Circulating tumor DNA is a sensitive marker for routine monitoring of treatment response in advanced colorectal cancer.

Accurate assessment of chemotherapy response provides the means to terminate ineffective treatment, trial alternative drug regimens or schedules and reduce dose to minimize toxicity. Here, we have compared circulating tumor DNA (ctDNA) with carcinoembryonic antigen (CEA) for the cycle by cycle assessment of chemotherapy response in 30 patients with metastatic colorectal cancer. CtDNA (quantified using individualized digital droplet PCR (ddPCR) assays) and CEA levels were determined immediately prior to each chemotherapy cycle over time periods ranging from 42-548 days (average of 10 time points/patient). Twenty-nine/thirty (97%) patients had detectable ctDNA compared with 83% whose tumors were CEA-positive (>5 ng/ml) during the monitoring course. Over the course of treatment, 20 disease progression events were detected by computed tomography; ctDNA predicted significantly more of these events than CEA (16 (80%) versus 6 (30%), respectively; P-value = 0.004). When progression was detected by both ctDNA and CEA, the rise in ctDNA occurred significantly earlier than CEA (P-value = 0.046). Partial responses to chemotherapy were also detected more frequently by ctDNA, although this was not significant (P-value = 0.07). In addition, another 28 colorectal cancer patients who underwent potentially curative surgery and showed no evidence of residual disease were monitored with ctDNA for up to 2 years. Clinical relapse was observed in 6/28 (21%) patients. Four out of 6 of these patients showed a significant increase in ctDNA at or prior to relapse. Overall, ctDNA analyses were able to be performed in a clinically relevant timeline and were a more sensitive and responsive measure of tumor burden than CEA.

Genome-wide methylation analysis identifies a core set of hypermethylated genes in CIMP-H colorectal cancer.

BACKGROUND: Aberrant DNA methylation profiles are a characteristic of all known cancer types, epitomized by the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC). Hypermethylation has been observed at CpG islands throughout the genome, but it is unclear which factors determine whether an individual island becomes methylated in cancer. METHODS: DNA methylation in CRC was analysed using the Illumina HumanMethylation450K array. Differentially methylated loci were identified using Significance Analysis of Microarrays (SAM) and the Wilcoxon Signed Rank (WSR) test. Unsupervised hierarchical clustering was used to identify methylation subtypes in CRC. RESULTS: In this study we characterized the DNA methylation profiles of 94 CRC tissues and their matched normal counterparts. Consistent with previous studies, unsupervized hierarchical clustering of genome-wide methylation data identified three subtypes within the tumour samples, designated CIMP-H, CIMP-L and CIMP-N, that showed high, low and very low methylation levels, respectively. Differential methylation between normal and tumour samples was analysed at the individual CpG level, and at the gene level. The distribution of hypermethylation in CIMP-N tumours showed high inter-tumour variability and appeared to be highly stochastic in nature, whereas CIMP-H tumours exhibited consistent hypermethylation at a subset of genes, in addition to a highly variable background of hypermethylated genes. EYA4, TFPI2 and TLX1 were hypermethylated in more than 90% of all tumours examined. One-hundred thirty-two genes were hypermethylated in 100% of CIMP-H tumours studied and these were highly enriched for functions relating to skeletal system development (Bonferroni adjusted p value =2.88E-15), segment specification (adjusted p value =9.62E-11), embryonic development (adjusted p value =1.52E-04), mesoderm development (adjusted p value =1.14E-20), and ectoderm development (adjusted p value =7.94E-16). CONCLUSIONS: Our genome-wide characterization of DNA methylation in colorectal cancer has identified 132 genes hypermethylated in 100% of CIMP-H samples. Three genes, EYA4, TLX1 and TFPI2 are hypermethylated in >90% of all tumour samples, regardless of CIMP subtype.

Synthesis, Crystal Structure and Catalytic Activity of Dioxidomolybdenum(VI) Complex with Tridentate ONO Aroylhydrazone Ligand.

A novel dioxidomolybdenum complex, [MoO(2)L], derived from the tridentate aroylhydrazone 2-amino-N'-(2-hydroxy-3-methylbenzylidene)benzohydrazide (H(2)L), has been prepared and characterized by elemental analysis, FT-IR, (1)H NMR, and single crystal structural X-ray diffraction. The complex has distorted square pyramidal structure in which the aroylhydrazone ligand behaves as a binegative donor one, leaving the amino-N uncoordinated towards the metal center. Single crystal structure analysis reveals that the Mo(VI) center is coordinated by the donor atoms of the binegative aroylhydrazone ligand and two oxido groups. Crystal of the complex is stabilized by hydrogen bonds. The complex functions as an effective olefin epoxidation catalyst with hydrogen peroxide as terminal oxidant and sodium hydrogen carbonate as a co-catalyst.

Synthesis, X-Ray Crystal Structures and Catalytic Epoxidation of Oxidovanadium(V) Complexes with Aroylhydrazone and Ethyl Maltolate Ligands.

Two oxidovanadium(V) complexes, [VOL1L] (1) and [VOL2L] (2) (L = ethyl maltolate), derived from the aroylhydrazones 4-bromo-N'-(2-hydroxy-5-methylbenzylidene)benzohydrazide (H2L1) and N'-(3,5-dibromo-2-hydroxybenzylidene)-4-methoxybenzohydrazide (H2L2), respectively, have been synthesized and characterized by elemental analysis, infrared and electronic spectroscopy. Structures of the complexes were further confirmed by single crystal X-ray determination. The V atoms in the complexes are coordinated by the ONO donor atoms of the aroylhydrazone ligand, OO donor atoms of the ethyl maltolate ligand, and one oxido O atom, forming octahedral coordination. The complexes function as effective olefin epoxidation catalysts with hydrogen peroxide as terminal oxidant and sodium hydrogen carbonate as a co-catalyst.

Epigenetic silencing in non-neoplastic epithelia identifies E-cadherin (CDH1) as a target for chemoprevention of lobular neoplasia.

Invasive lobular carcinoma (ILC) of the breast is believed to develop from in situ lesions, atypical lobular hyperplasia (ALH), and lobular carcinoma in situ (LCIS). Down-regulation of the cell-cell adhesion protein E-cadherin is a defining feature of lobular breast cancer (LBC) and already occurs in ALH and LCIS. Apart from mutational mechanisms, epigenetic silencing of the E-cadherin gene (CDH1) is thought to be involved in E-cadherin down-regulation and has been observed at a high frequency in ILC. Whether CDH1 promoter methylation is already present in in situ lesions and thus contributes to the initiation of LBC is not established. We thus examined microdissected archived tissue from 20 LBCs by methylation-specific PCR to determine the CDH1 methylation status of lobular lesions. Nineteen of the 20 LBCs had a hypermethylated CDH1 promoter, including 13/14 ILCs and 13/13 ALHs or LCIS. Bisulphite sequencing indicated that methylation was complete within the investigated promoter fragment. Intriguingly, CDH1 methylation was likewise present in 8/8 adjacent non-neoplastic epithelia, but not in 6/6 mammary epithelia from healthy subjects. E-cadherin protein and mRNA were down-regulated in in situ lesions relative to adjacent epithelia. Together, these results indicate that CDH1 promoter methylation occurs in LBC prior to E-cadherin down-regulation and neoplastic formation. We thus propose that epigenetic silencing represents the first of the two hits required to silence both CDH1 alleles for LBC to develop. Because promoter methylation is in principle reversible, our findings suggest that chemoprevention of LBC by epigenetic drugs should be feasible. Furthermore, the presence of CDH1 methylation in pre-neoplastic epithelia suggests the existence of mammary regions with increased disease susceptibility, providing an explanation for the often multifocal presentation of LBC.

Increased levels of active c-Src distinguish invasive from in situ lobular lesions.

INTRODUCTION: Mounting molecular evidence suggests that invasive lobular carcinoma (ILC) is developing from in situ lesions, atypical lobular hyperplasia (ALH), and lobular carcinoma in situ (LCIS). However, little is known about the mechanisms promoting the progression of lobular breast cancer (LBC) to invasive disease. Here, we investigated whether c-Src kinase, an established inducer of invasive states, contributes to the progression from ALH/LCIS to ILC. METHODS: Immunochemistry for c-Src and other cancer-related molecules was performed on archived tissue specimens from 57 LBC patients. Relative c-Src activity was estimated by comparing fluorescence intensity of ILC with that of adjacent ALH/LCIS and nonneoplastic epithelia after staining with an antibody against active c-Src. Expression of active c-Src was correlated with markers of invasion and malignancy and with relapse among LBC patients. RESULTS: Levels of activated c-Src were increased in ILC relative to ALH/LCIS (1.63-fold +/- 0.24 SD) and nonneoplastic epithelia (1.47 +/- 0.18 SD). Increased c-Src levels correlated with the activation of c-Src downstream targets (Fak, Stat-3) and the expression of mesenchymal markers. ILC cells with activated c-Src co-expressed metastatic markers (Opn, Cxcr4) and included cells positive for the cancer stem cell marker Aldh1. A tendency for high c-Src levels (P = 0.072) was observed among the seven LBC patients with relapsed disease. CONCLUSIONS: Our data indicate elevated c-Src activity in ILC relative to noninvasive neoplastic tissue. The associated molecular changes suggest that c-Src promotes LBC invasiveness by inducing an epithelial-mesenchymal transition. Therefore, c-Src antagonists might counteract the acquisition of invasiveness during LBC progression. Inhibition of c-Src may also affect ILC cells thought to have a high metastatic potential and to be capable of initiating/maintaining tumor growth. Together with the possible association between high c-Src levels and disease recurrence, our findings encourage the evaluation of c-Src antagonists for the treatment of LBC.

N'-(2,4-Dichloro-benzyl-idene)-4-methoxy-benzohydrazide methanol solvate.

In the title compound, C(15)H(12)Cl(2)N(2)O(2)·CH(3)OH, the hydrazone mol-ecule displays an E configuration about the C=N bond. The dihedral angle between the two benzene rings is 4.6 (2)°. In the crystal structure, the hydrazone and methanol mol-ecules are linked into a chain propagating along the a axis via N-H⋯O and O-H⋯O hydrogen bonds.

2-Chloro-N'-(2-chloro-benzyl-idene)benzohydrazide.

The mol-ecule of the title compound, C(14)H(10)Cl(2)N(2)O, adopts an E configuration about the C=N bond. The dihedral angle between the two benzene rings is 79.7 (2)°. In the crystal structure, mol-ecules are linked by inter-molecular N-H⋯O, C-H⋯Cl and C-H⋯O hydrogen bonds, forming chains running along the b axis.

Comparison of Roche Cell-Free DNA collection Tubes ® to Streck Cell-Free DNA BCT ® s for sample stability using healthy volunteers.

OBJECTIVES: To compare the Roche Cell-Free DNA Collection Tubes® against the Streck Cell-Free DNA BCT®s for sample stability using Cell Free DNA (cfDNA) from healthy volunteers (n = 20). DESIGN & METHODS: Whole blood was drawn into five Roche and five Streck tubes per volunteer, stored at room temperature and processed at five different time points (Days 0, 4, 7, 10 and 14). One volunteer had blood drawn into ×10 K3EDTA tubes to observe the effect of no preservation buffer on White Blood Cell (WBC) lysis. DNA was extracted from the plasma and the concentration (ng/µL) measured using the Qubit Fluorometer® at each time point. The eluted DNA was further analysed by capillary electrophoresis to determine the proportion of cfDNA and gDNA contamination in the samples over the 14 days. RESULTS: There was no difference in individual (p = 0.097) and median paired (p = 0.26) DNA concentration across the five time points between the two tubes. However, a difference was observed for samples in the Roche tubes for pair days 0-7 (p = 0.01), 0 to 10 (p = 0.046) and 0 to 14 (p = 0.0016) in contrast to the Streck tubes after adjustment for multiple testing. CONCLUSION: The findings of this study indicate that the Roche Cell-Free DNA Collection Tubes® are a suitable alternative for sample collection and storage at room temperature, albeit for a duration of less than 7 days.

Synthesis, Crystal Structures and Catalytic Property of Oxidovanadium(V) Complexes with Similar Aroylhydrazones.

A pair of new oxidovanadium(V) complexes, [VOL1L]·EtOH (1) and [VOL2L]·EtOH (2) (L = acetohydroxamate), derived from the aroylhydrazones N'-(5-bromo-2-hydroxybenzylidene)-4-methoxybenzohydrazide (H2L1) and N'-(5-bromo-2-hydroxybenzylidene)-4-methylbenzohydrazide (H2L2), have been prepared and characterized by elemental analyses, FT-IR, 1H and 13C NMR spectroscopy and single-crystal structural X-ray diffraction. The complexes have octahedral structures in which the aroylhydrazone ligands behave as binegative donors. Single-crystal structure analyses reveal that the V centers are coordinated by the donor atoms of the aroylhydrazone ligands, the acetohydroxamate ligands and the oxido groups.Crystal structures of the complexes are stabilized by hydrogen bonds. The complexes function as effective olefin epoxidation catalysts.