Scientific Paper Critique

Article: Canine scent detection in the diagnosis of lung cancer: revisiting a puzzling phenomenon

Authors: R. Ehmann, E. Boedeker, U. Friedrich, J. Sagert, J. Dippon, G. Friedel and T. Walles

 Introduction

           

Lung cancer is the second most common cancer and the most common cause of death from cancer. While early detection is key, issues arise in that its early stages are asymptomatic and there is currently no screening method. As well, modern techniques are not ideal for discriminating between healthy and lung cancer patients and so a better method of detection, preferably one that can detect the cancer early on is desired.

Most cancers are associated with volatile organic compounds (VOC's). These are compounds that patients breath out during the early stages of cancer. “Electronic noses” are the newest technology used to detect these however there are limitations to this procedure including very sensitive instruments, the patients cannot smoke or eat before the procedure, it takes a long time to get results, etc. As well, there have been no VOC’s identified for lung cancer.

Methods

           

The researchers obtained ethical permission and collected breath samples of healthy individuals (Group A), lung cancer patients (Group B) and chronic obstructive pulmonary disease patients (Group C) from volunteers who provided signed consent. Their medical history, including medications they were taking, was recorded to control for any confounding variables. The breath samples were taken by the volunteer breathing into a cylindrical glass tube, lined with polypropylene fleece, five times. The tube was sealed and handled by all individuals to avoid “scent labeling”.

            

Four house dogs were trained in a separate room dedicated solely to said task and were taught to lie on the floor with their nose on the tube when cancer was detected.

            

Three tests were performed within two days:

1)   Identifying healthy controls from lung cancer patients

2)   Identifying lung cancer from COPD

3)   A mixed study of COPD and healthy patients

Two people observed the dogs and were blinded to avoid bias. Following this a variety of statistical tests on the data were performed.

Results:

           

The official number of volunteers were 110 healthy individuals, 60 lung cancer patients and 50 COPD patients. The researchers found that the overall sensitivity of the dogs detection was 71% and the specificity was 93%. The dogs had the greatest success in the mixed study (COPD and healthy patients) and had the least success in identifying lung cancer versus healthy controls. They also discovered that the dogs were not more accurate in identifying advanced stages tumors and in fact the highest accuracy was for stage I tumors (100%) and the lowest for stage VI (63%). From this data they concluded that there must be some kind of stable, detectable marker found in the breath of lung cancer patients which is independent from that of COPD. As well, this marker must be present in the initial stages of lung cancer and thus it may prove to be a good, non-invasive screening test for lung cancer. Further work needs to be done to determine what specific compound this VOC is.

Personal Critique:

            I found this article to be very interesting. It is amazing to think that dogs have such an acute sense of smell to be able to detect cancer and that they may be used in medicine.

            

The authors did a fantastic job, in my opinion, of explaining their research and findings. Step by step descriptions of each aspect of the experiment was given- from the initial collection of patients, to training the dogs, analyzing the findings, accounting for bias and confounding variables and discussing the results and their implications. They also included many headings to divide the article into sections which made the experiment easy to follow and understand.

           

I found the researchers to be very thorough in their experiment and it seems as though they tried to account for all possible variables. They were sure to obtain signed consent from the patients and collected their medical histories and medications to prevent confounding variables. 

They then performed statistical tests on any possible confounders to determine if they influenced the results.  In addition, they handled their instruments with care- being sure not to introduce any outside scents that may affect the results.  They used multiple statistical models to evaluate their results (these could have been explained in more detail) to ensure accuracy. Their results were then presented clearly- both in words, figures and tables- and conclusions clearly drawn. I felt as though all the necessary information and details were included to give a comprehensive picture of the experiment.

One thing that I believe could have been improved would be to obtain patients from different areas. The researchers used volunteers from only two facilities in Germany so the questions of generalizability and bias can be raised.

Overall I found this article very informative, well conducted and written. It has a very wide audience since lung cancer is such a common disease and I believe it was written in such a way that many people could read and understand it. A very important aspect of this article is that it provides direction for future research- research must now be conducted to determine what the specific VOC found in lung cancer patients is. 

Article Citation:

Ehmann, R., Boedeker, E., Friedrich, U., Sagert, J., Dippon, J., Friedel, G., & Walles, T. (2012). Canine scent detection in the diagnosis of lung cancer: revisiting a puzzling phenomenon. European Respiratory Journal, 39(3), 669-676.

This blog was designed as an assignment for the BIOL 3500 course at Memorial University of Newfoundland.

Fig 1

It’s no secret that dogs have an excellent sense of smell! It's what makes them a great asset to police forces, hunters, search and rescue teams and now-a-days even scientists researching cancer and bacteria (as you will see in this blog). 

But have you ever wondered why they have this amazing sense? What makes them different from other animals and even humans? This blog aims to answer these questions and present the science behind the common knowledge (with some fun facts thrown in for good measure). 

Fig 2

Let's start at the beginning… how does a dog breathe?

Air takes a different journey through the dog nose than it would traveling through the human nose and many of these differences account for the dog’s superior sense of smell.

To fully understand the  anatomy of the dog nose, one must realize that inhaling air is not just important for identifying odors, but also important in respiration to obtain oxygen from the environment and release carbon dioxide back out. Thus, the anatomy of the dog nose is specialized to maximize both of these functions. (Lawson et al. 2012)

The dog nose has two main passages that are separated by the lamina transversa. One of these leads to the lungs for gas exchange while the other goes to the olfactory area.  This is the  first  difference we see from  humans who have only one passage for both functions with the olfactory epithelia found on the roof of the nose. (Craven et al. 2009)

Firstly, the air surrounding the nose enters the nasal vestibule and gets mixed up to ensure that the olfactory epithelia gets a “representative sample” containing all the different chemicals in the air. The air splits here and travels down each passageway.

Fig 3: The flow of air through the dog nose.
(Lawson et al. 2012)

The respiratory pathway is the least complicated of the two. The air leaves the nasal vestibule, goes through the maxilloturbinate to the nasopharynx then the lower respiratory tract. (Lawson et al. 2012.) As it travels this route the air gets warmed/cooled, moistened and filtered. (Craven et al. 2007.

The more complicated pathway is the olfactory pathway. The air entering this pathway leaves the nasal vestibule and travels through the dorsal meatus which leads to the olfactory region. Travelling through this airway, the air bypasses the maxilloturbinates and instead goes to the olfactory recess, turns 180° and goes forward through the ethmoturbinates and then on to the nasopharynx (Lawson et al. 2012). Since the ethmoturbinates have a scroll-like appearance, they provide a large surface area for odors to be absorbed (Craven 2009) and the air spends more time in this region than any of the other turbinates (Lawson et al. 2012).

Histology of the Dog Nose:
(Craven et al. 2007)

There are 4 main types of epithelium in the nose!

1) Simple squamous found in the nasal vestibule

2) Respiratory pseudo-stratified columnar epithelium in the maxilloturbinate and frontal sinuses

3) Olfactory pseudo- stratified columnar epithelium (contains olfactory cells) in the ethmoturbinates

4) Transitional epithelium in the region of the posterior nasal vestibule to the anterior maxilloturbinate

* If you notice, the nasal vestibule has simple squamous epithelium and the maxilloturbinates, which it leads into, is covered in respiratory pseudo-stratified columnar epithelium. The transitional epithelium thus allows the switch between the two.

Fig 4: Olfactory and respiratory epithelium
(Lawson et al. 2012)

As well, most of the interior of the nose is covered in a mucous secretion which serves many purposes such as removing odorous molecules, aiding in heat transfer and keeping the cilia alive, just to name a few. 

What is the difference between respiratory and olfactory epithelium?
(Craven et al. 2007)

It is hard to distinguish where the respiratory epithelium ends and the olfactory epithelium begins as there seems to be some olfactory cells mixed in amongst the non-sensory cells of the respiratory epithelium. 

Two of the main functions of the respiratory epithelium - filtration and warming/cooling - are accomplished by a couple of structural features. Firstly, the lamina propria (connective tissue) found underneath the epithelial layer contains an abundance of blood vessels which can dilate and constrict to aid in warming and cooling respectively. Secondly, filtration is aided by the motile cilia projecting from the surface. One note worth mentioning is that the respiratory epithelium does not participate in olfaction.

Fig 5: Olfactory and Respiratory Epithelium
(Lawson et al. 2012)

Olfactory epithelium differs from respiratory epithelium in a couple of ways. Although it also has a layer of lamina propria underneath its epithelial layer, unlike that of the respiratory epithelium it is not vascularized. It also has cilia but they also differ. These cilia are non-motile and actually extend from the dendrites of the olfactory receptor cells.

How does the olfactory epithelium detect scents?

At the surface of the olfactory epithelium lies many olfactory receptor neurons (ORN's) to which odors bind and are recognized. However, these are specific in that only certain odors bind to certain ORN’s. Dividing them into two main categories makes it easier to predict what odors bind to which receptor. Type 1 ORN’S bind hydrophilic molecules- those that are soluble in the mucous membrane of the nasal cavity while Type 2 bind hydrophobic particles. (Lawson et al. 2012).

As well, not only do dogs have more genes dedicated to encoding for olfactory receptors than humans (1,300 versus the 650-900 for humans) (Quignon et al. 2005) but a larger percentage of them are true genes (Quignon et al. 2003). For canines, 18% of these are pseudogenes compared to the 63% for humans!

What about sniffing?

Fig 6

I’m sure you’ve all seen a dog sniffing when trying to locate something, when it encounters something new or it’s favorite food, etc. But does sniffing really enhance olfaction? Yes! A dog will move its nose independently to sniff, dilate its nostrils, breathe more frequent and have higher air flow rates than during normal respiration. Seems obvious this would increase olfaction, right? But something else quite interesting happens when a dog sniffs which increases sensitivity. (Craven et al. 2009). The mechanism of sniffing actually opens a direct passage in the nose to the dorsal metus which leads straight to the olfactory region. Here, the odours concentrate and interact with the receptors on the olfactory epithelium (Craven 2007).

Just how much better smellers are dogs than humans?

(PBS Article)

*Their nose contains up to 300 million olfactory receptors in their nose compared to our ~6 million

*They can detect odor concentrations at 1-2 parts per trillion (10 000- 100 000 times that of humans)

*The area of their brain devoted to smells is about 40 times greater than ours when looked at in proportion

The Vomeronasal Organ
(PBS Article) 

Fig 7: Vomeronasal Organ

Dogs possess a vomeronasal organ (Jacobson’s organ)- located on the bottom of the nasal passage (above the roof of the mouth, behind the upper incisors) devoted to identifying pheromones. Nerves lead from here to the brain. 

NEW SCIENCE! 

Detecting Bacteria

            The canine’s sense of smell is so astonishing that they can be trained to detect bacteria! In one study, a 2 year old beagle was trained to sit or lie down when it detected Clostridium difficle in both stool samples and patients. The dogs sensitivity and specificity was both 100% in the stool samples and it correctly identified 25 of the 30 cases of patients with the bacteria and 365 out of 370 controls (Bomers et al. 2011). 

Detecting Cancer

Dogs are now being trained to detect cancers in humans! In one study a Labrador Retriever was trained to detect colorectal cancer in exhaled breath and stool samples. In the breath samples there was a 0.91 sensitivity and 0.99 specificity. In the stool samples, there was sensitivity of 0.97 and specificity of 0.99. This supports the claim that there actually are certain specific compounds present in the body of those with cancer (Sonoda et al. 2011).

Bloodhounds!

Fig 8

 The bloodhound has long flappy ears that sweep the odors up towards its nose making it a more efficient tracker! 

And to end, here's a picture of my dog, Milo ...

References:

Bomers, M. K., van Agtmael, M. A., Luik, H., van Veen, M. C., Vandenbroucke-Grauls, C. M., & Smulders, Y. M. (2011). Using a dog's superior olfactory sensitivity to identify Clostridium difficile in stools and patients: proof of principle study. BMJ (Clinical research ed.), 345, e7396-e7396.

Craven, B. A., Neuberger, T., Paterson, E. G., Webb, A. G., Josephson, E. M., Morrison, E. E., & Settles, G. S. (2007). Reconstruction and morphometric analysis of the nasal airway of the dog (Canis familiaris) and implications regarding olfactory airflow. The Anatomical Record, 290(11), 1325-1340.

Craven, B. A., Paterson, E. G., & Settles, G. S. (2009). The fluid dynamics of canine olfaction: unique nasal airflow patterns as an explanation of macrosmia. Journal of The Royal Society Interface, 7(47), 933-943.

Lawson, M. J., Craven, B. A., Paterson, E. G., & Settles, G. S. (2012). A Computational Study of Odorant Transport and Deposition in the Canine Nasal Cavity: Implications for Olfaction. Chemical senses, 37(6), 553-566.

Sonoda, H., Kohnoe, S., Yamazato, T., Satoh, Y., Morizono, G., Shikata, K., ... & Maehara, Y. (2011). Colorectal cancer screening with odour material by canine scent detection. Gut, 60(6), 814-819.

Quignon, P., Giraud, M., Rimbault, M., Lavigne, P., Tacher, S., Morin, E., ... & Galibert, F. (2005). The dog and rat olfactory receptor repertoires. Genome biology, 6(10), R83.

Quignon, P., Kirkness, E., Cadieu, E., Touleimat, N., Guyon, R., Renier, C., ... & Galibert, F. (2003). Comparison of the canine and human olfactory receptor gene repertoires. Genome biology, 4(12), R80-R80.

http://www.pbs.org/wgbh/nova/nature/dogs-sense-of-smell.html