quinta-feira, 18 de junho de 2015

Liquid Gold


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Thu, 06/18/2015 - 12:30pm

George Karlin-Neumann, the Digital Biology Center at Bio-Rad Laboratories

Droplet Digital PCR enables liquid biopsy for monitoring cancer treatment. Image: Bio-Rad Laboratories

Droplet Digital PCR enables liquid biopsy for monitoring cancer treatment. Image: Bio-Rad LaboratoriesBlood is the great aggregator of the body’s physiology. Many tumors slough off fragments of DNA into the bloodstream, which can be detected with a minimally invasive blood draw using advanced DNA tests—also known as a liquid biopsy. One of the challenges preventing liquid biopsy from becoming a clinical reality has been reliably finding the cancerous DNA in the vast sea of healthy DNA.

Breast cancer researchers are among those who are harnessing sensitive molecular tools such as Droplet Digital PCR (ddPCR) technology to improve cancer detection and track tumor progression in vivo. Numerous studies are underway to determine how we can use these cell-free circulating tumor DNA (ctDNA) clues to expand our understanding of cancer.

This is important because researchers are now re-envisioning cancers based on what mutations drive them, rather than the tissue of origin. In fact, tumor genotyping is rapidly becoming the standard of care in some areas of oncology where chemotherapy-based therapies target specific mutations. However, the standard surgical biopsies used to track a tumor’s ever-changing DNA can be costly and dangerous. Some cancers simply don’t provide enough tissue for conventional analysis. Additionally, the genetic heterogeneity of the evolving tumor means mutations present in only a portion of the tumor may be not be sampled and, thus, not detected in the slices of tumor tested.

Using ddPCR technology to reveal biomarkers in liquid biopsies holds the promise of helping overcome barriers to identifying and generating treatments for specific mutations.

How droplet digital PCR works

In digital polymerase chain reaction (dPCR), a sample is subdivided into many smaller microreactions. This partitioning increases the concentration of the rare target (tumor DNA) so it can amplify sufficiently and be specifically detected. Once PCR runs to end point in each aliquot, the original sample can be read essentially as a series of ones and zeros—individual partitions either containing a targeted sequence or not. From this tally, researchers can extrapolate the exact total molecular counts of tumor DNA for absolute quantification, an advantage over quantitative real-time PCR (qPCR) which requires a standard curve to achieve absolute quantification.

Various technologies offer different methods of partitioning and detection. Bio-Rad’s technology mixes the DNA sample with an oil, which is then partitioned into 20,000 evenly sized one-nanoliter droplets. Analytical Chemistry first published this technique in 2011. As a result of the highly uniform droplets, large number of partitions, as well as robust chemistry and engineering, ddPCR provides a high level of sensitivity, precision and reproducibility. In fact, clinical oncologist and researcher Lao Saal chose Bio-Rad’s ddPCR system for its “very good performance characteristics.”

Detecting recurrence of breast cancer post-treatment

In a paper published in EMBO Molecular Medicine in May (2015), a group led by Saal at Lund Univ. in Sweden found the emergence of circulating tumor DNA (ctDNA) in plasma after breast cancer surgery was a strong signal the cancer would return and metastasize, and its absence indicated long-term disease-free survival. They found 93% of individuals with tumor DNA in their bloodstream after breast cancer surgery eventually relapsed, while the ctDNA-negative individuals were disease-free a median of 9.2 years later. What’s more, for 86% of study participants whose cancers returned, Saal’s group could detect ctDNA an average of 11 months before conventional imaging and symptom-based diagnosis. In two patients, detection occurred three years before clinical diagnosis. The stage I-III tumors were primarily hormone receptor negative and HER2 negative.

In their retrospective study, Saal’s group used low-pass next-generation sequencing (NGS) to identify early chromosomal rearrangements in tumor tissue, effectively fingerprinting the DNA unique to that cancer. But to determine if circulating DNA with those rearrangements were present after surgery, they analyzed plasma taken at intervals with highly sensitive and specific ddPCR technology to quantify ctDNA. The simple, fast technology also proved more sensitive and much less costly than NGS for the repetitive measurements.

Saal’s group is currently engaged in a prospective study where they will administer chemotherapy in an attempt to shrink the breast tumor prior to surgery, and monitor for therapy response and recurrence via liquid biopsy.

Validating miRNA as a potential breast cancer biomarker

Another study published by Oncotarget in May (2015) used ddPCR technology to pursue a different path toward earlier breast cancer detection: microRNA (miRNA). Research suggests diseases such as cancer selectively impact the quantity of these small regulatory molecules found in the bloodstream. Concentrations of miRNA could therefore serve as valuable biomarkers and deliver insight into disease processes.

As proof of the concept, a team led by Manuela Ferracin and Massimo Negrini of the Univ. of Ferrara, identified significant down-regulation of miR-181a-5p—one of nearly 2,000 identified human miRNAs—in the serum of breast cancer patients compared to healthy controls. As a diagnostic tool, measuring this lone miRNA correctly identified nearly 70% of study participants with breast cancer with 70% specificity in two cohorts.

This advanced approach to detecting breast cancer follows and builds on a seminal 2013 study published in Nature Methods by Muneesh Tewari of the Fred Hutchinson Cancer Research Center that demonstrated the successful use of ddPCR to obtain reproducible, quantitative data for miRNA circulating in plasma and serum. By using ddPCR, Tewari found an alternative to the highly variable qPCR measurements that has been holding back the use of miRNA as clinical biomarkers.



In both of the newly published studies, ddPCR is used in concert with other diagnostic or discovery tools. As further confirmation studies take place, digital PCR technologies show promise to advance early breast cancer detection and improve long-term survival outlooks.

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