Exosomes Could Help Detect Early Pancreatic Cancer

Veronica Hackethal, MD

July 10, 2015

Exosomes, tiny particles shed by cells into the circulation, could be used to diagnose early pancreatic cancer, and could even be used to screen individuals at high risk for the disease, say researchers.

They report that exosomes containing the surface protein glypican-1 (GPC1) can be detected in as little as 150 to 200 microliters of blood, and in their study, this noninvasive test distinguished between healthy individuals and patients with pancreatic cancer with 100% accuracy; the results were also positive in some patients with breast cancer.

The findings were published online June 24 in Nature.

"We have identified a method to detect exosomes from cancer cells in the blood of cancer patients," commented senior author Raghu Kalluri, MD, PhD, a professor at the University of Texas MD Anderson Cancer Center, in Houston. "We showed that these exosomes are present in blood of patients with all stages of pancreatic cancer, including a few patients with early lesions."

"Using this discovery, we showed that such detection may offer a rapid method to detect cancer and the mutations associated with cancer," he added.

As many as 80% of patients with pancreatic cancer present with metastasis at diagnosis, and only 15% patients have resectable tumors at diagnosis, contributing to the grim prognosis of pancreatic cancer. The absence of specific biomarkers and the anatomic location of the pancreas hinders earlier diagnosis, according to background information in the article.

Exosomes are excess proteins secreted into the circulation from every cell type. These tiny particles measure about 100 nm and contain proteins and nucleic acids encased in a lipid bilayer. Although scientists have known about exosomes for decades, specific markers that can distinguish cancer exosomes from those formed by normal cells remain unknown.

GPC1 is a membrane-anchored protein that is overexpressed in many cancers, such as breast, colon, and lung cancer, but especially in pancreatic cancer. Studies have suggested that GPC1 may play a role in regulating cancer progression.

Study Results

In their study, Dr Kalluri and colleagues first isolated exosomes from cancer cells, fibroblasts, and nontumorigenic cells using ultracentrifugation. Further analyses identified 48 proteins unique to cancer exosomes.

Then, using mass spectrometry, they found that GPC1 was abundant on cells derived from cancer exosomes but was present at low levels on exosomes from normal cells.

Next, using flow cytometry, they isolated and monitored GPC1 CrExos (GPC1+) exosomes in the frozen serum from patients and mice with cancer. The study included patients with pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer.

Results showed consistently higher levels of GPC1+ exosomes in patients with pancreatic cancer when compared with healthy individuals and patients with benign pancreatic disease. Healthy individuals (n = 100) had GPC1+ exosomes at rates of 0.3% to 4.7%. All 190 pancreatic cancer patients had higher levels of GPC1+ exosomes than healthy donors (P < .0001).

However, it appears that the exosomes are not specific for pancreatic cancer but may be a signal generally for cancer. In this same study, 75% of patients with breast cancer (24/32) had GPC1+ levels higher than healthy donors (P < .0001).

But the results for pancreatic cancer were particularly impressive.

Measurement of GPC1+ exosome levels were "near perfect" ― with a sensitivity and specificity of 100% and a negative predictive value of 100% ― for distinguishing patients with all stages of pancreatic cancer from healthy individuals and patients with benign pancreatic disease, the researchers report.

Patients with pancreatic cancer precursor lesions (n = 5) had "consistently higher" levels of GCP1+ exosomes compared with healthy individuals and those with benign pancreatic disease (n = 26; pancreatitis, n = 18; cystadenomias, n = 8). Patients with benign pancreatic disease had similar GPC1+ exosome levels as healthy individuals.

Levels of GPC1+ exosomes also correlated with tumor burden. Patients with distant metastases had significantly higher GPC1+ exosome levels (average, 58.5%) compared with those with disease confined to the lymph nodes (average, 50.5%) and those with no metastases (average, 39.9%). After surgery, GPC1+ exosome levels significantly decreased in patients with pancreatic cancer (P < .0001) and pancreatic precursor lesions (P < .001).

GPC1+ exosome levels were found to be more reliable than CA19-9 levels, the clinical standard biomarker for PDAC. Roughly half of patients without cancer had elevated CA19-9 levels, but none had elevated GPC1+ exosome levels. CA19-9 levels were significantly elevated in patients with both benign pancreatic disease (P < .0001) and those with pancreatic cancer.

Similar results were found in an independent cohort of patients with chronic pancreatitis (n = 6), pancreatic cancer (n = 56), and healthy individuals (n = 20).

In animal studies, mice with implanted pancreatic cancer cells were found to have elevated GPC1+ exosome levels, and these levels increased with time, tumor burden, and severity of disease. GPC1+ exosome levels began to rise even before lesions became detectable on MRI.

In mice with spontaneous pancreatic cancer, and also in patients with pancreatic cancer, the GPC1+ exosomes carried specific KRAS mutations ― common mutations in pancreatic cancer that likely drive the disease. There was a 100% correlation between KRAS mutations in GPC1+ exosomes had KRAS mutations in tumors.

Although more research is needed in larger groups of patients, the authors suggest that GPC1+ exosomes are a "reliable" biomarker for early pancreatic cancer screening and diagnosis and are "superior" to CA19-9.

"This study provides an opportunity to perform more tests in patients with high risk for pancreatic cancer," Dr. Kalluri said. "This will allow us to examine whether this test is of any value for early detection in a population setting."

The potential implications of such a test are huge. Dr Clotilde Thery

"[T]he potential implications of such a test are huge. It would allow clinicians to decide whether or not to perform potentially debilitating surgery," wrote Clotilde Thery, PhD, of the Institut Curie, in Paris, France, in a linked commentary.

"The novelty of [the]...report resides in the presence of GPC1 in circulating vesicles in serum, and in the striking value of this molecule as a biomarker," she emphasized. "[T]his work demonstrates for the first time that circulating vesicles in blood can be a source of specific and reliable diagnostic biomarkers for cancer."

One advantage of the technique is that it uses equipment — an ultacentrifuge and flow cytometry — that is in widespread use. This means that the technique could potentially be easily adopted in clinical laboratories.

But Is It Specific?

Concerns hinge, however, on the specificity of the test: whether it can potentially be a generic cancer marker, or whether its usefulness is confined to early diagnosis of pancreatic cancer.

"I don't think GPC1 CrExos is a cancer-specific biomarker," commented Nicola Schultz, MD, PhD, a consultant surgeon in the Department of Gastrointestinal Surgery and Transplantation at the Rigshospitalet, University of Copenhagen, in Denmark, who was approached by Medscape Medical News for comment.

In particular, Dr Schultz was dubious about the surprisingly high sensitivity and specificity of 100% in this study.

"This could be explained by the low number of patients with benign pancreatic disease or relevant symptoms, the true control group," she pointed out.

Next steps, according to Dr Schultz, would include a large-scale, prospective multicenter study. To tease out the cancer specificity of GPC1+ exosomes, these studies should include patients with relevant cancer and noncancer- related symptoms and those with related cancer types, such as biliary, duodenal, gastric, and colorectal cancer.

Nevertheless, Dr Schultz was enthusiastic about the potential for exosomes in cancer diagnosis.

"The idea of using serum exosomes as a circulating biomarker detectable in a blood test is the same idea that has inspired my studies of circulating microRNA in whole blood and serum," Dr Schultz said. Her team recently published research on developing a test for pancreatic cancer using microRNAs in whole blood.

Analysis of whole blood microRNA includes exosomal microRNA as part of the total circulating microRNA, she explained. If the signal is strong enough, the selection procedures for microRNA panels include potentially cancer-specific exosomal microRNA. In essence, analysis of cancer-specific exosomes gives the same information in an undiluted form as analysis of whole blood microRNA.

Cancer-specific exosomes are potentially a game changer. Dr Nicola Schultz

"Cancer-specific exosomes are potentially a game changer," Dr Schultz commented, "not only for pancreatic cancer but for several cancers with late and unspecific symptoms, like biliary cancer, gastric cancer, and lung cancer. The difficulty will be related to isolation of cancer-specific exosomes."

The authors, Dr Thery, and Dr Schultz report no relevant financial relationships.

Nature. Published online June 24, 2015. Abstract, Commentary


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