ctDNA guided adjuvant chemotherapy versus standard of care adjuvant chemotherapy after curative surgery in patients with high risk stage II or stage III colorectal cancer: a multi-centre, prospective, randomised control trial (TRACC Part C).

BACKGROUND: Circulating tumour DNA (ctDNA) to detect minimal residual disease (MRD) is emerging as a biomarker to predict recurrence in patients with curatively treated early stage colorectal cancer (CRC). ctDNA risk stratifies patients to guide adjuvant treatment decisions. We are conducting the UK's first multi-centre, prospective, randomised study to determine whether a de-escalation strategy using ctDNA to guide adjuvant chemotherapy (ACT) decisions is non-inferior to standard of care (SOC) chemotherapy, as measured by 3-year disease free survival (DFS) in patients with resected CRC with no evidence of MRD (ctDNA negative post-operatively). In doing so we may be able to spare patients unnecessary chemotherapy and associated toxicity and achieve significant cost savings for the National Health Service (NHS). METHODS: We are recruiting patients with fully resected high risk stage II and stage III CRC who are being considered for ACT into the study which uses results from a plasma-only ctDNA assay to guide treatment decisions. Eligible patients are randomised 1:1 to receive ctDNA-guided chemotherapy versus SOC chemotherapy. The primary endpoint is the difference in DFS at 3 years between the trial arms. Secondary endpoints include the proportion of patients in the ctDNA-guided arm who are ctDNA negative post-operatively and receive de-escalated ACT compared to the standard arm, the difference in overall survival (OS), neurotoxicity and quality of life between the arms, and the cost-effectiveness of ctDNA-guided therapy compared to SOC treatment. We hypothesise that using a ctDNA-guided approach to ACT decisions is non-inferior to SOC. Target accrual is 1621 patients over 4 years, which will provide a power of 80% with an alpha of 0.1 to demonstrate non-inferiority with a margin of 1.25 in survival of the ctDNA-guided approach compared to SOC. We anticipate approximately 50 UK centres will participate. The study opened with the Guardant Reveal plasma-only ctDNA assay in August 2022. DISCUSSION: The trial will determine whether ctDNA guided ACT is non-inferior to SOC ACT in patients with fully resected high risk stage II and stage III resected CRC, with the potential to significantly reduce unnecessary ACT and the toxicity associated with it. TRIAL REGISTRATION: NCT04050345.

Tissue-free liquid biopsies combining genomic and methylation signals for minimal residual disease detection in patients with early colorectal cancer from the UK TRACC Part B study.

PURPOSE: Absence of post-operative circulating tumour DNA (ctDNA) identifies resected colorectal cancer (CRC) patients with low recurrence risk for adjuvant chemotherapy (ACT) de-escalation. We present the largest resected CRC cohort to date with tissue-free minimal residual disease (MRD) detection. DESIGN: TRACC included stage I-III resectable CRC patients. Prospective longitudinal plasma collection for ctDNA occurred pre- and post-surgery, post-ACT, every 3m for year 1 and every 6m in years 2 and 3 with imaging annually. The Guardant Reveal assay evaluated genomic and methylation signals. The primary endpoint was 2yr recurrence free survival (RFS) by post-operative ctDNA detection. (NCT04050345) Results: Between December 2016 and August 2022, 1203 were patients enrolled. Plasma samples (n=997) from 214 patients were analysed. 143 patients were evaluable for the primary endpoint; 92 (64.3%) colon, 51 (35.7%) rectal; 2 (1.4%) stage I, 64 (44.8%) stage II, 77 (53.8%) stage III. Median follow-up was 30.3m (95% CI: 29.5-31.3). 2yr RFS was 91.1% in patients with ctDNA not detected post-operatively and 50.4% in those with ctDNA detected (HR 6.5 [2.96-14.5] p<0.0001). Landmark negative predictive value (NPV) was 91.2% (95% CI 83.9-95.9). Longitudinal sensitivity and specificity were 62.1% (95% CI 42.2-79.3) and 85.9% (95% CI 78.9-91.3) respectively. Median lead-time from ctDNA detection to radiological recurrence was 7.3m (IQR 3.3-12.5; n=9). CONCLUSIONS: Tissue-free MRD detection with longitudinal sampling predicts recurrence in stage I-III CRC without need for tissue sequencing. NPV is high supporting ACT de-escalation in patients with ctDNA not detected post-operatively, now being investigated in the UK TRACC Part C study.

The role of small ubiquitin-like modifier-interacting motif in the assembly and regulation of metal-responsive transcription factor 1.

Metal-responsive transcription factor 1 (MTF-1) is an essential protein required for mouse embryonic development. We report here the occurrence of sumoylation on MTF-1. Mutational studies demonstrated that sumoylation occurs on the lysine residue at position 627 (Lys(627)) of mouse MTF-1. Small ubiquitin-like modifier (SUMO)-1 was fused to the C terminus of MTF-1 to mimic the sumoylated form of the protein and it suppressed the transcriptional activity of MTF-1. The nuclear translocation activity, DNA-binding activity, and protein stability of SUMO-fused MTF-1 are similar to that of wild type MTF-1. The level of sumoylation was reduced by metal in a dose- and time-dependent manner. The fact that zinc reduces MTF-1 sumoylation makes the suppressive role of sumoylated MTF-1 in transcription physiologically less significant because the SUMO moiety of MTF-1 is removed when MTF-1 translocates into nucleus. We further identified a SUMO-interacting motif (SIM) on MTF-1. Remarkably, MTF-1 binds sumoylated MTF-1 and/or other cellular factors in a SIM-dependent manner. This interaction was disrupted by treating cells with zinc. Gel permeation chromatography demonstrated that MTF-1 forms SIM-dependent complexes. This cross-interaction transpires in the cytoplasm and markedly reduces upon nuclear translocation. It can therefore be concluded that SUMO conjugation and the SIM on MTF-1 do not play a critical role in suppressing transcriptional activity. Instead, MTF-1 forms complexes with cellular factors through SIM and SUMO moiety in the cytoplasm. The result explores a new understanding for the mode of MTF-1 assembly and regulation in cells.

LD2SNPing: linkage disequilibrium plotter and RFLP enzyme mining for tag SNPs.

BACKGROUND: Linkage disequilibrium (LD) mapping is commonly used to evaluate markers for genome-wide association studies. Most types of LD software focus strictly on LD analysis and visualization, but lack supporting services for genotyping. RESULTS: We developed a freeware called LD2SNPing, which provides a complete package of mining tools for genotyping and LD analysis environments. The software provides SNP ID- and gene-centric online retrievals for SNP information and tag SNP selection from dbSNP/NCBI and HapMap, respectively. Restriction fragment length polymorphism (RFLP) enzyme information for SNP genotype is available to all SNP IDs and tag SNPs. Single and multiple SNP inputs are possible in order to perform LD analysis by online retrieval from HapMap and NCBI. An LD statistics section provides D, D', r2, deltaQ, rho, and the P values of the Hardy-Weinberg Equilibrium for each SNP marker, and Chi-square and likelihood-ratio tests for the pair-wise association of two SNPs in LD calculation. Finally, 2D and 3D plots, as well as plain-text output of the results, can be selected. CONCLUSION: LD2SNPing thus provides a novel visualization environment for multiple SNP input, which facilitates SNP association studies. The software, user manual, and tutorial are freely available at http://bio.kuas.edu.tw/LD2NPing.

Combinational polymorphisms of four DNA repair genes XRCC1, XRCC2, XRCC3, and XRCC4 and their association with oral cancer in Taiwan.

BACKGROUND: Many single nucleotide polymorphisms (SNPs) have been found to be associated with oral cancer but the biological interactions through SNPs are seldom addressed. In this study, we focused on the joint effect for SNP combinations of four DNA repair genes, X-ray repair cross-complementing groups (XRCCs) 1-4, involved in major cancer-related pathways. METHODS: Single nucleotide polymorphism genotyping was determined using by polymerase chain reaction-restriction fragment length polymorphism in this study (case = 103, control = 98). Different numbers of combinational SNPs with genotypes called the pseudo-haplotypes from these chromosome-wide genes were used to evaluate their joint effect on oral cancer risk. RESULTS: Except for XRCC2 rs2040639-AG, none of these SNPs was found to individually contribute to oral cancer risk. However, for two combined SNPs, the proportion of subjects with oral cancer was significantly higher in the pseudo-haplotype with AG-CC genotypes in rs2040639-rs861539 (XRCC2-XRCC3) compared with those with non-AG-CC genotypes. Similarly, the pseudo-haplotype of rs2040639-rs861539-rs2075685 (XRCC2-XRCC3-XRCC4) and rs2040639-rs861539-rs2075685-rs1799782 (XRCCs 1-4) with specific genotype pattern (AG-CC-TG and CT-AG-CC-TG) among three and four combinational SNPs were significantly associated with oral cancer. After controlling for age, gender, smoking, drinking, and betel nut chewing, the estimated odds ratio of oral cancer were 2.45, 5.03, and 10.10 for two, three and four specific SNP combinations, respectively, comparing these specific pseudo-haplotypes to their corresponding non-pseudo-haplotypes. CONCLUSION: We have identified the potential combined XRCCs 1-4 SNPs with genotypes that were associated with oral cancer risk and may have an impact on identification of a high-risk population.

SNP combinations in chromosome-wide genes are associated with bone mineral density in Taiwanese women.

Osteoporosis is a major public health problem, mainly quantified by low BMD. Eleven polymorphisms were investigated in this study; TNFalpha-857 (rs1799724), TGFbeta1-509 (rs1800469), osteocalcin (rs1800247), TNFalpha-308 (rs1800629), PTH BstB I (rs6254), PTH Dra II (rs6256), IL-1ra (VNTR), HSP70 hom (rs2227956), HSP 70-2 (rs1061581), CTR (rs1801197), and BMP-4 (rs17563). The relationship between the combined polymorphisms in different genomic regions and BMD variation was investigated. Among the female subjects, the proportion of subjects with low BMD in low BMI group (< or = 18.50) was significantly higher than that of the middle (18.51-22.99) and high (> or = 23.00) BMI groups (P < 0.05). In post-menopausal women, there was a significant association between low BMD and genotypes ranging from 2 to approximately 7 SNPs. For two combined SNPs, the portion of subjects with low BMD was significantly higher in those with CC-AA genotypes in rs1799724-rs1800629, compared to those with non-CC-AA genotypes in post-menopausal women and the combination of all women. Similarly, part of the combined SNPs with rs1799724-rs1800629-rs6254-rs6256-IL-1ra-rs2227956-rs1801197 was significantly associated with reduced BMD. After controlling for age and BMI, post-menopausal women with certain specific SNP combination had a 3.54- to 4.68-fold increased risk for low BMD, comparing to other SNP combinations. In conclusion, our data suggest that several gene polymorphisms may be cooperatively involved in the development of osteoporosis.

[Program for lowering the incidence of pressure sores in neurosurgical patients].

Pressure sores are one of the well known problems that occur in hospitals. As the literature on the subject indicates, a lot of money is expended in managing this problem every year, and 12-66% of pressure sores are caused during surgery. Patients who undergo neurosurgical procedures are susceptible to pressure sores because of lengthy operations. We collected data on patients with pressure sores who underwent surgery between May 2004 and August 2004, and found that the incidence of pressure sore in neurosurgical patients was 9.5%, which was the highest among all surgical patients. This project was developed to solve the problem of pressure sores by setting up standard preventive procedures, a nursing follow up system and continuing education courses, and utilizing cotton rolls to pad sites of pressure sores. The incidence of pressure sore in neurosurgical patients was reduced from 9.5% to 7% after the improvement project was carried out. The more concerned nurses are about pressure sores, the better the quality of operative nursing care.

The preventive role of breadfruit against inflammation-associated epithelial carcinogenesis in mice.

Artocarpus communis has been identified as a rich source of flavonoids and has been gaining attention for its potential chemopreventive abilities. In this study, methanol extracts from the fruit of A. communis (MEFA) and leaf of A. communis (MELA) were prepared, and their effects on inflammation-associated skin tumorigenesis were assessed using mouse models, including 12-O-tetradecanoylphorbol-13-acetate (TPA) induced cutaneous inflammation as well as 7,12-dimethylbenz[α]anthracene (DMBA) initiated and TPA-promoted skin tumorigenesis. According to the results, both MEFA and MELA decreased the intensity of leukocyte infiltration in mouse dorsal skin and cutaneous edema induced by TPA, which appeared to be mediated by inhibition of proinflammatory genes (inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), IL-1ß, and IL-6) and proinflammatory mediators (TNF-α, IL-1ß, and Prostaglandin E2 ). In addition, topical application with MEFA or MELA effectively attenuated tumor incidence, multiplicity, volume, malignancy as well as angiogenesis of TPA-stimulated skin tumor promotion in DMBA-initiated mice. Notably, immunohistochemical stain showed that MEFA and MELA attenuated COX-2 expression of both skin and tumor tissues in different animal tests, which may be closely related to the suppression of nuclear factor kappa B/activator protein signaling networks. These findings first demonstrate that flavonoid-rich A. communis may exert potent anti-inflammatory activity through modulation of COX-2 in TPA-activated skin and tumor tissues.