Accuracy of Canine Scent Detection of Non–Small Cell Lung Cancer in Blood Serum

Dogs

Four 2-year-old beagles were trained for the study. None of the beagles had been previously trained to detect lung cancer samples. The dogs were selected because of their impressive olfactory system and because they are also relatively small, calm, highly trainable, and highly sociable.¹² One dog, Canine No. 4, was eliminated from the study because she was poorly motivated by food and did not respond well to any training method used.

Samples

The serum samples of patients with newly diagnosed lung cancer were purchased from Boston BioSource. The serum was selected because of its commercial availability and confidence level that the serum was from patients who recently received a new diagnosis of NSCLC but had not started any treatment. The positive samples were collected from patients before surgery and chemotherapy. Control samples were obtained from healthy male and female donors through Boston BioSource. Data regarding sex, age, and race/ethnicity were collected on all donors to minimize confounding factors, but the smoking status of donors was unknown.

Non–small cell lung cancer was chosen for this study because it is more common and easier to treat if detected early, as opposed to small cell lung cancer, which is more aggressive. The samples were identified with an identification number, logged into a computer, and stored in a freezer at −30°C for 1 week before first use. Prior to training, each sample was stored in 2 mL of Cryovial (Thomas Scientific), freestanding polypropylene vials with external threaded caps. Researchers used 250 μL of each blood serum sample for training. During the training phase, the samples were thawed for 15 minutes, and the vials were placed in new 12-mL syringe cases. The syringes with the samples were placed in metal canisters with multiple perforations, and the canisters with the cancer and control samples were evenly placed at the dogs’ eye level. The dogs went from sample to sample and were given the opportunity to smell each sample individually (Figure 1).

Figure 1.

The blood serum samples were placed in metal canisters with multiple perforations and positioned at the dog's eye level.

Dog Training

Blood serum samples from patients with newly diagnosed NSCLC and matching controls were used to train and test each dog. Operant conditioning, specifically the “clicker-training method,” was used to train the dogs to positively identify samples from patients with lung cancer in the presence of control samples. During training and testing, the dogs were followed by a handler as they evaluated 5 evenly spaced canisters. Four canisters contained a control sample and 1 canister contained a positive lung cancer serum sample. Dogs were permitted to take as much time as they needed to evaluate each sample. The dog indicated a correct response by sitting in front of the canister. A correct response to a control sample was given by ignoring the sample and continuing on to the next canister. If the dogs sat in front of a control sample, they were told “no” and encouraged to move on to the next sample. If the dogs correctly indicated a positive serum sample, they were rewarded with the sound of a clicker and a training treat.

Training was done in 3 phases over 8 weeks. Phase 1 took place during the first 2 weeks, and the dogs were trained to correctly identify the presence of a treat in a canister. Phase 2 took place during the second 2 weeks of training, and a positive serum sample containing lung cancer was paired with a treat. As the dogs successfully identified the station with the positive sample and the treat, the amount of treat was weaned down until the dog was able to successfully identify the positive sample in the absence of the treat. Phase 3 took place during the final 4 weeks of training, and no treat was paired with the positive lung cancer serum samples. After successful completion of 3 training phases, the testing phase began.

Testing Design

A double-blind procedure was used to minimize the possibility of experimenter bias. The samples were prepared the same way as the training samples. During the testing phase, 10 new samples were used that were positive for NSCLC and had not been encountered during training. Additionally, the control samples that were used in the testing phase came from different donors from the control samples in the training phase. Each dog performed 10 test runs, and each test run was set up like the training runs. The dogs were allowed to sniff each canister for 5 to 10 seconds. A positive response was indicated by the dog sitting in front of the canister. A correct response to control samples was to ignore the sample and to move on to the next sample. All of the testing was performed on the same day. A 10-minute rest period occurred each time the samples were changed out, which allowed the odor from the previous test to dissipate and the scent from the new samples to accumulate in the area.

Statistical Analysis

The P value was calculated using the Easy Fisher Exact Test calculator (Social Science Statistics) and a 2 × 2 table setting the statistically significant value at P=.05. All other statistics, meaning sensitivity/specificity and positive/negative predictive values, were calculated using MedCalc (MedCalc Software).

Limitations

This is a pilot study with a relatively low sample size; after 1 dog was eliminated for poor response to training, there were only 30 positive and 120 negative samples used. Although the P value of the data was significant, additional studies are needed to verify the efficacy of the data. Another limitation was the narrow scope of the study that only determined whether the dogs could detect the cancer. Future studies are needed to determine whether dogs can detect cancer at an earlier stage than the current methods of chest x-ray and CT imaging. Studies should also determine whether dogs can detect recurrence of lung cancer while patients are undergoing chemotherapy and whether they can detect cancer in the presence of concurrent diseases such as diabetes. Larger and more focused studies are needed to expand on the preliminary findings of this study.

Future Research

At the time of publication, our team was in the early stages of conducting a larger study that used canine scent detection to isolate and identify the biomarkers in positive NSCLC and breast cancer samples. We plan to fractionate the cancer samples based on chemical and physical properties and then present the fractions to the trained dogs for evaluation. The fractions that the dogs identify as containing cancer biomarkers will be fractionated further and presented to the dogs again. Once sufficiently fractionated, the resulting fractions will be evaluated using liquid chromatography–tandem mass spectrometry to determine the identity of the molecules present. The end goal of this larger study is to identify the minimum number of biomarkers necessary in a sample for a positive identification of cancer. Once identified, biomedical researchers can work toward developing a simplified and cost-effective, non–canine scent detection system.

Canine No. 1 has been bred with a beagle who has been equally impressive in scent detection of breast cancer, and the resulting litter of 6 puppies are now being trained in an effort to produce a genetic line predisposed to scent detection of cancer. At a later time, these same dogs, will be trained to detect lung cancer in exhaled breath condensate and saliva from donors.