Spit-and-Stick Test Could Be Biomarker for HNSCC

Spit-and-Stick Test Could Be Biomarker for HNSCC

Neil Osterweil

July 01, 2015

It's early days yet, but a simple test that looks for tumor DNA shed in saliva and plasma could serve as a biomarker and screening tool for head and neck squamous cell carcinoma (HNSCC), reports a team of investigators.

Somatic mutations or human papillomavirus (HPV) — collectively referred to as tumor DNA — were identified in saliva samples from 71 of 93 patients with HNSCC (76%) and in the blood of 41 of 47 (87%) of those who also provided plasma samples.

Tumor DNA was detected in at least one of the samples of 45 of the 47 patients (96%) who provided both plasma and saliva samples.

The study, by Nishant Agrawal, MD, from the Johns Hopkins University School of Medicine in Baltimore, and colleagues, was published online June 24 in Science Translational Medicine.

"Going forward, what we're most excited about is trying to identify premalignant changes and high-risk changes as a screening tool. We may be able to do that in the very near future, possibly after deciding the false-positive rate among normal patients," Dr Agrawal said in an interview with Medscape Medical News.

This study was not designed to evaluate the test as a surveillance tool. However, the nine patients with pretreatment samples positive for tumor DNA who had samples collected at various times after surgery were assessed. In the five patients with no tumor recurrence, no further mutations could be identified, suggesting that the mutation-based assay is highly specific.

"It was also encouraging that we identified tumor DNA in the saliva of patients whose tumors were found to recur at the clinical level only months later, suggesting that these tests could provide a clinically meaningful lead time," the investigators write.

Dr Agrawal said that the proof-of-principle study yielded encouraging results, but that more work needs to be done before a simple spit-and-stick test is ready for the clinic.

"We can definitely improve the performance, maybe by varying the saliva collection methodology, possibly increasing or changing the volume of both saliva and plasma. Going forward, we're going to use a panel of genes and continue to study HPV," he said.

The investigators also plan to validate the test in large numbers of individuals without known cancers to rule out false-positive and false-negative results and establish the actual sensitivity and specificity of the assay, he reported.

Assay Details

Recent advances in technology have allowed the detection of mutations found only in small concentrations in bodily fluids, which prompted the investigators to study whether easily acquired samples could yield diagnostic and prognostic information.

The patients in the study were a small but representative sample of HNSCC patients; mean age was 60 years and 83% of the patients were male. Twenty patients had stage I or II disease and the remaining 93 had stage III or IV (advanced) disease.

The investigators attempted to identify at least one genetic alteration in each tumor, starting with HPV type 16 or type 18 sequences. They found HPV 16 DNA, but not HPV 18 DNA, in 30 tumors,. In the remaining 63 tumors, they looked for somatic mutations in genes or in gene regions that are associated with cancer, and found driver mutations in 58. For the remaining five samples, they performed genome-wide sequencing and found at least one genetic alteration of interest in each of the tumors.

They then looked for tumor DNA in saliva collected with oral rinses and in plasma. As already noted, they found tumor DNA in 76% of saliva samples and in 87% of plasma samples. And it was found in 96% of samples from patients who provided both saliva and plasma samples.

In the 47 patients who provided plasma samples, 21 had tumors positive for HPV. Of this subgroup, 18 had detectable HPV DNA in their plasma and/or saliva.

"Because HPV 16 is rarely found in oral specimens of healthy individuals, we analyzed 10 saliva or plasma samples from patients whose tumors were not HPV-positive as controls. As expected, no HPV was detected in any of these samples, confirming the specificity of the test," they write.

In the 26 of 47 patients whose tumors were HPV-negative, all had endogenous DNA mutations in either saliva or plasma, primarily in TP53, indicating that both somatic mutations and HPV sequences are useful as biomarkers for malignancy.

Of the 46 patients with cancers of the oral cavity, all had detectable tumor DNA in saliva, but the sensitivity of the test was considerably lower in patients with cancers not reached by the oral rinse used to collect saliva samples. The sensitivity was only 47% in patients with oropharyngeal cancers, 70% in those with laryngeal cancers, and 67% in those with hypopharyngeal cancers. For these patients, plasma DNA sensitivity was lower than saliva sensitivity for cancers of the oral cavity (80% vs 100%), but was more sensitive than saliva for oropharyngeal cancers (91%), laryngeal cancers (86%), and hypopharyngeal cancers (100%).

Saliva was better than plasma for identifying early-stage disease (20 of 20 cases vs 7 of 10 cases). In contrast, plasma was better than saliva for identifying advanced cancers (34 of 37 cases vs 51 of 73 cases).

Plasma was also better than saliva for detecting HPV-positive tumors, probably because most HPV-related HNSCCs occur in the oropharynx, which is not easily reached by oral rinses.

The study was supported by the Virginia and D.K. Ludwig Fund for Cancer Research, the Conrad R. Hilton Foundation, the Banyan Gate Foundation, Swim Across America, the Sol Goldman Sequencing Facility at Johns Hopkins, and the National Institutes of Health. Dr Agrawal has disclosed no relevant financial relationships. Several of his coauthors at Johns Hopkins may receive a portion of the royalties awarded to the university for the sale of products related to genes and technologies described in the study.

Sci Transl Med. Published online June 24, 2015. Abstract

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