Molecular Pathology Selected Abstracts

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Editors: Donna E. Hansel, MD, PhD, chair of pathology, Oregon Health and Science University, Portland; Richard D. Press, MD, PhD, professor and director of molecular pathology, OHSU; James Solomon, MD, PhD, assistant professor, Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York; Sounak Gupta, MBBS, PhD, senior associate consultant, Mayo Clinic, Rochester, Minn.; Tauangtham Anekpuritanang, MD, molecular pathology fellow, Department of Pathology, OHSU; Hassan Ghani, MD, molecular genetic pathology fellow, Department of Pathology, OHSU; and Fei Yang, MD, assistant professor, Department of Pathology, OHSU. 

Potential of mitigating bottlenecks in COVID-19 testing by spitting into a cup

June 2020—The COVID-19 pandemic has focused the world’s attention on using sensitive high-throughput molecular diagnostic testing for the SARS-CoV-2 virus as a public health tool for “flattening the curve” of the infection. Although initial shortages of specialized polymerase chain reaction (PCR) testing reagents that plagued the early weeks of the pandemic have slowly improved (as of CAP TODAY press time), an obstacle to universal testing continues to be the first-step bottleneck of collecting respiratory tract samples for virus-specific reverse transcriptase-PCR (RT-PCR) testing. The traditional gold standard sample for COVID-19 testing has been a nasopharyngeal (NP) swab. However, nationwide shortages of NP swabs, personal protective equipment (PPE), and viral transport media have intermittently delayed the testing process. In an attempt to alleviate these critical sample-collection issues and promote more widespread testing, the medical community and other entities have been investigating alternative sample-collection procedures. The use of saliva as a routine viral testing sample is particularly promising because it can be painlessly self-collected by patients in the safety of their homes without potentially exposing health care providers and others to the virus. It also frees busy health care workers from the task of collecting samples and from consuming precious PPE, swabs, or viral transport media. A Yale University research team has shown that saliva is a more sensitive and consistent type of sample for COVID-19 testing compared with standard NP swab samples. This finding may support the use of saliva in large-scale screening efforts. The Yale investigators found that in 38 patient-matched NP swab and saliva samples from SARS-CoV-2–infected hospital inpatients with severe disease, the viral load in the saliva samples was higher by an average of fivefold than that in the NP swabs collected concomitantly. In addition, eight (21 percent) of the patient-matched samples collected concomitantly were positive in the saliva but negative in the NP swabs, while three (eight percent) of the samples were negative in the saliva but positive in the NP swabs. Moreover, when studying samples collected serially, whereby the viral load typically declines after the initial COVID-19 diagnosis, the authors found five instances in which a negative NP swab sample was subsequently followed by a positive saliva test result, indicating a likely false-negative initial NP swab result. No such instance of serial negative to positive result generation was observed with saliva samples, suggesting that not only are saliva samples more analytically sensitive than NP swabs, but they are more consistent. To confirm this increased analytic sensitivity, the authors also collected serial samples every three days from 98 asymptomatic health care workers at moderate- to high-risk of COVID-19 exposure. Two of the health care workers had detectable SARS-CoV-2 virus (low titer) in a saliva sample but not in an NP swab sample. The saliva samples in this study were self-collected by patients spitting into a sterile cup. This important convenience factor for collecting saliva samples will likely be a key driver of the medical community’s ability to offer rapid, sensitive high-throughput COVID-19 testing to a larger fraction of the population than is possible using NP swabs.

Wyllie AL, Fournier J, Casanovas-Massana A, et al. Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs. Preprint at Med­Rxiv. Posted online April 22, 2020. doi:10.1101/2020.04.16.20067835.

Correspondence: Dr. Anne Louise Wyllie at anne.wyllie@yale.edu, or Dr. Nathan Grubaugh at nathan.grubaugh@yale.edu, or Dr. Albert Ko at albert.ko@yale.edu

Feasibility of a blood-based multi-cancer circulating tumor DNA screening test

The holy grail of population-based cancer screening is a blood-based test that can detect cancers at multiple sites in the early stages. A significant step toward this goal has been realized with publication of the DETECT-A (Detecting Cancers Earlier Through Elective Mutation-based Blood Collection and Testing) study conducted by a team of investigators at Johns Hopkins University. The prospective interventional study, the largest liquid biopsy study of an asymptomatic screening population, evaluated 10,000 women between the ages of 65 and 75, who had no known history of cancer, using a previously validated blood test. The blood test was designed to detect cancer-specific mutations in cell-free DNA released into the circulation from cancer cells located at heterogeneous sites throughout the body. The test targets DNA mutations from 16 genes and nine protein biomarkers to screen for the early presence of multiple types of cancer, with the exception of cancers of the skin and central nervous system and leukemias. To increase specificity in this study, if the initial screening blood test detected a possible circulating tumor marker, a confirmatory second blood test was performed. If this test was also positive, a PET-CT (positron emission tomography-computed tomography) imaging study was offered to confirm the cancer diagnosis. Ninety-six of the 9,911 asymptomatic women who were screened in the study were found to have cancer after one year of follow-up. The new blood test detected 26 of these cancers in 10 different organs. Twenty-four additional cancers—20 breast, three lung, and one colon—were detected through conventional screening methods, and 46 were not detected through either approach. Among the 26 cancers detected by the new blood test were cancers of the lung (nine), ovary (six), colon (two), uterus (two), breast (one), thyroid (one), kidney (one), and appendix (one), as well as lymphoma (two) and carcinoma of unknown primary (one). Seven of these cancer sites—all except breast, colon, and lung—lack approved screening tests and, therefore, often have high mortality rates as they are less often diagnosed at an early stage. Seventeen of the 26 women whose cancers were identified through blood tests had early stage disease that was local or regional. Most of the 26 had surgery with intent to cure or underwent other treatment. Twelve of them went into remission, suggesting that they benefited from the screening blood test. The combined sensitivity of the new circulating tumor DNA blood test plus standard screening was 52 percent, compared with 25 percent sensitivity for standard screening alone. However, the significant number of cancers not identified with the blood test suggests that it will not be used as a sole screening method but as a complement to established screening methods. As with most screening tests, this new multi-cancer blood test was falsely positive in 101 women in whom subsequent imaging failed to confirm a cancer diagnosis (specificity, 98.9 percent). Twenty-two of the women with a false-positive blood screen underwent an invasive test such as endoscopy. Longer-term follow up of this test will be required to critically analyze its possible benefits to overall survival. Nevertheless, this important proof-of-principle study has confirmed that a novel multicancer liquid biopsy screening blood test is safe and feasible. Therefore, it may become an important tool to assist in detecting many cancers at an earlier stage.