Paired exosomal mRNA, tumoral somatic DNA sequencing and DNA methylation profiling in patients with primary and / or metastatic brain tumors on radiology, to develop a liquid biopsy test.

One of the hallmarks of cancer biology, and indeed, its most dreaded complication, is metastasis to distant sites via the lymphatic and hematogenous systems [1]. In 2018, an estimated 632,405 patients in the United States were living with metastatic disease arising mainly from the six most common cancer types (breast, prostate, lung, colorectal, bladder and melanoma) [2]. Traditionally, over 90% of cancer deaths have been attributed to metastatic cancer, and a population-based cancer registry in Norway identified metastatic disease as a contributory factor in two-thirds of deaths in patients with solid tumors [3].

Brain metastases (BM) are the most common central nervous system tumor, occurring nearly 10 times as frequently as primary CNS malignancies [4] and peaks in incidence between the ages of 65 to 80 years. 2 percent of all cancer patients and 12.1 percent of patients with metastatic disease at the time of diagnosis have detectable BM [5]. Some patients may be asymptomatic at the time of diagnosis and  BMs are detected following screening, but up to 75% may present with neurological symptoms including seizures, focal deficits, altered sensorium, headaches, nausea or vomiting and somnolence [6-7]]. Symptomatic BM may be the presenting feature of metastatic cancer in up to 19% of patients [14]. The Graded Prognostic Assessment (GPA) score is used to estimate survival in patients diagnosed with BM [8]. Patients with BM require multidisciplinary input, with treatment options including surgical resection (metastatectomy), whole brain radiation therapy (WBRT) or stereotactic radiosurgery/radiotherapy (SRS and SRT), and systemic therapy has increasingly proven effective in certain subtypes of non-small cell lung cancer (NSCLC), breast cancer and melanoma [9]. Despite these advances, the presence of BM confers a median survival of 12 months or less across nearly all primary cancers [5].

The differentiation of primary and secondary malignant lesions of the brain is critical in the management of the patients. Currently, the only method of differentiating between primary and metastatic lesions is a biopsy, which itself comes with all its risks and pitfalls. In addition, a number of patients with a known malignancy develop a second lesion in the brain that is also a primary tumour. Even for these lesions there is no easy way of diagnosis[2].

The development of reliable liquid biopsies using blood or other body fluids for the diagnosis and evaluation of different cancers as an alternative to standard tissue biopsies is an area of active research [10]. Every tumor secretes within the blood (and sometimes other bodily fluids in contact with the tumor) endovesicles (small ‘bubbles’ that get detached from the cell carrying with it a representative sample of its cargo), cell-free DNA (DNA of dead cells released into the blood) and circulating tumor cells [11]. As the amount of extra cellular vesicles, circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) are usually directly proportional to the grade of tumor, the highest grade is better represented in the blood.  Liquid biopsy tests have been approved by the USFDA as companion diagnostic tests for the targeted treatment of NSLC (Cobas EGFR mutation Test v2), breast cancer (therascreen PI3LA RGQ PCR Kit) and even pan-cancer testing covering ovarian cancer, prostate cancer in addition to NSCLC and breast cancer (Guardant 360 CDx and FoundationOne Liquid CDx) [12].

Exosomes or extracellular vesicles are increasingly implicated in cancer development and progression, with a growing interest in their potential as diagnostic and therapeutic biomarkers [13]. In a pilot study we conducted on gliomas, we have already established an ~80% sensitivity and specificity of the exosomal assay in testing for the cancer marker epidermal growth factor (EGFR) receptor [14]. By increasing the number of markers tested, it may be possible to improve the sensitivity and specificity of liquid biopsies using the exosomal platform to a degree that can obviate the need for a tissue biopsy. By characterizing the exosomal profiles of patients diagnosed with BM as well as the primary cancers responsible for the majority of BM, it also may be possible to develop a single blood test capable of distinguishing between primary CNS tumors and brain metastases and identifying the primary cancer.

Hypothesis: The exosomal platform provides an ideal stage to be able to screen, diagnose and prognosticate brain tumors for treatment decisions without the need for a tissue biopsy in both primary (CNS) and secondary (metastatic) tumors.

References:

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