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Liquid Biopsy of Melanoma Surgery Drainage Fluid Shows Promise for Recurrence Monitoring

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NEW YORK (GenomeWeb) – An international team led by investigators from the Spanish National Cancer Research Centre (CNIO) are pushing forward with efforts to validate a liquid biopsy technique  involving the extraction and analysis of extracellular vesicles from wound drainage fluid in melanoma patients who have a lymph node surgically removed.

The technique joins a growing cadre of liquid biopsy methods looking beyond blood and urine to a variety of "peripheral" sources such as spinal fluid, lavage washings from various body sites, pap smears, and even menstrual blood.

The CNIO team's new effort, which authors described in a publication last month in the Journal of Experimental Medicine, focused on the exudative seroma, a fluid that is collected from the drainage tube inserted after surgery and normally disposed of.

Héctor Peinado, Head of the Microenvironment and Metastasis Group at CNIO, said this week that the current study was a preliminary proof of concept, showing that exosomes in these normally discarded fluid samples could be both a useful surrogate of melanoma progression and offer the opportunity to measure informative genomic biomarkers. But the results were strong enough that the team is now working with pharmaceutical partners to extend the study toward possible clinical implementation.

"This was a pre-clinical study, but it is setting the field for the potential use not only in melanoma and also in [other cancers]," Peinado said. "What we want to do now is follow 60 to 90 patients over several years."

In their JEM report, Peinado and colleagues described their effort to glean signals of lingering or recurring cancer in lymphatic fluid samples from a small cohort of melanoma patients.

Investigators started by characterizing the number of circulating exosomes in matched exudative seroma (ES) and plasma samples from a small group of melanoma patients, concluding that the drainage fluid was much more highly enriched in exosomes compared to plasma. The size of particles in ES was also significantly larger than that of plasma EVs, and electron microscopy showed that ES-derived exosomes displayed a more heterogeneous size distribution than their plasma counterparts.

The team then used liquid chromatography-tandem mass spectrometry to analyze the protein cargo of EVs from ES and plasma, finding a higher number of exosomal proteins in the drainage fluid with a "significant enrichment in several pathways … related to antigen presentation, endoplasmic reticulum (ER)-phagosome pathway, G2/M transition, and IL-12 family signaling."

Looking at two proteins, HSP90 and TRP-2 — previously described as enriched in plasma exosomes from melanoma patients and correlated with progression — the team found that while TRP-2 was present in both fluids, HSP90 was only detected in ES-derived exosomes. Comparing the liquid biopsy protein results with data from melanoma cell lines, 605 proteins were recapitulated in ES exosomes, but only 256 in plasma vesicles.

In samples from 14 melanoma patients who had a range of different levels of cancer lymph node involvement, the team found 17 pathways significantly enriched in N3 versus N1a patients, several of which remained significantly associated after limiting the analysis to proteins common to the cell-line directed exosome dataset.

Finally, the group assessed whether DNA in seroma-derived EVs could yield genomic markers of disease progression or recurrence risk. BRAF, because of its commonality in melanomas, was the chosen target. Combining exosomal RNA/DNA and cell-free DNA, the group analyzed samples from 17 patients, 10 of whom also had tissue samples that were tested for BRAF using quantitative PCR.

Cell-free DNA from the seroma seemed to be a poor source of signal, because of a large background of wild-type BRAF, the authors wrote. But using EV samples worked much better.

"All patients found to be BRAF-negative in tumor samples were also BRAF-negative in ES-EVs, indicating that specificity was 100 percent," the authors wrote. In four out of the eight tissue-positive cases, the mutation was also detected in ES-derived EVs. Importantly, all four of these patients relapsed with distal metastases between 56 and 463 days after lymphadenectomy, while only two cases without evidence of BRAF in their ES Evs progressed, and at a much further timepoint.

Among patients diagnosed as BRAF mutants by tissue biopsy, those who were also mutation-positive in seroma EVs had a median survival of just 146 days compared to 715 days for those who were negative. Although tumor burden was also predictive, it did not reach statistical significance, the authors wrote.

Among the team's conclusions were that the study provides evidence that extracellular vesicles in lymphatic drainage fluid may offer proteomic and genomic signals of melanoma recurrence risk. BRAF detection in these samples, specifically, could be a powerful tool for identifying at-risk patients based on evidence of residual disease — offering the chance to identify and treat such patients with adjuvant therapy.

Melanomas have a high recurrence rate — up to 50 percent after lymph node removal — and clinicians have practically no effective means to predict which individuals will recur or to catch those recurrences early. As a result, Peinado and colleagues are not the only academic team hoping to find a way to use liquid biopsy to help guide care for melanoma patients post-surgery, with other groups focusing on blood-based analyses.

According to Peinado he and his team didn't see much success with cell-free DNA in their analyses of lymphatic fluid, but agreed that much work remains to see which signals will be most predictive, and whether there could be additive value in combining testing of different compartments.

The team also believes that their seroma drainage method could be useful in other tumor types, where lymphadenectomy is common, and hope to extend the research into other areas like breast cancer.

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