What is the Best way to Diagnose

Transitional Cell Carcinoma in Dogs

as Early as Possible?
Nathan Baker
Independent Research G/T
April 23, 2018

Advisor: Dr. Shay Bracha

1
 Abstract:

Transitional cell carcinoma (TCC) is a form of bladder cancer in dogs that forms
malignant tumors. Transitional cell carcinoma mostly affects older dogs and at-risk breeds.
Transitional cell carcinoma is a highly aggressive disease and a late diagnosis leads to a low
chance of survival. Unfortunately, TCC is almost always diagnosed in its intermediate-later
stages (Glickman, et al., 2004). Biopsies cannot diagnose TCC in its early stages and the Bladder
Tumor Antigen (BTA) test is not specific enough to diagnose TCC over similar bladder
conditions such as Urinary Tract Infections. It has been discovered that TCC secretes 96 unique
proteins (Bracha, et al.,2014). Identification of these proteins could be used to diagnose TCC
accurately and specifically in its early stages. ​This method has proved to be a viable diagnostic
tool due to its accuracy, specificity, moderate expense, ability to be done in any stage of the
cancer, and its ability to be regularly screened. ​Multiplex protein identification​ is the best way to
diagnose TCC in its early stages.

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 Table of Contents

I. Abstract…………………………………………………………………………………... 2

II. Introduction ……………………………………………………………………………… 4

III. Literature Review ……………………………………………………………………….. 5

IV. Data Collection ………………………………………………………………………… 14

1. Rationale .....……………………………………………………………………………. 14

2. Articles ………………………………………………………………………………… 15

3. Analysis ………………………………………………………………………………… 15

4. Graphs ………………………………………………………………………………….. 16

5. Data Collection Conclusion ……………………………………………………………. 19

V. Conclusion ………………………………………………………………………………….. 19

VI. References ………………………………………………………………………………….. 20

VII. Appendix 1 ………………………………………………………………………………... 22

VIII. Appendix 2 ……………………………………………………………………………….. 22

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 Introduction

​Transitional cell carcinoma is the most common urinary bladder cancer in the dogs

(Knapp, et al., 2013). The increase in carcinogens1 to which dogs are exposed to is causing it to

be a growing problem, “the prevalence of TCC in dogs examined at veterinary teaching hospitals

in North America has increased >600% between 1975 and 1995” (Glickman, et al., (2004).

Transitional cell carcinoma is a highly aggressive disease, which can spread throughout the

layers of the bladder and can metastasize2 to other organs. The ideal time to treat TCC is when it

is in its early stages, before it metastasizes. However, the disease is often diagnosed with the

appearance of clinical symptoms and at a progressive stage. In addition, TCC has similar

symptoms to a Urinary Tract Infection (UTI) and cannot be specifically diagnosed by the

standard diagnostic bench top tests such as the Bladder Tumor Antigen (BTA) test. This puts

veterinarians in a challenging situation, where invasive and expensive tests are needed for further

diagnosis. In recent years, novel technologies emerged allowing the identification of proteins

secreted by TCC, extending our understanding of the disease etiology3 and advancing our

diagnostic options. This paper is to inform the reader about why multiplex protein identification

is a reliable and robust diagnostic approach for diagnosing TCC and why older dogs and at-risk

breeds should be regularly screened for TCC.

1
​a
substance capable of causing cancer in living tissue.
2
​spread
to other sites in the body by metastasis
3
​the cause, set of causes, or manner of causation of a disease or condition
4
 Literature Review

Scottish Terriers, Shetland Sheepdogs, Beagles, West Highland White Terriers, and Wire

Hair Fox Terriers are genetically predisposed to TCC due to their capacity to metabolize certain

carcinogens. Shetland Sheepdogs, Beagles, West Highland White Terriers, and Wire Hair Fox

Terriers are 3-5 times as likely to get TCC, and Scottish Terriers are approximately 18 times as

likely to get TCC (Glickman, et al., 2004). Though some breeds of dogs are genetically

predisposed to TCC, certain carcinogens have been shown to induce the disease in the

predisposed breeds and others. Transitional cell carcinoma can be caused by repeated mutations

from carcinogens. Because TCC is caused by repeated mutations, it can take several years of

exposure to carcinogens for TCC to form. For this reason, TCC affects mainly older dogs “with

o​ver 95% of cases occurring in dogs age 6 years and older” (Breen, et al).

Transitional cell carcinoma is a highly aggressive bladder cancer. Its tumors can invade

the bladder wall and often metastasize to other distant organs (Kay, 2014). Transitional cell

carcinoma can be formed in the kidneys, ureters, urinary bladder, prostate, or urethra. In all dogs,

there are transitional cells normally lining the bladder and if there is an accumulation of critical

mutations they become malignant, causing TCC. The disease in dogs is usually

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intermediate-high grade, meaning it develops and spreads quickly (Knapp, et al., 2013). ​In fact,

at the time of diagnosis over 90% of cases are of intermediate to high-grade invasive TCC

(Patrick, et al., ​2006​).​ Due to late diagnosis and the aggressive nature of the disease, TCC in

dogs is associated with poor prognosis4 (Knapp, et al., 2013). An early diagnosis can greatly

improve the success of treatment. In TCC’s later stages its tumors can invade nearby organs and

can metastasize to distant organs.

The common clinical symptoms of TCC are: ​Hematuria5, Dysuria6, Pollakiuria7,

Stranguria8, and sometimes Tenesmus9, (CriticalCareDVM, 2015). The majority of these

symptoms are not pathognomonic10 and can be evident with UTIs. Symptoms usually appear in

the later stages of TCC, when the tumor is large and/or has metastasized. Dysuria, Pollakiuria,

Tenesmus, and Stranguria can be caused by a mass11 effect.​ ​“Cancerous growth [of TCC] has a

propensity for growing within the trigone region of the bladder” which is the entry point of the

ureters and where the ureter is descended (Kay, 2014). The trigone region is a common location

where masses can accumulate and create a mass effect.

In addition, in rare cases lameness, limb swelling, and lethargy caused by hypertrophic

osteopathy occur in dogs with TCC that has already metastasized. Hypertrophic osteopathy is

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​the likely course of a disease or ailment
5
​blood in the urine
6
​painful urination
7
​frequent urination
8
​straining to urinate
9
​the constant feeling of needing to defecate
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​specifically characteristic or indicative of a particular disease or condition.
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​accumulation of a mass that causes an obstruction
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new bone formation on the outside of the diaphyses12 bones of the limbs and it is ​caused by TCC

metastasizing to the bones or lungs (Withers, et al.,2013).

The three common diagnostic tools analyzed in this paper are cystoscopic biopsies, BTA,

and ​S100A8/A9USG. These tools will be compared to ​identification of TCC’s unique protein

biomarkers through a benchtop assay that will be developed. The benchtop assay will be

developed based on the discriminating proteins identified via the ​liquid chromatography tandem

mass spectrometry ​(LC-MS/MS) approach (Bracha).​ Mass Spectrometry of ​TCC’s unique

protein biomarkers is an investigational method, rather than a diagnostic tool. ​For this reason,

when comparing the advantages and disadvantages of protein biomarker identification as a

diagnostic tool, both theoretical data from the mass spectrometry investigational method and the

characteristics of a benchtop assay that would identify the protein biomarkers are considered.

During cystoscopic endoscopies and biopsy collection, the ​dog is placed under

anesthesia13. Then a fiberoptic scope is inserted through the dog’s urethra. Generally, a rigid

scope is used for females and a flexible scope is used for males. Biopsy forceps are inserted

through the scope and tissue samples are collected. The collected tissues are then processed and

histological14 preparations are assessed by a pathologist to see if the mass is a TCC tumor

(​Procedures for Urinary Disorders​, 2015).

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​the shaft or central part of a long bone.
13
​insensitivity to pain, especially as artificially induced by the administration of gases or the injection of
drugs before surgical operations.
14
l​ ooking at a masses structure under a microscope to identify it
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 The BTA test is an enzyme linked immunosorbent assay (ELISA), upon which urine

samples are placed. The test plate is coated with monoclonal antibodies that detect if

complement factor H-related proteins are present. A color change indicates if these proteins are

present to indicate TCC (Steinberg, et al., 2015).

For the S100A8/A9USG test, urine samples are prepared and put through a mass

spectrometer. Specific proteins are identified based on their mass - charge ratio ​(Han, et al.,

2008). ​The proteins calgranulin A/B or calprotectin and calgranulin C are measured. High

calgranulin A/B or calprotectin to calgranulin C ratios indicate TCC (Heilmann, et al., 2014).

The investigational method of LC-MS/MS identified 96 TCC unique protein biomarkers.

For the research model to identify proteins that could discriminate TCC over UTIs or a healthy

control “​Urine was collected from 12 dogs in three cohorts (healthy, urinary tract infection,

TCC) and analyzed using liquid chromatography tandem mass spectrometry… of the total 379

proteins identified, 96 were unique to the TCC group” (Bracha, et al., 2014). [See Appendix 1]

When specific clusters of these protein biomarkers are identified TCC is indicated [See

Appendix 2]. A benchtop assay should be developed based on the discriminating proteins

identified via the LC-MS/MS approach. The benchtop assay would identify the presence of a

cluster of specific discriminating proteins to indicate the presence of TCC.

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 The most important criteria, as decided upon by the researcher, for the diagnostic tools of

TCC were invasiveness, ability to be tested in any stage of TCC, ability to be regularly screened,

expense, accuracy, and specificity over similar diseases.

Invasiveness is an important aspect to diagnosis because it is associated with higher stage

of disease and poor prognosis. ​The only tool discussed in this paper that is invasive is

cystoscopy. Cystoscopy is somewhat invasive and may cause ​haematuria or a UTI (King, 2018).

Another important aspect of diagnosis of TCC is the stage of progression that the

diagnosis can be completed. The stage of disease is critical for the clinician to assess the

prognosis of the patient and identify an optimal therapy and can be determined by cystoscopy

and advanced imaging (Radiographs, CT, ultrasound). Cystoscopy can be challenging to perform

in the early stages of TCC because the smaller the tumor is, the harder it is to obtain a sample.

Protein biomarker identification, BTA, and the ​S100A8/A9USG test ​can be done anytime in

TCC’s progression as long as urine samples are available, but data is not available on how the

disease’s progression level correlates to the proteins abundance.

Ability to be regularly screened is especially important to TCC because regular screening

could identify TCC in its early stages when it is easier to treat. ​Cystoscopies should not be

performed regularly, due to its invasiveness and expense. It should only be done when symptoms

are present or other screening tests such as the BTA suggest the presence of disease.

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​BTA, Mass Spectrometry, and S100A8/A9USG​ ​can be tested as often as needed if the dog

owner is willing to pay for it due to their non-invasive nature and the fact that they do not need

masses to test.

One of the most important factors in choosing the best tool is the specificity and

accuracy of the test. All data will be explained more thoroughly in the data collection section.

Cystoscopies are on average 80.5% accurate for diagnosing TCC. They are 96% accurate in

females and 65% accurate in males (Childress, et al., 2011). This is somewhat misleading,

because the accuracy is dependent on the quality of the samples collected which can be more

challenging with males due to anatomy of their urinary tract. Any form of biopsy when tissues

are collected successfully is very reliable. For identification of unique protein biomarkers, “a

statistical model, designed to evaluate the accuracy of this multiplex biomarker approach for

diagnosis of TCC, predicted the presence of disease with 90% accuracy” (Bracha, et al., 2014).

The BTA test is 90% accurate; however, the rate of false positive in UTI cases reduces the

accuracy of this test to less than 70% (Henry, et al., 2007). Finally, the S100A8/A9USG test is

96% accurate (Heilmann, et al., 2014).

Specificity in diagnosis means how often the test indicates the disease is not present

when it truly is not, or how often it produces true negative results. Specificity over similar

diseases is important to the diagnosis of TCC in particular because the TCC shares many

symptoms with UTIs, so if a test indicates TCC is present when it is not it creates a lot of

confusion. Cystoscopies do not produce false positives, meaning they are 100% specific. Mass

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Spectrometry for TCC unique proteins is highly specific, >90% because the proteins for TCC are

unique to this condition. The specificity of mass spectrometry relies on methods of sample

collection, sample processing, sensitivity of the mass spectrometer used, algorithm and

parameters for analysis, and proficiency of the operator.​ ​BTA is​ ​78% specific over other

diseases. Hematuria, a common symptom of a UTI, can cause the BTA test to give a false

positive result, because there are ​complement factor H-related ​proteins in the blood (Steinberg, et

al., 2015). ​Centrifugation of the sample separates debris that may cause false positives.

S100A8/A9USG is​ ​66% specific over other diseases because calgranulin A/B or calprotectin to

calgranulin C ratios can often be high in dogs with UTIs and dogs with Haematuria (McNiel, et

al., 2017).

Cost is an important factor in patients and veterinarians choosing a diagnostic test,

especially screening tests which are done regularly for a tight follow up. Some tests can be too

expensive for a patient, so a less effective, but cheaper tool is used or no diagnostic tool is used

at all. A cystoscopy is approximately $1,800 - $1,900 including the cost of anesthetics and

antibiotics (​Estimated Costs of Common Procedures,​ 2016). Sometimes cystoscopies do not

collect the proper tissue and have to be done multiple times, thus increasing the price. BTA is

approximately $16 per test (Simon, et al., 2003). S100A8/A9USG​ ​costs about $260 average

(Duke Center for Genomic and Computational Biology, 2017). The benchtop assay that would

identify TCC’s unique protein biomarkers would be similar to the cost of the BTA test because it

will not require being run through a machine or being sent to a lab. One thing that makes the

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BTA test a very popular screening method is its very low cost, because it is a benchmark assay,

meaning it just needs a urine sample placed on it instead of being run through a machine. While

its sensitivity is high, it has low specificity which makes it a good screening test, but a poor

diagnostic test. If the BTA test shows positive results a cystoscopy is often followed to confirm

the presence of disease. Obviously, an expensive confirmatory test such as cystoscopy required

alongside a screening test needs to be considered when looking at cost.

Detection of the unique protein biomarkers of TCC is the best way to diagnose TCC in its

early stages. This method has proved to be a viable diagnostic tool due to its accuracy,

specificity, moderate expense, ability to be done in any stage of the cancer, and its ability to be

regularly screened. It is 90% accurate (Bracha, et al., 2014), which is tied for the second highest

accuracy among the other tests, only behind S100A8/A9USG (Heilmann, et al., 2014). It is very

specific, which is a very important aspect to the diagnosis of TCC because how similar TCC and

UTI’s are. Detection of protein biomarkers can be done in every stage of TCC so it can be

diagnosed in its early stages, when it is easy to treat. It can be regularly screened, so it can be

diagnosed in its early stages before symptoms appear and it is easy to treat. Finally, it will be

relatively inexpensive to screen regularly because it will be done in a simple benchtop assay such

as an ELISA.

Detection of the unique protein biomarkers of TCC is the best way to screen dogs for

TCC. This method has proved to be a viable tool due to its accuracy, specificity, moderate

expense, ability to be done in any stage of the cancer, and its ability to be regularly screened.

12
with the proteins detected with the LC-MS/MS a future bench top test can be developed to

facilitate a quick, specific and affordable diagnostic test for TCC . Ultimately, this test will most

likely be set as an ELISA detection kit instead of through mass spectrometry, which would make

it much less expensive and available in most animal clinics, and not just in labs. The breeds at

risk and older dogs should have their urine checked regularly for protein biomarkers of TCC so if

it is developed, it can be diagnosed early. Biopsies are still the gold standard for diagnosing TCC

and should follow any positive results of the screening tests. If TCC can be diagnosed early it

may mark the downfall of an epidemic.

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 Data Collection

​Rationale

Transitional Cell Carcinoma (TCC) is a form of bladder cancer that mostly affects older

dogs. Transitional cell carcinoma is the most common cancerous condition affecting the urinary

tract of dogs, and the increase in carcinogens to which dogs are exposed is causing it to be a

growing problem. In addition, TCC has similar symptoms to a Urinary Tract Infection (UTI) and

cannot be specifically diagnosed by the standard diagnostic bench top tests such as the Bladder

Tumor Antigen (BTA). As of now, TCC is almost never discovered in its early stages in dogs, so

a better form of diagnosis needs to be used. New knowledge is emerging on a way to use the

proteins TCC produces to indicate its existence. In summary, the best chance of survival from

TCC involves a early diagnosis so the need for a better diagnostic tool is essential.

The researcher decided to look at the accuracy and specificity of common diagnostic

tools for dogs with TCC versus the accuracy and specificity of the investigational method of

LC-MS/MS of identifying clusters of​ protein biological markers that TCC secretes to predict the

presence of the disease. To do this the researcher looked at four different experiments testing the

accuracy and specificity of each tool. These tools are cystoscopic biopsies, the BTA enzyme

linked immunosorbent assay (ELISA), the ​S100A8/A9USG test, and the protein biomarker

identification method. ​The researcher​ did meta analysis of 4 experiments to see what tool was the

most effective for diagnosing TCC in dogs. ​The researcher​ could not run these experiments

personally, due a to lack of funding, materials, and equipment.

14
Articles
BTA test:
(Henry, et al., 2007)
Cystoscopic Biopsy:
(Childress, et al., 2011)
Mass Spectrometry of TCC
unique proteins:
(Bracha, et al., 2014)
S100A8/A9USG:
(​Heilmann, et al., 2014)
​Analysis
The researcher compiled experiments that tested the accuracy and specificity of the 4
tools previously mentioned. Accuracy is how often the test correctly identifies the presence of
About study
Urine samples from 20
histologically confirmed with
TCC, 19 healthy, and 26
other bladder diseases are
placed on BTA dipstick and
accuracy and specificity are
recorded.
Cystoscopic biopsies
performed on 92
histologically confirmed
canines. Accuracy is
recorded.
Urine from 12 canines per
cohort of healthy, urinary
tract infection, and
histologically confirmed TCC
are prepared for mass
spectrometry and run through
mass spectrometer. Accuracy
is recorded.
Urine samples from 164
canines with urinary tract
issues (including TCC) and
75 healthy canines are run
through mass spectrometer
and ​S100A8/A9USG
comparisons are done.
Findings
Accuracy: 90%
Specificity: 78%
Accuracy: 80.5% (96%
females 65% males)
Specificity: 100%
Accuracy: 90%
Specificity: very high (>90%)
Dependant on ​methods of
sample collection, sample
processing, sensitivity of the
mass spectrometer used,
algorithm and parameters for
analysis, and proficiency of
the operator.
Accuracy: 96%
Specificity: 66%
15
TCC , or a true positive. Specificity is how often the test correctly shows the absence of TCC, or

a true negative. The results are:

(Specificity for Mass Spectrometry is listed as 99%, this may vary)

16
 The results are not very surprising, because the more complex the collection and the more

broad the substance being identified, the less effective the test.

The biopsy is 100% specific, because if no mass or a non-TCC mass is collected it will

not be histologically confirmed. Biopsies are only 80.5% accurate for TCC, due to the fact that

sometimes no mass or a non-TCC mass is collected. This data is misleading because technically

cystoscopy is 100% accurate, but the success rate of propor tissue collection is only 80.5%

accurate. In males, a flexible scope is needed, because a rigid scope cannot be inserted through

their urethra. The flexible scopes make it harder to collect a mass, making the accuracy lower.

This is most likely why cystoscopies in male dogs are only 65% accurate and in females they are

96% accurate.

Mass spectrometry is 90-99% specific, because it identifies TCC unique proteins. The

1-10% margin of error in specificity is dependant on ​methods of sample collection, sample

17
processing, sensitivity of the mass spectrometer used, algorithm and parameters for analysis, and

proficiency of the operator.​ It is 90% accurate. The 10% margin of error in accuracy is dependent

on ​methods of sample collection, sample processing, sensitivity of the mass spectrometer used,

algorithm and parameters for analysis, and proficiency of the operator as well.

The BTA test is 90% accurate, because sometimes the monoclonal antibodies do not

bond with the complement factor H-related proteins due to lower expression of the marker in the

urine​, however the rate of false positive in UTI cases reduces the accuracy of this test to less than

70%.​ BTA test has only 78% specificity, because when blood is present in the urine, as is

common in canines with UTIs or other similar diseases, the proteins in the blood can cause false

positive results.

The ​S100A8/A9USG test has 96% accuracy, because the ratio of calgranulin A/B or

calprotectin to calgranulin C are usually high is dogs with TCC. Specificity is 66%, but it is 75%

specific after excluding dogs with a UTI (McNeil). Calgranulin A/B or calprotectin to

calgranulin C may be high in dogs with haematuria, a common symptom of UTIs, because of the

level of these proteins in the blood.

It should be noted that the data does have some flaws, because the researcher could not

make the procedures personally. For example, the ​S100A8/A9USG and BTA experiments

looked at many conditions where blood was present in the urine and/or glycoprotein

concentration was expected to be high, but the mass spectrometry of TCC unique proteins looked

only at UTIs. In addition, the urine collection process and urine processes could be slightly

different between the studies for mass spectrometry studies. These flaws may have slightly

changed the data, but the researcher still thinks the results would be similar enough to prove

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protein biomarker identification is an effective diagnostic tool. The biopsy study is somewhat

misleading because histological confirmations are nearly perfect, but if the mass is not gathered

properly the biopsy must be repeated. This is why the accuracy for cystoscopic biopsies was

seemingly low, even though it is a very reliable confirmatory test.

Data Collection Conclusion

Protein Identification of TCC’s unique protein biomarkers has proven itself as an

accurate and specific screening test for TCC. With >90% specificity, it is the second most

specific tool out of the 4 tools analyzed, only behind cystoscopy. With 90% accuracy, it is tied

for the second most accurate tool out of the 4 tools analyzed. In conclusion, the data supports the

fact that protein identification of TCC’s unique protein biomarkers is both an accurate and

specific tool for diagnosing TCC. Due to it’s high accuracy and specificity it is a great screening

and diagnostic test. However, it’s accuracy and specificity are not as high as cystoscopy with

propor tissue collection so cystoscopy is still a superior confirmatory test.

Conclusion

There is significant evidence to routinely use TCC unique protein biomarker

identification along with other confirmatory tests like cystoscopic biopsies. Not only does the

mass spectrometry of TCC unique protein biomarkers have a very high specificity (>90%) and

the second highest accuracy (90%), it also has other benefits. The most significant benefits are

the moderate expense, ability to be done in any stage of TCC, the non-invasive process, and the

option for regular screening. A benchtop test should be developed based on the discriminating

proteins identified via the LC-MS/MS approach so older and at-risk breeds can be regularly

screened for TCC.

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Appendix 1

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4874471/figure/F2/

(Bracha, et al., 2014)

Appendix 2

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4874471/table/T1/?report=objectonly

(Bracha, et al., 2014)

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