Carol Loi

Genome‐wide scan identifies a copy number variable region at 3q26 that regulates PPM1L 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. However, APC mutations are not detected in 10–50% of FAP patients. We searched for a new cancer gene by performing genome‐wide genotyping on members of an APC mutation‐negative FAP variant family and ethnicity‐matched healthy controls. No common copy number change was found in all affected members using the unaffected members and healthy controls as baseline. A 111 kb copy number variable (CNV) region at 3q26.1 was shown to have copy number loss in all eight polyps compared to matched lymphocytes of two affected members. A common region of loss in all polyps, which are precursors to CRC, is likely to harbor disease‐causing gene in accordance to Knudsens “two‐hit” hypothesis. There is, however, no gene within the deleted region. A 2‐Mb scan of the genomic region encompassing the deleted region identified PPM1L, coding for a novel serine‐threonine phosphatase in the TGF‐β and BMP signaling pathways. Real‐time PCR analyses indicate that the 3′UTR of PPM1L transcript was down‐regulated more than two‐folds in all six polyps and tumors compared to matched mucosa of the affected member. This down‐regulation was not observed in APC mutation‐positive FAP patients. Our results suggest that the CNV region at 3q26 harbors an element that regulates the expression of an upstream candidate tumor suppressor, PPM1L, thus providing a novel mechanism for colorectal tumorigenesis in APC mutation‐negative familial CRC patients.

Singapore Familial Adenomatous Polyposis (FAP) Patients with Classical Adenomatous Polyposis but Undetectable APC Mutations Have Accelerated Cancer Progression

OBJECTIVES:Germline mutation in adenomatous polyposis coli (APC) is detected in up to 80% of familial adenomatous polyposis (FAP) patients worldwide. In this study, we evaluated clinical features and APC mutations of Singapore FAP patients and contrasted genotype–phenotype correlation with Caucasians from other regions of the world and between FAP patients with and without detectable APC mutations.METHODS:We screened 242 members from 57 unrelated FAP families using a combination of cDNA protein truncation test, multiplex ligation-dependent probe amplification, and differential expression techniques.RESULTS:APC germline mutations were detected in 50 families. In contrast to Caucasians, fundic gland polyposis in Singapore patients was associated with APC mutations throughout the coding region and osteomas were also not confined to codon 767–1573. There was also no FAP-associated hepatoblastoma or medullablastoma. APC mutation-negative patients from four families with mixed (adenomatous/hyperplastic/atypical juvenile) polyps were subsequently reclassified as hereditary mixed polyposis syndrome (HMPS) patients. APC mutation-negative patients with classical adenomatous polyposis were negative for MYH, β-catenin, and Axin 1 mutations. These patients had a significantly older age at diagnosis (P < 0.001) and more colorectal cancers (P = 0.017) than patients with APC mutations.CONCLUSIONS:We achieved a 94% (50/53) APC mutation detection rate via a combination of techniques, suggesting that the current detection rate is probably not exhaustive. Singapore patients have some features similar to and other features distinct from Caucasians. Furthermore, APC mutation-negative patients have accelerated cancer progression that merits closer surveillance.

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.

Critical appraisal of laparoscopic vs open rectal cancer surgery

AIM To evaluate the long-term clinical and oncological outcomes of laparoscopic rectal resection (LRR) and the impact of conversion in patients with rectal cancer. METHODS An 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. RESULTS Among 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. CONCLUSION LRR 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.

Preliminary Results of Mismatch Repair Deficiency Screening via Immunohistochemical Staining in Young Asian Colorectal Cancers

Background: The incidence of mismatch repair (MMR) deficiency in young colorectal cancers (CRC) remains unknown in Asians. This preliminary study assessed the clinicopathological features and efficacy of screening for MMR protein deficiency in young Asian CRC patients. Methods: From January 2006 to October 2009, patients under the age of 50 with immunohistochemical (IHC) staining for MMR proteins in resected CRC specimens were retrieved from a prospective computerised database. Results: Eighty unrelated patients comprising predominantly 80% Chinese (n = 64), with median age of diagnosis at 41 years (range 22–50 years) had IHC performed. Twenty-three per cent (n=18) of the patients had abnormal IHC staining. Loss of staining for MLH1, MSH2 and MSH6 proteins were observed in 18%, 2% and 6% of tumours respectively. Of the 15 patients who had abnormal staining of MLH1, three had concomitant equivocal staining for MSH6. One tumour specimen had abnormal staining in all 3 proteins. Multivariate analysis revealed that family history was the only significant predictive factor for defective MMR detection (OR 8.06, 95% CI 1.69–38.35, p=0.002). However if Amsterdam criteria alone were to be used, 72% (n=12) of the cohort would have not been detected for MMR gene defects. Conclusion: The overall burden of germline MMR deficiency in the Singapore population may be as high as 23%. Amsterdam criteria alone are insufficient to detect hereditary non-polyposis colorectal cancer (HNPCC) related patients. The use of IHC staining of at least 3 MMR proteins is a useful screening strategy for HNPCC diagnosis and routine screening of mismatch repair deficiency may be recommended for all young Asian CRC patients.

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.