Unlocking Cancer’s Genetic Secrets with Digital PCR

In this episode of Absolute Gene-ius, we explore how digital PCR (dPCR) is transforming leukemia research with Valeria Rangel, a PhD candidate at the University of California, Irvine. Her work focuses on Philadelphia chromosome-like (Ph-like) B-cell acute lymphoblastic leukemia (ALL), a rare, aggressive subtype that disproportionately affects Hispanic populations. By leveraging dPCR and CRISPR-Cas9, Rangel’s lab is uncovering how genetic mutations, methylation patterns, and chromosomal translocations contribute to cancer progression.

To hear the full conversation, visit thermofisher.com/absolutegeneius.

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Using Digital PCR to Decode Leukemia

Q: Could you tell us about your research at UC Irvine and how it’s advancing our understanding of leukemia?
Valeria Rangel: “Our lab focuses primarily on rare hematological malignancies, and my work has centered on unraveling the genetic and epigenetic factors driving a subset of blood cancer called Philadelphia chromosome-like B-cell acute lymphoblastic leukemia (Ph-like ALL). This particular subtype disproportionately affects Hispanics. Using digital PCR assays, we discovered that increased levels of activation-induced cytidine deaminase—an enzyme expressed in B cells—can drive acute lymphoblastic leukemia health disparities in Latin American populations.”

Q: What makes Ph-like ALL unique in terms of genetic drivers?
Valeria Rangel: “There’s a very particular chromosomal rearrangement between the CRLF2 locus and IGH that can lead to this rare subtype of ALL. We study this translocation because it’s known to affect different Hispanic and Latino populations.”

Q: How do single nucleotide polymorphisms (SNPs) factor into your research?
Valeria Rangel: “SNPs are mutations that happen at a single-nucleotide position and vary from person to person. We’re trying to pinpoint why this population is predisposed to this cancer, and we believe that a single variant could be enough to make that change or make them predisposed.”

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Precision Detection and Innovation with Digital PCR

Q: What led you to introduce digital PCR into your workflow?
Valeria Rangel: “We started off by doing amplicon sequencing on these different subjects, as well as the different cell lines that we had in the lab. However, we quickly realized that there was no real way of quantifying the events that we were trying to amplify up and look at. We really just fell in love with the way that digital PCR allows us to use a very small amount of genomic material into our reactions, be able to load multiple subjects or cell lines onto a single plate and then get results within a matter of hours.”

Q: Your team developed an innovative drop-off probe assay. How does that work?
Valeria Rangel: “We wanted to quantify how many times we could get an amplicon to amplify with all our probes annealing perfectly, our wild-type amplicons. We designed a multiplex assay with four probes within a 300 base pair region. Three probes were designed to anneal to regions where we expected mutations, and one served as a control probe. This setup lets us see if a mutation prevents a probe from binding, which shows up as a loss of signal in the assay.”

Q: How did CRISPR-Cas9 fit into your validation strategy?
Valeria Rangel: “We used CRISPR-Cas9 methods to validate the assay as a proof of concept. We designed single guide RNAs that would create a Cas-9 break at one or several of the binding sites. We were able to detect amplicons where the control probe binded, but not the mutation probe—showing that the assay was sensitive enough to pick up those mutations.”

Q: What surprising results did you encounter?
Valeria Rangel: “We saw activity at sites in subjects that had been diagnosed with other subtypes of cancer. Activity that was missed by other sequencing methods but detected using digital PCR. That was pretty amazing.”

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Exploring Epigenetics and Next Steps

Q: What’s next for your research?
Valeria Rangel: “We’re now tying in the epigenetic side, seeing how methylation plays a role in how this enzyme in B cells contributes to mutations at the CRLF2 locus. I’ve been designing a digital PCR assay specific to bisulfite-converted DNA to examine methylation patterns. It’s been challenging but exciting to see digital PCR’s power in detecting these changes.”

Q: Could you explain the link between methylation and mutation formation?
Valeria Rangel: “The enzyme we study, activation-induced cytidine deaminase (AID), can deaminate both methylated and unmethylated cytosines. We believe that with transient AID activity in places where it shouldn’t be active, it could potentially create more chaos, issues that lead to chromosomal translocations forming. So we’re asking, ‘How does methylation play a role, and does it influence how often these translocations form?’”

Q: What best practices do you recommend for designing multiplex assays?
Valeria Rangel: “We design primers and probes with similar melting temperatures to minimize annealing adjustments. I test primers with regular PCR first to ensure specificity, then move into digital PCR. It’s important to consider concentration differences between PCR and dPCR when using online design tools.”

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Listen and Learn More

To learn more about how digital PCR is helping decode cancer’s genetic and epigenetic foundations, listen to the full episode of Absolute Gene-ius featuring Valeria Rangel, PhD Candidate, University of California, Irvine, at thermofisher.com/absolutegeneius.

Discover additional episodes to hear from other scientists using digital PCR to advance research in oncology, molecular biology, and beyond.