BALTIMORE & HOUSTON—On the hunt for better cancer screening tests, Johns Hopkins scientists led a proof-of-principle study that successfully identified tumor DNA shed into the blood and saliva of 93 patients with head and neck squamous cell carcinomas (HNSCCs). A report on the findings is published in the June 24 issue of Science Translational Medicine under the title “Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas.”
For the study, 93 patients with newly diagnosed and recurrent HNSCCs gave saliva samples, and 47 of them also donated blood samples before their treatment at The Johns Hopkins Hospital in Maryland and MD Anderson Cancer Center in Texas. The scientists detected tumor DNA in the saliva of 71 of the 93 patients (76 percent) and in the blood of 41 of the 47 (87 percent). In the 47 who gave both blood and saliva samples, scientists were able to detect tumor DNA in at least one of the body fluids in 45 of them (96 percent).
When the scientists analyzed how well their tumor DNA tests found cancers in certain regions of the head and neck, they found that saliva tests fared far better than blood tests for oral cavity cancers. All 46 oral cavity cancers were correctly identified through saliva tests (100 percent), compared with 16 of 34 oropharynx cancers (47 percent), seven of 10 larynx cancers (70 percent) and two of three hypopharynx cancers (67 percent).
“We have shown that tumor DNA in the blood or saliva can successfully be measured for these cancers,” according to Dr. Nishant Agrawal, associate professor of otolaryngology-head and neck surgery and oncology at The Johns Hopkins University School of Medicine. “In our study, testing saliva seemed to be the best way to detect cancers in the oral cavity, and blood tests appeared to find more cancers in the larynx, hypopharynx and oropharynx. One reason that saliva tests may not have been as effective for cancer sites in the back of the throat is because we didn’t ask patients to gargle; we only asked them to rinse their mouths to provide the samples. However, combining blood and saliva tests may offer the best chance of finding cancer in any of those regions.”
Since detection of oral cavity squamous cell carcinoma was 100-percent effective in saliva, the focus will be on saliva as a testing method for the oral cavity, Agrawal tells DDNews. “Saliva testing is more specifically oriented for head and neck cancers that affect the upper aerodigestive tract. Early-stage head and neck cancers (stage I and II) have not metastasized to lymph nodes or other parts of the body. They are localized to the lining of the upper aerodigestive tract, which is usually bathed in saliva. So we are just directly sampling the affected area by analyzing saliva. We are currently investigating the difference between oral rinse and gargling to determine if our test performance is improved. If there is benefit, we will collect saliva by a combination of gargling and oral rinse.”
The saliva for testing was collected by rinsing the oral cavity with saline, or by passive saliva collection (drooling). Agrawal explains that for blood tests, the study is specifically testing plasma, which is isolated from whole blood by centrifugation. They have also previously demonstrated that tumor DNA can be identified in plasma from many solid malignancies.
Blood tests correctly identified tumor DNA more often in 20 of 22 oropharynx cancers (91 percent), six of seven larynx cancers (86 percent) and all three hypopharynx cancers. Taken together, blood and saliva tests correctly identified all oral cavity, larynx and hypopharynx cancers and 20 of 22 oropharynx cancers (91 percent).
The sensitivity of the tests overall depended on the cancer site, stage and HPV status, ranging between 86 to 100 percent. Agrawal also reports that saliva tests performed better for early-stage cancers, finding all 20 cancers, compared with blood tests that correctly identified seven of 10. He and his team found the opposite was true for late-stage cancers: Blood tests found more late-stage cancers (34 of 37) than saliva tests did (51 of 73). Blood tests also correctly identified HPV-related tumors, occurring in 30 of the 93 patients, more often than saliva tests, probably because HPV-related tumors tend to occur in the back of the throat, which may not have been reached with the saliva rinse.
DDNews learns from Agrawal that HPV-associated squamous cell carcinomas generally involve the oropharynx (tonsils and base of tongue), and are associated with high-risk HPV type 16. A study by Gillison in 2012 published in the Journal of the American Medical Association reported an approximate rate of 7 percent for the overall prevalence of HPV infection. “We will further investigate in larger experiments, with appropriate controls, how HPV will affect the assay,” Agrawal says.
In the case of head and neck cancers associated with HPV—tumors that are on the rise among Americans—Agrawal and his colleagues searched patients’ blood and saliva samples for certain tumor-promoting HPV-related DNA. HPV is an etiologic agent for a growing subset of HNSCCs, specifically oropharyngeal SCC, according to the study paper. For non-HPV-related cancers, which account for the worldwide majority of head and neck tumors, the study looked for mutations in cancer-related genes that included TP53, PIK3CA, CDKN2A, FBXW7, HRAS and NRAS.
“Our ultimate goal is to develop better screening tests to find head and neck cancers among the general population and improve how we monitor patients with cancer for recurrence of their disease,” says Dr. Bert Vogelstein, the Clayton Professor of Oncology at the Johns Hopkins Kimmel Cancer Center, co-director of the Ludwig Center at Johns Hopkins and a co-author of the study.
According to the study’s paper, follow-up samples of saliva and/or plasma were taken after patients had their tumors surgically removed. In three cases, patients had tumor DNA in their saliva or plasma after surgery, but before clinical evidence of disease recurrence was found. So there is promise in this area that, with regular testing, any tumor recurrence may be caught many months earlier than it might have been previously.
The scientists caution that further study of their tumor DNA detection method in larger groups of patients and healthy people is needed before clinical effectiveness can be determined, and that refinements also may be needed in methods of collecting saliva and the range of cancer-specific genes in the gene test panel. In addition, Agrawal says, “We don’t yet have definitive data on false-positive rates, and won’t until there are more studies of the tests in healthy people.” However, he notes, the formulas used to analyze their blood and saliva tests are designed to weed out questionable results.
