroblasts, endothelial cells, leukocytes). In conclusion, we identify novel recurrent amplifications with impact on gene expression in CRC and provide the first evidence that CMS2 may have a stronger copy-number related genetic basis than subtypes more heavily influenced by gene expression signals from the tumor microenvironment.

Serum protein profiling in mice: identification of Factor XIIIa as a potential biomarker for muscular dystrophy.

Protein profiling in blood serum by fractionation and MS analysis has been applied in mice to assess its applicability as a fast, economical alternative to current DNA and RNA analyses for diagnosis of neuromuscular disorders. Mass spectra of peptides and proteins were generated using serum from dystrophin-deficient mdx and control mice by WCX ClinProt bead fractionation, followed by MALDI-MS. Double cross-validatory linear discriminant and logistic regression data analysis methods were compared with a new Bayesian logistic regression method. These were evaluated on their ability to discriminate between healthy and dystrophic samples, and to identify the discriminatory peaks in the mass spectra. All three approaches classified the spectra with comparable misclassification rates (between 18.4 and 20.6%), with much overlap between the differential peaks identified between the methods. The differential peak pattern from the Bayesian method was sparser and easier to interpret than from the other two methods, without compromising classifying strength. One of the two main differentiating peaks at m/z 3908 was identified as an N-terminal peptide of coagulation Factor XIIIa, previously identified in human serum. This work underlines the translational aspect of serum protein profiling in mice and supports a further study with serum from patients with neuromuscular disorders.

Identification of Novel Fusion Genes in Testicular Germ Cell Tumors.

Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men ages 15 to 44 years. Embryonal carcinomas (EC) comprise a subset of TGCTs that exhibit pluripotent characteristics similar to embryonic stem (ES) cells, but the genetic drivers underlying malignant transformation of ECs are unknown. To elucidate the abnormal genetic events potentially contributing to TGCT malignancy, such as the existence of fusion genes or aberrant fusion transcript expression, we performed RNA sequencing of EC cell lines and their nonmalignant ES cell line counterparts. We identified eight novel fusion transcripts and one gene with alternative promoter usage, ETV6. Four out of nine transcripts were found recurrently expressed in an extended panel of primary TGCTs and additional EC cell lines, but not in normal parenchyma of the testis, implying tumor-specific expression. Two of the recurrent transcripts involved an intrachromosomal fusion between RCC1 and HENMT1 located 80 Mbp apart and an interchromosomal fusion between RCC1 and ABHD12B. RCC1-ABHD12B and the ETV6 transcript variant were found to be preferentially expressed in the more undifferentiated TGCT subtypes. In vitro differentiation of the NTERA2 EC cell line resulted in significantly reduced expression of both fusion transcripts involving RCC1 and the ETV6 transcript variant, indicating that they are markers of pluripotency in a malignant setting. In conclusion, we identified eight novel fusion transcripts that, to our knowledge, are the first fusion genes described in TGCT and may therefore potentially serve as genomic biomarkers of malignant progression.

A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: structural determinants of redox potential.

Flavodoxin II from Azotobacter vinelandii is a "long-chain" flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 A by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five-stranded parallel beta-sheet flanked on either side by alpha-helices. An eight-residue insertion, compared with other long-chain flavodoxins, forms a short 3(10) helix preceding the start of the alpha3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent-accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular.

Novel RNA variants in colorectal cancers.

With an annual estimated incidence of 1.4 million, and a five-year survival rate of 60%, colorectal cancer (CRC) is a major clinical burden. To identify novel RNA variants in CRC, we analyzed exon-level microarray expression data from a cohort of 202 CRCs. We nominated 25 genes with increased expression of their 3' parts in at least one cancer sample each. To efficiently investigate underlying transcript structures, we developed an approach using rapid amplification of cDNA ends followed by high throughput sequencing (RACE-seq). RACE products from the targeted genes in 23 CRC samples were pooled together and sequenced. We identified VWA2-TCF7L2, DHX35-BPIFA2 and CASZ1-MASP2 as private fusion events, and novel transcript structures for 17 of the 23 other candidate genes. The high-throughput approach facilitated identification of CRC specific RNA variants. These include a recurrent read-through fusion transcript between KLK8 and KLK7, and a splice variant of S100A2. Both of these were overrepresented in CRC tissue and cell lines from external RNA-seq datasets.

Molecular characteristics of malignant ovarian germ cell tumors and comparison with testicular counterparts: implications for pathogenesis.

This review focuses on the molecular characteristics and development of rare malignant ovarian germ cell tumors (mOGCTs). We provide an overview of the genomic aberrations assessed by ploidy, cytogenetic banding, and comparative genomic hybridization. We summarize and discuss the transcriptome profiles of mRNA and microRNA (miRNA), and biomarkers (DNA methylation, gene mutation, individual protein expression) for each mOGCT histological subtype. Parallels between the origin of mOGCT and their male counterpart testicular GCT (TGCT) are discussed from the perspective of germ cell development, endocrinological influences, and pathogenesis, as is the GCT origin in patients with disorders of sex development. Integrated molecular profiles of the 3 main histological subtypes, dysgerminoma (DG), yolk sac tumor (YST), and immature teratoma (IT), are presented. DGs show genomic aberrations comparable to TGCT. In contrast, the genome profiles of YST and IT are different both from each other and from DG/TGCT. Differences between DG and YST are underlined by their miRNA/mRNA expression patterns, suggesting preferential involvement of the WNT/ß-catenin and TGF-ß/bone morphogenetic protein signaling pathways among YSTs. Characteristic protein expression patterns are observed in DG, YST and IT. We propose that mOGCT develop through different developmental pathways, including one that is likely shared with TGCT and involves insufficient sexual differentiation of the germ cell niche. The molecular features of the mOGCTs underline their similarity to pluripotent precursor cells (primordial germ cells, PGCs) and other stem cells. This similarity combined with the process of ovary development, explain why mOGCTs present so early in life, and with greater histological complexity, than most somatic solid tumors.

Transforming pluripotency: an exon-level study of malignancy-specific transcripts in human embryonal carcinoma and embryonic stem cells.

To circumvent difficulties of isolating pure populations of cancer stem cells (CSCs) for the purpose of identifying malignancy-specific gene expression, we have compared exon-resolution transcriptomic profiles of 5 embryonal carcinoma (EC) cell lines, a histological subtype of germ cell tumor (GCT), to their nonmalignant caricature, specifically 6 human embryonic stem (ES) cell lines. Both cell types are readily accessible, and were purified for undifferentiated cells only. We identified a set of 28 differentially expressed genes, many of which had cancer and stemness roles. Overexpression of the recently discovered pluripotency gene NR5A2 in malignant EC cells revealed an intriguing indication of how WNT-mediated dysregulation of pluripotency is involved with malignancy. Expression of these 28 genes was further explored within 2 publically available data sets of primary EC tumors and normal testis. At the exon-level, alternative splicing events were detected in ZNF195, DNMT3B, and PMF1, and alternative promoters were detected for ASH2L and ETV5. These events were validated by reverse transcriptase-polymerase chain reaction-based methods in EC and ES lines, where the alternative splicing event in the de novo DNA methyltransferase DNMT3B may have functional consequences. In conclusion, we have identified malignancy-specific gene expression differences within a rigorous pluripotent stem cell context. These findings are of particular interest for both GCT and ES cell biology, and, in general, to the concept of CSCs.

BCL-XL mediates the strong selective advantage of a 20q11.21 amplification commonly found in human embryonic stem cell cultures.

Human embryonic stem cells (hESCs) regularly acquire nonrandom genomic aberrations during culture, raising concerns about their safe therapeutic application. The International Stem Cell Initiative identified a copy number variant (CNV) amplification of chromosome 20q11.21 in 25% of hESC lines displaying a normal karyotype. By comparing four cell lines paired for the presence or absence of this CNV, we show that those containing this amplicon have higher population doubling rates, attributable to enhanced cell survival through resistance to apoptosis. Of the three genes encoded within the minimal amplicon and expressed in hESCs, only overexpression of BCL2L1 (BCL-XL isoform) provides control cells with growth characteristics similar to those of CNV-containing cells, whereas inhibition of BCL-XL suppresses the growth advantage of CNV cells, establishing BCL2L1 as a driver mutation. Amplification of the 20q11.21 region is also detectable in human embryonal carcinoma cell lines and some teratocarcinomas, linking this mutation with malignant transformation.

Probing the reactivity of different forms of azurin by flavin photoreduction.

The reactivity of a variant of the blue copper protein, azurin from Pseudomonas aeruginosa, was investigated with laser flash photolysis and compared with the reactivity of the wild-type (WT) protein. The variant was obtained by changing the Cu ligating His117 for a glycine. The mutation creates a gap in the ligand shell of the Cu that can be filled with external ligands or water molecules. The crystal structure of the H117G variant is reported. It shows that the immediate surrounding of the Cu site in the variant exhibits less rigidity than in the WT protein and that the loop containing the Cu ligands Cys112, His117 and Met121 in the WT protein has gained flexibility in the H117G variant. Flash photolysis experiments were performed with 5-deazariboflavin and 8α-imidazolyl-(N-propylyl)-amino riboflavin as electron donors to probe the reactivity of WT and H117G azurin, and of H117G azurin for which the gap in the Cu co-ordination shell was filled with imidazole. 8α-Imidazolyl-(N-propylyl)-amino riboflavin appears one to two orders less efficient as a photo-flash reductant than 5-deazariboflavin. The reactivity of the H117G variant in the absence of external ligands appears to be 2.5-fold lower than the WT reactivity (second-order rate constants of 51 ± 2 × 10(7) m(-1) ·s(-1) versus 21 ± 1 × 10(7) m(-1) ·s(-1) ), whereas the addition of imidazole restores reactivity to above the WT level (71 ± 4 × 10(7) m(-1) ·s(-1) ). The differences are discussed in terms of structural modifications and changes in reorganizational energy and electronic coupling. Database Structural data are available in the Protein Data Bank under the accession number 3N2J.

TPD52, a candidate gene from genomic studies, is overexpressed in testicular germ cell tumours.

Several genomic regions are recurrently over- or underrepresented in testicular germ cell tumours (TGCTs), but only a fraction of their genes change their expression accordingly. Two publications to date have studied DNA copy numbers and associated gene expression changes on a genome-wide level to identify key players in TGCT tumorigenesis. Here, we compare lists of significant genes in these studies, and show that 17 genes are common to both. These include concomitant gain and over-expression of JUB, NRXN3, and TPD52, and loss and under-expression of C11orf70 and CADM1, in addition to 12 overexpressed genes located on the chromosome arm 12p. We performed immunohistochemical analysis of TPD52 on a tissue microarray, which showed complete absence of TPD52 protein in normal germ cells and most intratubular germ cell neoplasias. TPD52 was expressed in two-thirds of seminomas and embryonal carcinomas, and at intermediate frequencies in the more differentiated non-seminomas.

Sensitive detection of the redox state of copper proteins using fluorescence.

The blue copper protein azurin from Pseudomonas aeruginosa has been covalently labelled with the fluorescing dye Cy5. The optical spectrum of the azurin changes markedly with its redox state. These changes are reflected in the fluorescence intensity of the dye through fluorescence resonance energy transfer (FRET). This provides a sensitive way to monitor biological redox events. The method shown to work in the nanomolar range of protein concentrations, can be easily extended into the sub-nanomolar regime and holds promise for single-molecule detection.