Detection of MGMT methylation status using a Lab-on-Chip compatible isothermal amplification method

Abstract

The growing cancer burden necessitates the development of cost-effective solutions that provide rapid, precise and personalised information to improve patient outcome. The aim of this study was to develop a novel, Lab-on-Chip compatible method for the detection and quantification of DNA methylation for MGMT, a well-established molecular biomarker for glioblastoma, with direct clinical translation as a predictive target. A Lab-on-Chip compatible isothermal amplification method (LAMP) was used to test its efficacy for detection of sequence-specific methylated regions of MGMT, with the method's specificity and sensitivity to have been compared against gold-standards (MethyLight, JumpStart). Our LAMP primer combinations were shown to be specific to the MGMT methylated region, while sensitivity assays determined that the amplification methods were capable of running at clinically relevant DNA concentrations of 0.2 -20 ng/µL. For the first time, the ability to detect the presence of DNA methylation on bisulfite converted DNA was demonstrated on a Lab-on-Chip setup, laying the foundation for future applications of this platform to other epigenetic biomarkers in a point-of-care setting. †M.J. and B.F.S. have contributed equally to this work.

Cancer Methylation Biomarker Detection in an Automated, Portable, Multichannel Magnetofluidic Platform

“…ACCESS overcame these challenges and thus offers a more advanced and suitable diagnostic solution. Other existing microfluidic-enabled methylation detection devices − have explored various alternative techniques such as methylation-specific endonuclease digestion , and isothermal amplification. − Importantly, however, none of these devices to date have integrated important cell digestion steps or tested clinical samples. Taken together, ACCESS currently represents the standard of assay integration, point-of-care amenability, and clinical sample testing for microfluidic-enabled methylation detection devices.…”

Revisiting Temozolomide's role in solid tumors: Old is gold?

“…It is recommended to use a biological cutoff of 10% or 21% of the receiver operating characteristic. Other approaches that show promise for MGMT promoter methylation detection include endonuclease-resistant DNA methylation quantification, Lab on Chip compatible isothermal amplification, and two probe quantification of MSB [40][41][42]. In terms of defining the ideal cutoff, research indicates that, for CpG sites 74-78, a cutoff of 9% is preferable to a higher cutoff of 28% or 29% [38].…”

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“…This is generated through sequence-specific amplification reactions, all achievable without the need for a thermal cycler, thus allowing the chemistry to function within a miniaturised lab-on-chip device. Demonstration of this work has been shown in a range of applications in the field of diagnostics [81][82][83][84] and particularly in cancer, with the latest features of its capability to be presented for the detection of clinically validated cancer-specific mutations in breast cancer (ESR1, PIK3CA) [85][86][87], circulating mRNA biomarkers in prostate cancer (TMPRSS2-ERG, YAP1, AR-V7) [88], tumour-specific markers (HPV-16/18, hTERT mRNA) in cervical cancer [89], and DNA methylation biomarkers present in several cancer types [90][91][92]. A summary of all the emerging technologies that have been discussed can be found in Table 2 and an image illustrating these technologies can be seen in Figure 2.…”