Researchers at Case Western Reserve University discovered that the ratio between two proteins, human beta defensin 3 (hBD‐3) and human beta defensin 2 (hBD‐2), is an early biomarker of oral cancer in suspicious oral cavity lesions. The team validated a new non-invasive oral cancer test based on the ratio of these proteins, claiming it can reduce biopsies by 95%. Their findings are published in Cell Reports Medicine.
The test was created to meet the demand for a low-cost, non-biopsy method for screening individuals with oral cavity lesions. Currently, biopsies are the gold standard tool for diagnosing oral cancers and precancerous mouth lesions. This new method is designed to detect oral cancer, monitor precancerous lesions, and determine when a biopsy is warranted.
Their findings are based on a scoring system linked to the levels of the two proteins in cells brushed from suspicious oral lesions of patients. HBD-3, an epithelial cell-derived anti-microbial protein is pro-tumorigenic and expressed at high levels in early-stage oral squamous cell carcinoma while the second (hBD-2) is low or unchanged.
The laboratory-based test uses a cytobrush to collect samples from a suspected lesion and healthy tissue on the opposite side. ELISA tests measure the presence of the hBDs. The ratio of the two—over the ratio of the two proteins on the opposite, normal site—generates a score, called the beta defensin index (BDI). A score above a predetermined threshold implies cancer; anything below does not.
The team validated the BDI method using clinical samples from three non-overlapping patient cohorts. The study produced sensitivity and specificity values 98.2% and 82.6%, respectively.
When the team first studied hBD-3 and hBD-2, they believed that hBD-3 rose in early oral cancer acting as a wound-healing compound, since it was known to be a potent anti-microbial peptide of the innate immune system that plays a role in normal wound healing. But they subsequently learned that it is regulated in the same way certain cells grow uncontrollably.
HBD-3 selectively attracts and activates tumor-associated macrophages (TAMs). In turn, TAMs produce pro-inflammatory cytokines and growth factors that facilitate tumor development and progression. In addition, the mouse ortholog of hBD-3, mBD-14, induces regulatory T cell (Treg) conversion, while hBD-3 promotes PDL-1 expression on tumor cells.
“These data collectively suggest that hBD-3 plays an important role in tumorigenesis associated with primary oral squamous cell carcinoma,” the authors write.
“Imagine our surprise when this Dr. Jekyll turned out to be Mr. Hyde,” says Aaron Weinberg, chair of the Department of Biological Sciences at the Case Western Reserve School of Dental Medicine and the study’s lead researcher. “We found it was not only promoting tumor growth but was overexpressed in the early stages of the disease, while another member, hBD-2, wasn’t changing. This difference in levels of expression of the two proteins compared to the opposite side in the same patient led us to examine the BDI’s ability to distinguish cancer from benign lesions.”
The study’s lab-based approach claims to reduce biopsies in primary care clinics by 95% because it can tell clinicians who needs a biopsy.
The positive data from the lab-based approach inspired the development of a point-of-care (POC) device that can be used performed in a dental or ENT clinic as part of oral health check-up. The team claims the device can provide results within 30 minutes. A proof-of-principle study showed that BDI is adaptable to a POC assay using a microfluidic intact cell assay (MICA). The MICA detection approach traps mucosal cells in a microfluidic chip. The cell membranes are permeabilized followed by incubating with anti-hBD-3 and anti-hBD-2 fluorescent antibodies to detect the hBDs and quantify the BDI. The team envisions this format can also be used in locations where pathology services are lacking.
