Microsatellite instability testing in colorectal cancer using the QiaXcel advanced platform

Förster, Isabel; Brockmann, Michael; Schildgen, Oliver; Schildgen, Verena

doi:10.1186/s12885-018-4400-z

Cited by 14 publications

(7 citation statements)

References 20 publications

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“…Ten microsatellites were included in the protocol, namely BAT25, BAT26, BAT40, APC, D17s250, D2s123, D13s153, D18s58, D10s197, and MycI. The protocol was de-multiplexed and established on the QiaXcel Advanced system (Qiagen, Hilden, Germany) ( Forster et al, 2018 ).…”

Section: Methodsmentioning

confidence: 99%

Association of the Human Bocavirus With Tonsil Squamous Cell Carcinomas

et al. 2018

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Background: The human bocavirus (HBoV) is known to persist latently in the infected host cells and seems to replicate its DNA via the DNA damage response system, which is frequently defect in tumors and correlates with microsatellite instability (MSI). Because HBoV is able to persist in the infected tissues, induces pro-fibrotic and pro- cancerogenic cytokines in vivo and in vitro, and is detected in colorectal and lung tumors, the virus may be involved in cancerogenesis at least as a cofactor. Recently it was shown that the adenotonsillar tissue is an important site of HBoV1 persistence and replication. Considering the background that approximately 60% of oropharyngeal cancers were thought to be attributable to a HPV infection, a co-participation of HBoV in terms of a chronic virus infection might play a role in the cancerogenesis of tonsil tumors.Methods: Formalin-fixed, paraffin-embedded tonsil tumor samples were screened for HBoV and HPV DNA. Positive tissue sections were afterward subjected to fluorescence in situ hybridization (FISH) analysis to identify HBoV and HPV infected cells. By use of an in vitro cell culture model with primary tonsil fibroblasts, keratinocytes, and lymphocytes infected by HBoV we tried to find the target cells of virus replication. MSI testing was based on a previously published protocol using a de-multiplexed PCR followed by fluorescent detection of PCR products in a capillary sequencing device.Results: In total 62 of 103 (60, 19%) of the tonsil squamous cell carcinomas tested positive for HBoV DNA and 66 of 103 (66%) samples were identified as HPV positive. The FISH analysis revealed both double infection of HPV and HBoV in the same cells as well as single infections of both viruses within the tumor tissue. Twenty-two of 62 HBoV positive tumors tested HPV negative, 40 of 62 tissue sections were HBoV and HPV positive. We analyzed 21 out of the 62 HBoV positive tumors for MSI. Of those four tonsils displayed MSI in at least 1 of 10 microsatellite markers.Conclusion: Our findings support the hypothesis that human bocavirus infections as a cofactor may have an impact on tumor development in tonsils, although it still remains possible that HBoV solely displays a tumor tropism.

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Section: Methodsmentioning

confidence: 99%

Association of the Human Bocavirus With Tonsil Squamous Cell Carcinomas

et al. 2018

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“…The PCR products were analysed through capillary electrophoresis using the QIAxcel DNA screening kit and Advanced System ( Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. The PCR products which showed inconclusive smearing patterns in the screening gel were re-run in a high-resolution gel by using the QIAxcel Advanced System according to the manufacturer’s instructions ( Fig 1A ) [ 29 ].…”

Section: Methodsmentioning

confidence: 99%

High concordance rate of capillary electrophoresis workflow for microsatellite instability analysis and mismatch repair (MMR) immunostaining in colorectal carcinoma

Huang

Lee

et al. 2023

PLoS ONE

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Microsatellite instability (MSI) is the primary predictive biomarker for therapeutic efficacies of cancer immunotherapies. Establishment of the MSI detection methods with high sensitivity and accessibility is important. Because MSI is mainly caused by defects in DNA mismatch repair (MMR), immunohistochemical (IHC) staining for the MMR proteins has been widely employed to predict the responses to immunotherapies. Thus, due to the high sensitivity of PCR, the MSI-PCR analysis has also been recommended as the primary approach as MMR IHC. This study aimed to develop a sensitive and convenient platform for daily MSI-PCR services. The routine workflow used a non-labeling QIAxcel capillary electrophoresis system which did not need the fluorescence labeling of the DNA products or usage of a multi-color fluorescence reader. Furthermore, the 15 and 1000 bp size alignment markers were used to precisely detect the size of the DNA product. A cohort of 336 CRC cases was examined by MSI-PCR on the five mononucleotide MSI markers recommended by ESMO. The PCR products were analyzed in the screening gels, followed by high-resolution gel electrophoresis for confirmation if needed. In the MSI-PCR tests, 90.1% (303/336) cases showed clear major shift patterns in the screening gels, and only 33 cases had to be re-examined using the high-resolution gels. The cohort was also analyzed by MMR IHC is, which revealed 98.5% (331/336) concordance with MSI-PCR. In the five discordant cases, 4 (3 MSI-L and 1 MSS) showed MSH6 loss. Besides, one case exhibited MSI-H but no loss in the MMR IHC. Further NGS analysis, in this case, found that missense and frameshift mutations in the PMS2 and MSH6 genes occurred, respectively. In conclusion, the non-labeling MSI-PCR capillary electrophoresis revealed high concordance with the MMR IHC analysis and is cost- and time-effective. Therefore, it shall be highly applicable in clinical laboratories.

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“…Polymerase chain reaction (PCR) analyses were performed alongside DNA extract from a pure S. aureus isolate known to carry either the ermC or msr(A) gene (positive controls) [ 3 ]. The PCR products were analyzed using QIAxcel advanced Screen gel 1.5.0 (Qiagen) [ 13 ] with a tolerance rate of ±15% of the expected band size [ msr(A) 145 bp and ermC 398 bp]. Samples were categorized as being either positive or negative for each resistance gene.…”

Section: Methodsmentioning

confidence: 99%