N_LyST: a simple and rapid screening test for Lynch syndrome

Abstract

<jats:sec><jats:title>Aims</jats:title><jats:p>We sought to use PCR followed by high-resolution melting analysis to develop a single closed-tube screening panel to screen for Lynch syndrome. This comprises tests for microsatellite instability (MSI), MLH1 methylation promoter and <jats:italic>BRAF</jats:italic> mutation.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>For MSI testing, five mononucleotide markers (BAT25, BAT26, BCAT25, <jats:italic>MYB</jats:italic>, <jats:italic>EWSR1</jats:italic>) were developed. In addition, primers were designed to interrogate Region C of the MLH1 promoter for methylation (using bisulphite-modified DNA) and to test for mutations in codon 600 of BRAF. Two separate cohorts from Nottingham (n=99, 46 with MSI, 53 being microsatellite stable (MSS)) and Edinburgh (n=88, 45 MSI, 43 MSS) were tested.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>All the cases (n=187) were blind tested for MSI and all were correctly characterised by our panel. The <jats:italic>MLH1</jats:italic> promoter and <jats:italic>BRAF</jats:italic> were tested only in the Nottingham cohort. Successful blinded analysis was performed on the MLH1 promoter in 97 cases. All MSS cases showed a pattern of non-methylation while 41/44 cases with MSI showed full methylation. The three cases with MSI and a non-methylated pattern had aberrations in MSH2 and MSH6 expression. <jats:italic>BRAF</jats:italic> mutation was detected in 61% of MSI cases and 11% of MSS cases.</jats:p><jats:p>Finally, 12 cases were blind screened by using the whole panel as a single test. Of these, five were identified as MSS, four as MSI/non-LS and three as MSI/possible LS. These results were concordant with the previous data.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>We describe the Nottingham Lynch Syndrome Test (N_LyST). This is a quick, simple and cheap method for screening for Lynch syndrome.</jats:p></jats:sec>

Immuno-Interface Score to Predict Outcome in Colorectal Cancer Independent of Microsatellite Instability Status

“…A series of 99 cases of CRC which had undergone curative resection was retrieved from the archives of the Pathology Department at the Nottingham University Hospitals NHS Trusts. The series was selected to include 50 CRCs with MSI and 49 CRCs which were MSS and had been used to develop a screening test for Lynch Syndrome [ 57 ]. These cases had been tested by IHC for mismatch repair protein (MMR) for the purpose of either making a decision on adjuvant chemotherapy or for screening for Lynch Syndrome.…”

“…>50% of the tumor section showing mucinous histology, n = 4) since a specific tissue classifier is needed, appendiceal tumor (n = 1), and tumor section area below <4.5 mm 2 (n = 2) were excluded from further analyses. All tumor specimens were tested by IHC for expression of DNA MMR proteins, i.e., MLH1, PMS2, MSH2, MSH6, and by PCR followed by high-resolution melting analysis for MSI status and BRAF , KRAS , PIK3CA gene mutations, as described previously [ 57 , 58 ]. The MLH1 gene promoter methylation analysis revealed MSI tumors to be mainly sporadic [ 57 ].Clinicopathological parameters and follow-up data of the final CRC patient cohort were obtained from the pathology reports and the clinical records and are summarized in Table 1 .…”

“…IHC was performed on FFPE as previously described [ 57 ]. Four micrometer thick tissue sections were cut and stained for cytotoxic T cell marker CD8 (clone SP57, Roche Diagnostics, Mannheim, Germany), B cell marker CD20 (clone L26, Roche Diagnostics) and macrophage marker CD68 (clone KP1, Dako, Glostrup, Denmark).…”

A novel panel of short mononucleotide repeats linked to informative polymorphisms enabling effective high volume low cost discrimination between mismatch repair deficient and proficient tumours

“…Several groups have developed sequencing based approaches to identify microsatellite instability. These include methods utilising genome [ 23 ] or transcriptome [ 24 ] wide data, as well as sequences from target enriched libraries [ 25 , 26 ] and more recently, melt-curve analysis based testing [ 27 ]. In vitro amplification errors, which lead to the presence of variant read lengths in the PCR product, can complicate sequence-based approaches.…”

Positive association of PIK3CA mutation with KRAS mutation but not BRAF mutation in colorectal cancer suggests co-selection is gene specific but not pathway specific

“…Recently, we published a paper describing N_LyST, a screening panel for Lynch syndrome,5 which was developed using two data sets: cohort 1 (the Nottingham cohort) comprised 99 cases of CRC (46 MSI, 53 microsatellite stable (MSS)) and cohort 2 (the Edinburgh cohort) comprised 88 cases of CRC (45 MSI, 43 MSS). None of the cases used in this study had received any anti-epidermal growth factor receptor (EGFR) therapy at the time of molecular testing.…”