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During disease progression the cells that comprise solid malignancies undergo significant changes in gene copy number and chromosome structure. Colorectal cancer provides an excellent model to study this process. To indentify and characterize chromosomal abnormalities in colorectal cancer, we performed a statistical analysis of 299 expression and 130 SNP arrays profiled at different stages of the disease, including normal tissue, adenoma, stages 1-4 adenocarcinoma, and metastasis. We identified broad (> 1/2 chromosomal arm) and focal (< 1/2 chromosomal arm) events. Broad amplifications were noted on chromosomes 7, 8q, 13q, 20, and X and broad deletions on chromosomes 4, 8p, 14q, 15q, 17p, 18, 20p, and 22q. Focal events (gains or losses) were identified in regions containing known cancer pathway genes, such as VEGFA, MYC, MET, FGF6, FGF23, LYN, MMP9, MYBL2, AURKA, UBE2C, and PTEN. Other focal events encompassed potential new candidate tumor suppressors (losses) and oncogenes (gains), including CCDC68, CSMD1, POLR1D, and PMEPA1. From the expression data, we identified genes whose expression levels reflected their copy number changes and used this relationship to impute copy number changes to samples without accompanying SNP data. This analysis provided the statistical power to show that deletions of 8p, 4p, and 15q are associated with survival and disease progression, and that samples with simultaneous deletions in 18q, 8p, 4p, and 15q have a particularly poor prognosis. Annotation analysis reveals that the oxidative phosphorylation pathway shows a strong tendency for decreased expression in the samples characterized by poor prognosis.

If properly translated to clinical use, our knowledge about biomarkers may lead to a more effective way of combating colorectal cancer (CRC). Biomarkers are biomolecular, genetic, or cytogenetic attributes indicative of the disease’s progression, predisposition, prognosis, or therapeutic options. For CRC, these include chromosomal instability, mutations in KRAS and TP53 , loss of 18q, and elevated level of carcinoembryonic antigen (CEA), which are all associated with poor prognosis. The prognostic significance of 18q loss can be attributed to reduced expression of SMAD4 , or DCC , although the chromosomal arm is actually heavily populated by genes whose downregulation correlate to worse survival. Potentially, identification of prognostic biomarkers can help the oncologist decide whether adjuvant chemotherapy is necessary after surgery. Testing for therapeutic biomarkers can be necessary if targeted therapeutics are being considered. The identification of highly penetrant predisposition markers (such as mutations in APC and MLH1 ) can be a lifesaver for carrier individuals, who would then have to undergo colonoscopy at an earlier age. Even sporadic CRCs may have some hereditary components, according to recent studies. Genome-wide association studies (using SNP arrays) showed that polymorphisms of certain genes can have subtle influence on CRC predisposition. Our own SNP array-based analysis suggested that long stretches of germline homozygosity (autozygosity), indicative of consanguinity, may also factor in CRC predisposition.

Background Interrogation of site-specific CpG methylation in circulating tumor DNAs (ctDNAs) has been employed in a number of studies for early detection of breast cancer (BrCa). In many of these studies, the markers were identified based on known biology of BrCa progression, and interrogated using methyl-specific PCR (MSP), a technique involving bisulfite conversion, PCR, and qPCR. Methods In this report, we are demonstrating the development of a novel assay (Multiplex Bisulfite PCR-LDR-qPCR) which can potentially offer improvements to MSP, by integrating additional steps such as ligase detection reaction (LDR), methylated CpG target enrichment, carryover protection (use of uracil DNA glycosylase), and minimization of primer-dimer formation (use of ribose primers and RNAseH2). The assay is designed to for breast cancer-specific CpG markers identified through integrated analyses of publicly available genome-wide methylation datasets for 31 types of primary tumors (including BrCa), as well as matching normal tissues, and peripheral blood. Results Our results indicate that the PCR-LDR-qPCR assay is capable of detecting ~ 30 methylated copies of each of 3 BrCa-specific CpG markers, when mixed with excess amount unmethylated CpG markers (~ 3000 copies each), which is a reasonable approximation of BrCa ctDNA overwhelmed with peripheral blood cell-free DNA (cfDNA) when isolated from patient plasma. The bioinformatically-identified CpG markers are located in promoter regions of NR5A2 and PRKCB , and a non-coding region of chromosome 1 (upstream of EFNA3 ). Additional bioinformatic analyses would reveal that these methylation markers are independent of patient race and age, and positively associated with signaling pathways associated with BrCa progression (such as those related to retinoid nuclear receptor, PTEN, p53, pRB, and p27). Conclusion This report demonstrates the potential utilization of bisulfite PCR-LDR-qPCR assay, along with bioinformatically-driven biomarker discovery, in blood-based BrCa detection.

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).

We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein:protein interaction (PPI) target classes.

The p53 gene is a critical tumor suppressor that is inactivated in a majority of cancers. The central role of p53 in response to stresses such as DNA damage, hypoxia, and oncogene activation underlies this high frequency of negative selection during tumorigenic transformation. Mutations in p53 disrupt checkpoint responses to DNA damage and result in the potential for destabilization of the genome. Consistent with this, p53 mutant cells have been shown to accumulate genomic alterations in cell culture, mouse models, and some human tumors. The relationship between p53 mutation and genomic instability in human osteosarcoma is addressed in this report. Similar to some other primary human tumors, the mutation of p53 correlates significantly with the presence of high levels of genomic instability in osteosarcomas. Surprisingly, osteosarcomas harboring an amplification of the HDM2 oncogene, which inhibits the tumor-supressive properties of p53, do not display high levels of genomic instability. These results demonstrate that the inactivation of p53 in osteosarcomas directly by mutation versus indirectly by HDM2 amplification may have different cellular consequences with respect to the stability of the genome.

Array-based mutation detection methodology typically relies on direct hybridization of the fluorescently labeled query sequence to surface-bound oligonucleotide probes. These probes contain either small sequence variations or perfect-match sequence. The intensity of fluorescence bound to each oligonucleotide probe is intended to reveal which sequence is perfectly complementary to the query sequence 1 . However, these approaches have not always been successful, especially for detection of small frameshift mutations. Here we describe a multiplex assay to detect small insertions and deletions by using a modified PCR to evenly amplify each amplicon (PCR/PCR) 2 , followed by ligase detection reaction (LDR) 3 . Mutations were identified by screening reaction products with a universal DNA microarray 4 , which uncouples mutation detection from array hybridization and provides for high sensitivity. Using the three BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population ( BRCA1 185delAG; BRCA1 5382insC; BRCA2 6174delT) 5 as a model system, the assay readily detected these mutations in multiplexed reactions. Our results demonstrate that universal microarray analysis of PCR/PCR/LDR 2 products permits rapid identification of small insertion and deletion mutations in the context of both clinical diagnosis and population studies.

Point mutations in codons 12, 13, and 61 of the K-ras gene occur early in the development of colorectal cancer and are preserved throughout the course of tumor progression. These mutations can serve as biomarkers for shed or circulating tumor cells and may be useful for diagnosis of early, curable tumors and for staging of advanced cancers. We have developed a multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) method which identifies all 19 possible single-base mutations in K-ras codons 12, 13, and 61, with a sensitivity of 1 in 500 wild-type sequences. In a blinded study, 144 paraffin-embedded archival colon carcinomas were microdissected and K-ras mutations determined by both dideoxy-sequencing and multiplex PCR/LDR. Results were concordant for 134 samples. The ten discordant samples were re-evaluated using higher sensitivity uniplex PCR/LDR, and the original multiplex PCR/LDR result was confirmed in nine of these ten cases. Multiplex PCR/LDR was able to identify mutations in solid tumors or paraffin-embedded tissues containing a majority of wild-type stromal cells, with or without microdissection. The technique is well suited for large scale studies and for analysis of clinical samples containing a minority population of mutated cells.

Our understanding of metastatic prostate cancer (mPrCa) has dramatically advanced during the genomics era. Nonetheless, many aspects of the disease may still be uncovered through reanalysis of public datasets. We integrated the expression datasets for 209 PrCa tissues (metastasis, primary, normal) with expression, gene dependency (GD) (from CRISPR/cas9 screen), and drug viability data for hundreds of cancer lines (including PrCa). Comparative statistical and pathways analyses and functional annotations (available inhibitors, protein localization) revealed relevant pathways and potential (and previously reported) protein markers for minimally invasive mPrCa diagnostics. The transition from localized to mPrCa involved the upregulation of DNA replication, mitosis, and PLK1-mediated events. Genes highly upregulated in mPrCa and with very high average GD (~1) are potential therapeutic targets. We showed that fostamatinib (which can target PLK1 and other over-expressed serine/threonine kinases such as AURKA, MELK, NEK2, and TTK) is more active against cancer lines with more pronounced signatures of invasion (e.g., extracellular matrix organization/degradation). Furthermore, we identified surface-bound (e.g., ADAM15, CD276, ABCC5, CD36, NRP1, SCARB1) and likely secreted proteins (e.g., APLN, ANGPT2, CTHRC1, ADAM12) that are potential mPrCa diagnostic markers. Overall, we demonstrated that comprehensive analyses of public genomics data could reveal potentially clinically relevant information regarding mPrCa.

Background/Objectives: Bisulfite-based cell-free DNA (cfDNA) methylation assays enable the detection of clinically valuable epigenetic biomarkers but often cause DNA degradation and inconsistent conversion efficiency, limiting performance in low-input liquid biopsy samples. We aimed to develop and evaluate a fully enzymatic cfDNA methylation workflow that preserves DNA integrity and supports quantitative clinical detection. Methods: The assay integrates TET2-mediated oxidation and APOBEC3A deamination with RNase H2-guided primer design, uracil-DNA glycosylase error suppression, and dual-probe detection compatible with quantitative PCR (qPCR) and digital PCR (dPCR). Performance was assessed using serial dilutions of methylated HT29 DNA, unmethylated controls, and plasma cfDNA from colorectal cancer (CRC) patients and healthy donors. Analytical sensitivity, linearity, and concordance between platforms were evaluated. Results: The 40-marker panel demonstrated higher cumulative methylation scores and more frequent methylation-positive signals in CRC cfDNA compared to controls. dPCR confirmed single-molecule resolution and clear discrimination between methylated and unmethylated templates, with occasional double-positive partitions consistent with mixed allelic methylation. Signal intensity across the dilution series followed a four-parameter logistic model, achieving detection sensitivity below 0.2% methylated DNA. qPCR and dPCR results showed strong correlation across the HT29 dilution series (R2 = 0.80) and high concordance in classifying CRC and healthy samples. Conclusions: This TET2–APOBEC-based enzymatic cfDNA assay enables sensitive, quantitative, sequencing-free methylation detection under gentle conditions, supporting its application in early colorectal cancer screening and routine clinical liquid biopsy workflows.