Choong Leong Tang

Analysis of colorectal cancer glyco-secretome identifies laminin β-1 (LAMB1) as a potential serological biomarker for colorectal cancer

The high mortality rate in colorectal cancer is mostly ascribed to metastasis, but the only clinical biomarker available for disease monitoring and prognosis is the carcinoembryonic antigen (CEA). However, the prognostic utility of CEA remains controversial. In an effort to identify novel biomarkers that could be potentially translated for clinical use, we collected the secretomes from the colon adenocarcinoma cell line HCT‐116 and its metastatic derivative, E1, using the hollow fiber culture system, and utilized the multilectin affinity chromatography approach to enrich for the secreted glycoproteins (glyco‐secretome). The HCT‐116 and E1 glyco‐secretomes were compared using the label‐free quantitative SWATH‐MS technology, and a total of 149 glycoproteins were differentially secreted in E1 cells. Among these glycoproteins, laminin β‐1 (LAMB1), a glycoprotein not previously known to be secreted in colorectal cancer cells, was observed to be oversecreted in E1 cells. In addition, we showed that LAMB1 levels were significantly higher in colorectal cancer patient serum samples as compared to healthy controls when measured using ELISA. ROC analyses indicated that LAMB1 performed better than CEA at discriminating between colorectal cancer patients from controls. Moreover, the diagnostic performance was further improved when LAMB1 was used in combination with CEA.

Association of Caucasian-Identified Variants with Colorectal Cancer Risk in Singapore Chinese

Background Genome-wide association studies (GWAS) in Caucasians have identified fourteen index single nucleotide polymorphisms (iSNPs) that influence colorectal cancer (CRC) risk. Methods We investigated the role of eleven iSNPs or surrogate SNPs (sSNPs), in high linkage disequilibrium (LD, r2≥0.8) and within 100 kb vicinity of iSNPs, in 2,000 age- and gender-matched Singapore Chinese (SCH) cases and controls. Results Only iSNP rs6983267 at 8q24.21 and sSNPs rs6695584, rs11986063, rs3087967, rs2059254, and rs7226855 at 1q41, 8q23.3, 11q23.1, 16q22.1 and 18q21.1 respectively showed evidence of association with CRC risk, with odds ratios (OR) ranging from 1.13 to 1.40. sSNP rs827401 at 10p14 was associated with rectal cancer risk (ORu200a=u200a0.74, 95% CI 0.63–0.88) but not disease prognosis (ORu200a=u200a0.91, 95% CI 0.69–1.20). Interestingly, sSNP rs3087967 at 11q23.1 was associated with CRC risk in men (ORu200a=u200a1.34, 95% CI 1.14–1.58) but not women (ORu200a=u200a1.07, 95% CI: 0.88–1.29), suggesting a gender-specific role. Half of the Caucasian-identified variants, including the recently fine-mapped BMP pathway loci, BMP4, GREM1, BMP2 and LAMA 5, did not show any evidence for association with CRC in SCH (OR ∼1; p-value >0.1). Comparing the results of this study with that of the Northern and Hong Kong Chinese, only variants at chromosomes 8q24.21, 10p14, 11q23.1 and 18q21.1 were replicated in at least two out of the three Chinese studies. Conclusions The contrasting results between Caucasians and Chinese could be due to different LD patterns and allelic frequencies or genetic heterogeneity. The results suggest that additional common variants contributing to CRC predisposition remained to be identified.

Serum methylation levels of TAC1. SEPT9 and EYA4 as diagnostic markers for early colorectal cancers: a pilot study

Abstract Objective: To identify methylated genes in serum with diagnostic potentials for early colorectal cancer (CRC). Methods: Serum methylation levels of up to 12 genes were measured in two sets of serum samples with the second set from 26 stage I CRC patients and 26 age/gender-matched controls. Results: Serum methylation levels of TAC1, SEPT9, and EYA4 were significant discriminants between stage I CRC and healthy controls. Combination of TAC1 and SEPT9 rendered 73.1% sensitivity with 92.3% specificity. Conclusion: Serum methylation levels of TAC1. SEPT9 and EYA4 may be useful biomarkers for early detection of CRC though a validation study is necessary.

Amino-terminal p53 mutations lead to expression of apoptosis proficient p47 and prognosticate better survival, but predispose to tumorigenesis

Significance Mutations in the amino-terminal transactivation domain of the tumor-suppressor p53 are mostly insertions or deletions, and result in loss of full-length p53 expression. However, these changes concomitantly result in the expression of a truncated p47 isoform, which retains the ability to selectively transactivate some apoptotic target genes. The selectivity appears to be due to a default feature, stemming from the lack of acetylation on K382 at the carboxyl terminus, which requires the amino terminus. Consistently, expression of p47 could prognosticate better survival in sporadic cancer patients, corroborating with its ability to induce apoptosis. However, apoptosis proficiency appears to be insufficient for tumor suppression, because these amino-terminal mutations are found in the germ line, leading to the Li–Fraumeni syndrome. Whereas most mutations in p53 occur in the DNA-binding domain and lead to its functional inactivation, their relevance in the amino-terminal transactivation domain is unclear. We show here that amino-terminal p53 (ATp53) mutations often result in the abrogation of full-length p53 expression, but concomitantly lead to the expression of the amino-terminally truncated p47 isoform. Using genetically modified cancer cells that only express p47, we demonstrate it to be up-regulated in response to various stimuli, and to contribute to cell death, through its ability to selectively activate a group of apoptotic target genes. Target gene selectivity is influenced by K382 acetylation, which depends on the amino terminus, and is required for recruitment of selective cofactors. Consistently, cancers capable of expressing p47 had a better overall survival. Nonetheless, retention of the apoptotic function appears insufficient for tumor suppression, because these mutations are also found in the germ line and lead to Li–Fraumeni syndrome. These data from ATp53 mutations collectively demonstrate that p53’s apoptosis proficiency is dispensable for tumor suppression, but could prognosticate better survival.

Palliative surgical intervention in metastatic colorectal carcinoma: a prospective analysis of quality of life.

Quality of life (QOL) was assessed after palliative surgery for incurable metastatic colorectal cancer (CRC).

Chromosome 19q13 disruption alters expressions of CYP2A7, MIA and MIA-RAB4B lncRNA and contributes to FAP-like phenotype in APC mutation-negative familial colorectal cancer patients

Familial adenomatous polyposis (FAP) is an autosomal-dominantly inherited form of colorectal cancer (CRC) caused by mutation in the adenomatous polyposis coli (APC) gene. Our ability to exhaustively screen for APC mutations identify microsatellite-stable and APC-mutation negative familial CRC patients, enabling us to search for novel genes. We performed genome-wide scan on two affected siblings of one family and 88 ethnicity- and gender-matched healthy controls to identify deletions shared by the siblings. Combined loss of heterozygosity, copy number and allelic-specific copy number analysis uncovered 5 shared deletions. Long-range polymerase chain reaction (PCR) confirmed chromosome 19q13 deletion, which was subsequently found in one other family. The 32 kb deleted region harbors the CYP2A7 gene and was enriched with enhancer, repressor and insulator sites. The wildtype allele was lost in the polyps of the proband. Further, real-time RT-PCR assays showed that expressions of MIA and MIA-RAB4B located 35 kb upstream of the deletion, were up-regulated in the polyps compared to the matched mucosa of the proband. MIA-RAB4B, the read-through long non-coding RNA (lncRNA), RAB4B, PIM2 and TAOK1 share common binding site of a microRNA, miR-24, in their 3’UTRs. PIM2 and TAOK1, two target oncogenes of miR-24, were co-ordinately up-regulated with MIA-RAB4B in the polyps, suggesting that MIA-RAB4B could function as competitive endogenous RNA to titrate miR-24 away from its other targets. The data suggest that the 19.13 deletion disrupted chromatin boundary, leading to altered expression of several genes and lncRNA, could contribute to colorectal cancer via novel genetic and epigenetic mechanisms.

The classification of intestinal polyposis

1. Jaeger, E. et al. Nat. Genet. 44, 699–705 (2012). 2. Cheah, P.Y. et al. Am. J. Gastroenterol. 104, 3027– 3033 (2009). 3. Merg, A. & Howe, J.R. Am. J. Med. Genet. 129c, 44–55 (2004). 4. Whitelaw, S.C. et al. Gastroenterology 112, 327–334 (1997). 5. Cao, X. et al. J. Med. Genet. 43, e13 (2006). 6. Cheah, P.Y. Crit. Rev. Oncol. Hematol. 69, 45–55 (2009). 7. O’Riordan, J.M. et al. Colorectal Dis. 12, 570–573 (2012).

Critical appraisal of laparoscopic vs open rectal cancer surgery

AIMnTo evaluate the long-term clinical and oncological outcomes of laparoscopic rectal resection (LRR) and the impact of conversion in patients with rectal cancer.nnnMETHODSnAn analysis was performed on a prospective database of 633 consecutive patients with rectal cancer who underwent surgical resection. Patients were compared in three groups: Open surgery (OP), laparoscopic surgery, and converted laparoscopic surgery. Short-term outcomes, long-term outcomes, and survival analysis were compared.nnnRESULTSnAmong 633 patients studied, 200 patients had successful laparoscopic resections with a conversion rate of 11.1% (25 out of 225). Factors predictive of survival on univariate analysis include the laparoscopic approach (P = 0.016), together with factors such as age, ASA status, stage of disease, tumor grade, presence of perineural invasion and vascular emboli, circumferential resection margin < 2 mm, and postoperative adjuvant chemotherapy. The survival benefit of laparoscopic surgery was no longer significant on multivariate analysis (P = 0.148). Neither 5-year overall survival (70.5% vs 61.8%, P = 0.217) nor 5-year cancer free survival (64.3% vs 66.6%, P = 0.854) were significantly different between the laparoscopic group and the converted group.nnnCONCLUSIONnLRR has equivalent long-term oncologic outcomes when compared to OP. Laparoscopic conversion does not confer a worse prognosis.

A novel indel in exon 9 of APC upregulates a ‘skip exon 9’ isoform and causes very severe familial adenomatous polyposis

Germline mutation in the adenomatous polyposis coli (APC) gene causes the majority (80%) of familial adenomatous polyposis (FAP), an autosomal dominantly inherited form of colorectal cancer (CRC). Mutation in 5′end of exon 9 of APC usually results in an attenuated form of FAP (aFAP), characterized by later age of onset and fewer polyps. The presence of exon 9a, an in-frame isoform with exon 8 spliced to 3′end of exon 9, modulates any deleterious effect of the mutation. A third lowly expressed isoform that completely skips exon 9 is present in both healthy individuals and FAP patients. We report here an interesting case of a proband with an APC mutation in 5′end of exon 9 that presented with six synchronous advanced CRCs at age 37. The novel insertion–deletion (indel) at codon 409, c.1226-1229delTTTTinsAAA, caused upregulation of the ‘skip exon 9’ isoform, r934-1312del, resulting in a premature stop codon at exon 10 and a truncated protein that removed all of the β-catenin (CTNNB1) binding motifs, thus activating the downstream T-cell transcription factor (Tcf) pathway. Exon 9a isoform was concomitantly downregulated. This finding emphasizes the necessity of examining the various isoforms of exon 9 to avoid clinical mismanagement and counseling based on just the mutation site by genomic DNA sequencing alone.

Systematic study on genetic and epimutational profile of a cohort of Amsterdam criteria-defined Lynch Syndrome in Singapore.

Background Germline defects of mismatch repair (MMR) genes underlie Lynch Syndrome (LS). We aimed to gain comprehensive genetic and epigenetic profiles of LS families in Singapore, which will facilitate efficient molecular diagnosis of LS in Singapore and the region. Methods Fifty nine unrelated families were studied. Mutations in exons, splice-site junctions and promoters of five MMR genes were scanned by high resolution melting assay followed by DNA sequencing, large fragment deletions/duplications and promoter methylation in MLH1, MSH2, MSH6 and PMS2 were evaluated by multiplex ligation-dependent probe amplification. Tumor microsatellite instability (MSI) was assessed with five mononucleotide markers and immunohistochemical staining (IHC) was also performed. Results Pathogenic defects, all confined to MLH1 and MSH2, were identified in 17 out of 59 (28.8%) families. The mutational spectrum was highly heterogeneous and 28 novel variants were identified. One recurrent mutation in MLH1 (c.793C>T) was also observed. 92.9% sensitivity for indication of germline mutations conferred by IHC surpassed 64.3% sensitivity by MSI. Furthermore, 15.6% patients with MSS tumors harbored pathogenic mutations. Conclusions Among major ethnic groups in Singapore, all pathogenic germline defects were confined to MLH1 and MSH2. Caution should be applied when the Amsterdam criteria and consensus microsatellite marker panel recommended in the revised Bethesda guidelines are applied to the local context. We recommend a screening strategy for the local LS by starting with tumor IHC and the hotspot mutation testing at MLH1 c.793C>T followed by comprehensive mutation scanning in MLH1 and MSH2 prior to proceeding to other MMR genes.