Citation:

Pleil, J., M. Wallace, J. McCord, M. Madden, J. Sobus, AND G. Ferguson. How do cancer-sniffing dogs sort biological samples? Exploring case-control samples with non-targeted LC-Orbitrap, GC-MS, and immunochemistry methods. Journal of Breath Research. Institute of Physics Publishing, Bristol, Uk, 14(1):016006, (2019). https://doi.org/10.1088/1752-7163/ab433a

Impact/Purpose:

There is a lack of useful pre-clinical cancer diagnostic tests, and often cancer is not detected until it is in an advanced stage that is more difficult to treat. The development of sensitive pre-clinical cancer tests that are simple, non-invasive, and cost-effective are needed to improve early cancer diagnosis with the goal of improved prognosis. Dogs are capable of identifying cancer odors on hospital-style masks worn by individuals for 10 min at a time. However, the mechanism by which the dogs sort the masks into cancer/non-cancer categories is not fully understood. Therefore, we investigated whether high-resolution mass spectrometry techniques could be utilized to sort the mask samples into yes/no categories. Ten samples were acquired that the dogs identified as having odors consistent with cancer, and ten samples that were not identified as cancerous by the dogs were also obtained. The samples were extracted for both high-resolution orbitrap mass spectrometry, GC-MS analysis, and immunochemistry analysis. A model developed for the Orbitrap-MS results was able to separate the cancer/non-cancer samples with statistical significance. This work shows that analytical instrumentation can be used to categorize the samples, and that the dogs are likely sorting the masks based on the presence of larger, non-volatile compounds found in exhaled breath aerosol deposited on the masks. This is the first step in developing a future analytical pre-clinical cancer test using mask sampling and high resolution mass spectrometry analysis. These results will be of interest to the general public as well as collaborators at CancerDogs and other pre-clinical cancer researchers.

Description:

Early diagnosis of disease is regarded as the most important factor for successful medical intervention. However, cancer and other long-term latency diseases are rare and may take years to manifest clinically; as such, there are no “gold standards” to immediately validate proposed preclinical diagnoses. Herein, we examine hybrid methodology to gain a better understanding of preclinical bioindicators and their potential value for preserving health. The approach is to use dogs to sort biological samples into “yes/no” categories based on training sets, and then follow-up with detailed chemical studies. There is general consensus that dogs can sort samples reproducibly, but it is not known if they are right, or what they base their decisions on. The first step is to ascertain what the dogs actually detect. Only then could this capability be translated into analytically based diagnostic medical and public health applications. Because the dogs are sniffing air, the distinguishing chemicals must be either in the gas-phase or attached to aerosols and/or airborne particles. As such, the exhaled breath from human subjects suggest a good first choice as the biological medium for health state diagnosis. Recent biomonitoring research has shown how to extract and analyze semi- and non-volatile compounds from human breath in exhaled condensates and aerosols. Further research has shown that exhaled aerosols can be directly collected on standard “hospital-style” paper or propylene masks, and that these masks can be used as a simple sampling scheme for canine diagnostics. In this article, detailed liquid chromatography-high resolution mass spectrometry (LC-HR-MS) with Orbitrap instrumentation and gas chromatography mass spectrometry (GC-MS) analyses are performed on two sets of masks identified unambiguously by four-dog cohorts as falling into either cancer/not cancer categories. Specifically, after sorting by the dogs, sample masks were cut into multiple sections and extracted for LC-MS and GC-MS non-targeted analyses. Extracts were also analyzed for human cytokines to confirm human aerosol content. Preliminary evaluations show that a variety of organic chemicals are differentiated between the dog-sorted groups and that these may ultimately be used to develop metabolic pathways and analytical screening tests for pre-clinical cancers.

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