DEVELOPING AND TESTING THE EFFECTS OF A PSYCHOEDUCATION INTERVENTION ON PATIENTS WITH COLORECTAL CANCER: A MIXED METHOD STUDY

Ph.DDOCTOR OF PHILOSOPH

Systematic Study on Genetic and Epimutational Profile of a Cohort of Amsterdam Criteria-Defined Lynch Syndrome in Singapore

<div><p>Background</p><p>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.</p><p>Methods</p><p>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 <i>MLH1</i>, <i>MSH2</i>, <i>MSH6</i> and <i>PMS2</i> 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.</p><p>Results</p><p>Pathogenic defects, all confined to <i>MLH1</i> and <i>MSH2</i>, 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 <i>MLH1</i> (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.</p><p>Conclusions</p><p>Among major ethnic groups in Singapore, all pathogenic germline defects were confined to <i>MLH1</i> and <i>MSH2</i>. 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 <i>MLH1</i> c.793C>T followed by comprehensive mutation scanning in <i>MLH1</i> and <i>MSH2</i> prior to proceeding to other MMR genes.</p></div

Flow diagram of the analytical strategy.

<p>Fifty nine unrelated families were screened for genetic defects in common MMR genes with all tests performed in parallel without using any sets of results for subsequent assays. Germline gene status was investigated by utilizing DNA extracted from blood or normal mucosa. Additional analyses in tumor tissues, such as immunohistochemical (IHC) assessment, microsatellite instability (MSI) test and mutation detection of <i>BRAF</i> V600E were performed where tumor materials were available. When a pathogenic or suspected deleterious defect was identified, family relatives were screened for presence of the same defect. ^†, at least one CRC patient each from 59 unrelated families.</p

Pathogenic mutations identified in 17 Amsterdam-criteria-defined families in Singapore.

†<p>, LOVD, London open variation database (V2.0 build 35) maintained by International Society of Gastroenterological Hereditary Tumors (InSiGHT). Variants were classified according to Consensus InSiGHT Classification v.1.9: 5/09/2013.</p>‡<p>, HGMD, human gene mutation database. Dis causing, disease causative mutation; Our data were checked against this database as of 10 Jan 2014.</p><p>Putative novel mutations were highlighted in bold.</p

Correlation between tumor immunohistochemical (IHC) stains and germline mutations in 28 suspected Lynch Syndrome patients.

$<p>, Tumor IHC sensitivity for germline defects.</p>†<p>, LOVD, London open variation database (V2.0 build 35) maintained by International Society of Gastroenterological Hereditary Tumors (InSiGHT). Variants were classified according to Consensus InSiGHT Classification v.1.9: 5/09/2013.</p>‡<p>, HGMD, human gene mutation database. Dis causing, disease causative mutation; Our data were checked against this database as of 10 Jan 2014.</p><p>Novel mutations were highlighted in bold; VUS, variant of uncertain significance; NA, not applicable.</p

Correlation between tumor MSI status and germline mutations.

<p>Number in each box, case numeration. VUS, variant with uncertain significance.</p

Evaluation of cardinal clinico-pathological features of Lynch Syndromes (LS) as predictors of pathogenic germline mutations.

†<p>, CRC lesions include those from patients whose initial tumors were extracolonic malignancies.</p><p>NS, not significant, <i>P</i> >0.05.</p