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Harvard-MGH Liquid Biopsy Assay Targets Early Detection of Pancreatic Cancer


NEW YORK (360Dx) – A multiplexed nanoplasmonic assay designed specifically to fit into clinical workflows is taking aim at high-throughput detection of pancreatic ductal adenocarcinoma (PDAC), an aggressive and often inoperable form of pancreatic cancer.

Developed by researchers at Harvard Medical School and Massachusetts General Hospital, the assay employs a liquid biopsy approach, leveraging low blood volumes and the clinical potential of exosomes — tiny vesicles shed by all kinds of cells, including tumor cells.

The researchers said that Cambridge, Massachusetts-based Exosome Diagnostics has licensed the technology and is exploring integrating the test with its own portfolio of exosome tests to explore the potential for "doing concurrent testing of exosome-based proteins and mRNA."

Among other biological molecules freely circulating in the blood as cancer biomarkers, extracellular vesicles represent "a promising target for the minimally invasive diagnosis of pancreatic cancer based on liquid biopsy," said Giuseppe Spoto, a professor of chemical sciences at the University of Catania in Italy, and coordinator of a European Union-sponsored research project that integrates plasmonics in the detection of DNA, microRNA, and protein-based tumor autoantibody detection.

"Plasmonics offers new opportunities for the development of innovative approaches to cancer diagnosis with potential application to early diagnosis, as well as patient follow-up," he said, adding that it can be used to detect entities, such as circulating extracellular vesicles, with excellent limit of detection, which "translates into simplified assays that can operate with good sensitivity and specificity."

The Harvard-MGH group published research on Wednesday in the journal Science Translational Medicine, describing clinical testing and validation of the assay.

In its sensitivity and specificity, the Harvard-MGH test outperformed the best-established blood test currently available, which uses cancer antigen 19-9 as a PDAC tumor marker, said Cesar Castro, director of the cancer program at MGH Center for Systems Biology, and a coauthor of the study. He noted that the new assay may enable earlier detection for PDAC, a condition that is often untreatable.

Initially, the researchers had hypothesized that multiplexed markers would be needed for clinical use because pancreatic cancer tumors are so heterogenous, so they established an objective to "define protein signatures representative of epithelial and pancreatic cancers."

The researchers said that they surveyed about 50 proteins of potential interest and discarded 40 of them after feasibility studies in PDAC cell lines and patient-derived xenograft models.

The remaining 10 markers included seven tumor-derived extracellular vesicle markers (EGFR, EPCAM, MUC1, HER2, GPC1, WNT2, and GRP94) and three pan extracellular vesicle markers (CD63, CD9, and Rab5b). The pan extracellular vesicle markers were excluded from the tumor diagnostic marker panel and were solely used to confirm the presence of extracellular vesicles in each sample.

To further develop the panel, they collected plasma from 32 patients enrolled in a training cohort that included 22 patients with PDAC and 10 healthy patients, and they conducted receiver operating characteristic analyses to calculate "the sensitivity, specificity, and accuracy for each marker individually and in combination," tested in the patient samples.

As no single marker achieved sufficient sensitivity and specificity, they decided that multiplexing would be the best approach, Castro said.

The researchers validated the panel by analyzing a prospective cohort of 43 patients undergoing surgery for pancreatic or other abdominal indications. The assay diagnosed PDAC with 86 percent sensitivity and 81 percent specificity based on a signature of five tumor-based protein markers — EGFR, EPCAM, MUC1, GPC1, and WNT2.

The development work has culminated in a prototype assay for PDAC detection that can complete testing in about 10 minutes, Castro said. The system's materials costs about $60, which could be further reduced by batch-fabricating the sensor chip used in the assay, he said.

That would facilitate making the test accessible to a broad population of users, including resource-strapped geographies and clinical settings, Castro added.

To increase its accessibility even further, the team automated use of the assay and improved upon a first-generation prototype, described in the journal Nature Biotechnology in 2014.

Castro noted that it's an achievement to demonstrate that it's possible to develop a blood-based diagnostic test that uses signature markers for detecting cancers, but it's important also to develop a test that fits within an oncologist's clinical workflow, and has suitable levels of accuracy, sensitivity, and specificity.

As a result, this second-generation test employs a higher-density chip that enables the instrument to view additional markers and use a lower-volume sample as well as less reagents, he noted.

The assay runs on a nanoplasmonic sensor platform that MGH and Harvard are using to develop assays for several cancers.

The team has already reported using the platform to analyze ascites samples from ovarian cancer patients, and found that exosomes derived from ovarian cancer cells can be identified by CD24 and EpCAM expression.

Exosomes are valuable for diagnostics because they could be applied to identify virtually all types of cancers that have associated biomarkers, MGH codeveloper and coauthor Hakho Lee said recently.

The team designed the PDAC tumor assay so that it would not only be suitable for clinical workflows, but could also do high-throughput detection using blood volumes as low as 10 ml, Castro said.

Within the instrument, a sensor chip consists of periodically arranged nanopores that are 200 nanometers in diameter and patterned in a 100-nanometer-thin gold film.

Light shone onto the gold surface flows through the pores. Extracellular vesicles bound near the pores by PDAC-specific monoclonal antibodies shift the light's wavelength.

Sensors detect the shift in the light spectrum, enabling the instrument to calculate the quantity of antibody-bound, tumor-derived vesicles.

The researchers noted that the sensor chip could be easily scaled to larger arrays of more than 1,000 sensing sites. In their study, they prepared a customized chip with 100 detection sites.

They used molecular printing to attach the monoclonal antibodies on the sensor chip in 100 nl size spots. A piezoelectric microscope stage collected transmission spectra, and the overall system did its printing and measuring automatically, which improves assay throughput and reduces variation among users, the researchers said.

Because they used a smaller chip size, higher spot density, and smaller measurement volumes, the assay's sensitivity rose twenty-fivefold from the sensitivity of the previous prototype.

Pancreatic cancer

PDAC is difficult to diagnose and it's the fourth-leading cause of cancer death in the US. It has a five-year survival rate less than 10 percent. Tumors in more than 80 percent of new patients can't be removed by surgery, Castro said.

Earlier detection could increase survival by 30 percent to 40 percent, and more reliable and real-time assessment of the effect of treatment could prolong survival and improve quality of life, the researchers said.

Detecting serum concentrations of CA 19-9 is currently the best-established blood test for PDAC, and is often used to track a patient's response to treatment for PDAC, but it's relatively less effective for PDAC detection, and its signature commonly rises late in the onset of the disease, Castro said.

"There has been no perfect tumor marker for this condition; even the [CA 19-9] signature can be elevated in other medical conditions that are not related to cancer," Castro said.

He noted that clinicians would benefit from a method to screen, diagnose, and treat PDAC that's similar to how they are able to screen, diagnose, and treat colon cancer.

However, because of physical limitations, it's not possible for clinicians to interrogate the pancreas in the same way that they interrogate the colon, he said, adding that the new assay may give clinicians a route to early detection of PDAC similar to what colonoscopies have done for early detection of colon cancer.

Various approaches are being explored to achieve higher sensitivity and specificity to prolong survival in patients with PDAC. Besides exploring circulating tumor-derived extracellular vesicles, researchers are also exploring the use of circulating tumor cells as diagnostic markers.

Tumor-derived extracellular vesicles are relatively more abundant and more stable than other circulating biomarkers, and they consist of protein and mRNA profiles that reflect parental cancer cells, Castro said.

However, most existing assays require large amounts of extracellular vesicles that are relatively difficult to isolate and purify in preparation for measurement. A standard method of isolation involves using expensive ultracentrifuges that take hours to isolate extracellular vesicles and involve manual work.

Existing diagnostic assays also encounter challenges in effectively distinguishing between tumor-derived EVs and those derived from healthy cells. This is partly because the vesicles are so small, and most available diagnostic instruments are not sensitive enough to detect cancers with low volumes of a sample, or low volumes of vesicles, Castro said.

The MGH-Harvard team, by contrast, believes that their approach may eventually enable them to analyze a single extracellular vesicle. Achieving this may have implications for researchers looking to manipulate matter at the cellular level, though the clinical implications of achieving that are unclear, Castro said.

In addition to the methods employed by the MGH-Harvard team, other approaches have emerged that could lead to clinical use for exosome diagnostic tests. One such approach is to use surface markers to detect exosomes specific to a particular type of cancer and isolate them from the background of other circulating vesicles, cells, and cell-free nucleic acids.

Peregrine Pharmaceuticals recently reported on its efforts in this area after licensing IP from the University of Texas Southwestern last year for the detection of exosomes that express the protein phosphatidylserine.

To detect exosomes bearing PS on their surfaces, the UT group used an engineered, multivalent PS-specific antibody to create a highly sensitive and quantitative ELISA for the detection of picogram amounts of PS in plasma.

The study was "performed nicely in [a] small subset of ovarian cancer and controls," Castro said. However, phosphatidylserine is expressed in all cells, so the technique will need further validation he added.

"ELISAs are the type of conventional testing that requires larger clinical sample volumes and significant processing," he said, and the cost of their "specialized antibodies may be feasible for well-funded labs, but may be a challenge to others."

Separately, researchers from Arizona State University have developed a method for the detection of tumor-derived exosomes in circulation that could be turned into a simpler and potentially cheaper platform for cancer diagnostics development than other current approaches.

The ASU team's approach uses the interaction between two nanoparticles to detect exosomes that express a particular molecular target on their surface.

Castro noted that he appreciated the "cool nanotech modification," achieved by the team. However, his team has tested EphA2 "many times" and it "has not performed well," he added.

At Ulsan National Institute of Science & Technology in South Korea, a team of researchers is developing a urine liquid biopsy-based lab-on-a-disc that incorporates centrifugal force and nanoporous membranes to identify extracellular vesicles that carry potentially important biomarkers of cancer.

Castro said that although the technique focuses on urine, which is less viscous than serum or plasma, and it would need exploration in other media, it could "align nicely with our device once optimized."

A European Union-sponsored research project that integrates DNA, microRNA, and protein-based tumor autoantibody detection may lead to a liquid biopsy in vitro diagnostic test for colorectal cancer in 2018.

As part of that effort, several research groups and companies are teaming up to develop an ultrasensitive plasmonic devices for early cancer diagnosis. The system would detect protein-based tumor autoantibodies and recognize specific DNA and microRNA mutations, eliminating the requirement for preliminary amplification of nucleic acid sequences.

Castro said that exosome and microvesicle testing would be a next logical step for the European team to pursue, and that the work shows the power of multinational collaborators working together and rallying around a technology.

Exosome Diagnostics, licensee of the Harvard-MGH assay, recently entered into an agreement with Merck KGaA, Darmstadt, Germany, to help further the company’s drug development efforts in oncology and other therapeutic areas, "utilizing the full breadth of Exosome Diagnostics’ proprietary technology platforms." The platforms span across both nucleic acid and protein, and include Shahky, its exosomal protein capture and quantitative analysis instrument. The agreement represents the first publicly announced partnership that grants access to Exosome Diagnostics’ recently unveiled point-of-care protein detection instrument, the firm said in a statement

The instrument can generate a high signal above the noise "by selectively targeting disease-specific exosomes and removing background that is nonrelevant to the disease in question."

The firm said that the instrument was tested and its performance validated in an undisclosed "leading Boston Hospital in early January of 2017."