Usage of Droplet Digital PCR (ddPCR) Assays for T Cell Quantification in Cancer

Zoutman, Willem H.; Nell, Rogier J.; Velden, Pieter A. van der

doi:10.1007/978-1-4939-8885-3_1

Cited by 13 publications

(20 citation statements)

References 24 publications

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“…Digital PCR experiments were carried out using the QX200™ Droplet Digital™ PCR System (Bio-Rad Laboratories) following the protocols described earlier [ 6 , 16 , 17 ]. A methodical overview of all experimental setups is presented in Supplementary Data 1 .…”

Section: Methodsmentioning

confidence: 99%

Involvement of mutant and wild-type CYSLTR2 in the development and progression of uveal nevi and melanoma

et al. 2021

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Background Activating Gαq signalling mutations are considered an early event in the development of uveal melanoma. Whereas most tumours harbour a mutation in GNAQ or GNA11, CYSLTR2 (encoding G-protein coupled receptor CysLT2R) forms a rare alternative. The role of wild-type CysLT2R in uveal melanoma remains unknown. Methods We performed a digital PCR-based molecular analysis of benign choroidal nevi and primary uveal melanomas. Publicly available bulk and single cell sequencing data were mined to further study mutant and wild-type CYSLTR2 in primary and metastatic uveal melanoma. Results 1/16 nevi and 2/120 melanomas carried the CYSLTR2 mutation. The mutation was found in a subpopulation of the nevus, while being clonal in both melanomas. In the melanomas, secondary, subclonal CYSLTR2 alterations shifted the allelic balance towards the mutant. The resulting genetic heterogeneity was confirmed in distinct areas of both tumours. At the RNA level, further silencing of wild-type and preferential expression of mutant CYSLTR2 was identified, which was also observed in two CYSLTR2 mutant primary melanomas and one metastatic lesion from other cohorts. In CYSLTR2 wild-type melanomas, high expression of CYSLTR2 correlated to tumour inflammation, but expression originated from melanoma cells specifically. Conclusions Our findings suggest that CYSLTR2 is involved in both early and late development of uveal melanoma. Whereas the CYSLTR2 p.L129Q mutation is likely to be the initiating oncogenic event, various mechanisms further increase the mutant allele abundance during tumour progression. This makes mutant CysLT2R an attractive therapeutic target in uveal melanoma.

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Section: Methodsmentioning

confidence: 99%

Involvement of mutant and wild-type CYSLTR2 in the development and progression of uveal nevi and melanoma

et al. 2021

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“…Digital PCR experiments were performed using the QX200™ Droplet Digital™ PCR System (Bio-Rad) following the general experimental guidelines as described earlier (Zoutman, Nell, & van der Velden, 2019). In short, 20-ng incubated DNA was analyzed in a 22-µl experiment, using 11-µl ddPCR™ Supermix for Probes (No dUTP; Bio-Rad) and primers and probes in a final concentration of 900 and 250 nM, respectively.…”

Section: Msre Incubation and Digital Pcrmentioning

confidence: 99%

Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR

et al. 2020

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Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation. We designed an experimental setup combining efficient methylation-sensitive restriction enzymes with digital polymerase chain reaction (PCR) to quantify a targeted density of DNA methylation independent of bisulfite conversion. By using a stable reference and comparing experiments treated and untreated with these enzymes, copy number instability could be properly normalized. In silico simulations demonstrated the mathematical validity of the setup and showed that the measurement precision depends on the amount of input DNA and the fraction methylated alleles. This uncertainty could be successfully estimated by the confidence intervals. Quantification of RASSF1 promoter methylation in a variety of healthy and malignant samples and in a calibration curve confirmed the high accuracy of our approach, even in minute amounts of DNA. Overall, our results indicate the possibility of quantifying DNA methylation with digital PCR, independent of bisulfite conversion. Moreover, as the context-density of methylation can also be determined, biological mechanisms can now be quantitatively assessed.

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“…Nowadays, new techniques have become available that may help to further our knowledge of the immune system and improve cancer therapy. De Lange et al showed that droplet PCR that identifies DNA differences is a very useful technique to determine tumor heterogeneity; this technique is being used to determine the percentage tumor cells in a sample, as well as the percentage of T and B cells [88,89]. Mass cytometery (CyTOF) is another method which can assess different subpopulations of infiltrating immune cells in small tissue samples [90].…”

Section: New Technologies Might Serve To Improve Knowledge About mentioning

confidence: 99%

HLA Expression in Uveal Melanoma: An Indicator of Malignancy and a Modifiable Immunological Target

Souri

Wierenga

Mulder

et al. 2019

Cancers

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Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, and gives rise to metastases in 50% of cases. The presence of an inflammatory phenotype is a well-known risk factor for the development of metastases. This inflammatory phenotype is characterized by the presence of high numbers of lymphocytes and macrophages, and a high expression of the HLA Class I and II antigens. An abnormal expression of HLA Class I may influence cytotoxic T lymphocyte (CTL) as well as Natural Killer (NK) cell responses. We provide a comprehensive review regarding the inflammatory phenotype in UM and the expression of locus- and allele-specific HLA Class I and of Class II antigens in primary UM and its metastases. Furthermore, we describe the known regulators and the role of genetics (especially chromosome 3 and BRCA-Associated Protein 1 (BAP1 status)), and, last but not least, the effect of putative therapeutic treatments on HLA expression.

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Usage of Droplet Digital PCR (ddPCR) Assays for T Cell Quantification in Cancer

Cited by 13 publications

References 24 publications

Involvement of mutant and wild-type CYSLTR2 in the development and progression of uveal nevi and melanoma

Involvement of mutant and wild-type CYSLTR2 in the development and progression of uveal nevi and melanoma

Quantification of DNA methylation independent of sodium bisulfite conversion using methylation‐sensitive restriction enzymes and digital PCR

HLA Expression in Uveal Melanoma: An Indicator of Malignancy and a Modifiable Immunological Target

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