J. A. Cintolo-Gonzalez1, W. Michaud1, S. Cohen1, D. Plana1, D. J. Panka2, R. J. Sullivan3, G. M. Boland1 1Massachusetts General Hospital,Surgery,Boston, MA, USA 2Beth Israel Deaconess Medical Center,Hematology/Oncology,Boston, MA, USA 3Massachusetts General Hospital,Hematology/Oncology,Boston, MA, USA
Introduction:
Exosomes are extracellular microvesicles, which contain a variety of nucleic acids (DNA, messenger RNA (mRNA), microRNA (miRNA)) and proteins. Exosomes can be analyzed from archived serum/plasma samples making them ideal biomarkers to study tumor-based changes. We examined the feasibility and accuracy of exosomal mRNA analysis to assess disease burden in patients undergoing surgical resection for metastatic melanoma.
Methods:
Melanoma cell lines (A375, RPMI 7951, SK-MEL-30, and MeWo) were purchased directly from ATCC. Serial tumor and blood samples were collected from melanoma patients under IRB approved protocols. Exosomes were isolated using combined filtration and ultracentrifugation. Exosomal RNA was isolated using the exoRNA serum/plasma kit (Qiagen Inc).
Quantitative PCR (qPCR) was used to assess concordance of gene expression between paired exosomes and cell lines. Paired exosomal and tumor mRNA from patient samples underwent whole transcriptome sequencing using Affymetrix Whole Transcriptome Pico Array. Patient plasma-derived exosomes were analyzed via qPCR at pre-resection and post-resection time points.
Results:
Paired exosomes and their parental cell lines demonstrated concordance in gene expression as assessed by qPCR. Specifically, in cell lines harboring a BRAF V600E mutation, BRAF V600E was detected by qPCR in cell lines and paired exosomes. Likewise, whole transcriptome analysis of patient-derived exosomes and paired tumors demonstrated 80% concordance of gene expression. In patients undergoing resection of BRAF V600E mutant metastatic lesions, there was a significant decrease in exosomal BRAF V600E mRNA in patients rendered NED or with minimal residual disease but not in patients undergoing palliative resection with multiple residual sites of disease (Figure 1). Of the patients undergoing definitive resection, patient 410, who showed ongoing, albeit decreased, exosomal BRAF V600E expression, recurred 3 months following resection.
Conclusion:
Exosomal mRNA reflects parental tumor mRNA expression and accurately predicts the presence of residual disease after resection in melanoma. These findings support the use of exosomes as a biomarker for assessing tumor burden after surgery. Work is ongoing to assess the utility of exosomal RNA to assess minimal residual disease and risk of recurrence in high risk melanoma patients.
