# Blood biomarkers for canine cancer, from human to veterinary oncology

Philippe Colombe, Jérémy Béguin, Ghita Benchekroun, Delphine Le Roux

► To cite this version:

Philippe Colombe, Jérémy Béguin, Ghita Benchekroun, Delphine Le Roux. Blood biomarkers for canine cancer, from human to veterinary oncology. Veterinary and Comparative Oncology, 2022, 20 (4), pp.767-777. 10.1111/vco.12848. hal-04011686

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REVIEW

Veterinary and
Comparative Oncology

WILEY

# Blood biomarkers for canine cancer, from human to veterinary oncology

Philippe Colombe<sup>1,2</sup> | Jérémy Béguin<sup>2,3</sup> | Ghita Benchekroun<sup>2,4</sup> |
Delphine Le Roux<sup>1,5 ID</sup>

Correspondence

Delphine Le Roux, Ecole Nationale Vétérinaire d'Alfort, 7 avenue du Général de Gaulle, 94700 Maisons-Alfort, France.

Email: delphine.le-roux@vet-alfort.fr

Abstract

In recent decades, interest in circulating tumour biomarkers is increasing both in human and veterinary oncology. An ideal tumour biomarker would allow early diagnosis of neoplasia, identify it specifically, accurately, establish a prognosis and predict its behaviour, especially regarding different therapeutic solutions. It would also allow to monitor its evolution over time and all this in a non-invasive and inexpensive way. Actually, no biomarkers meeting all of these criteria have been identified in veterinary medicine, particularly due to a lack of specificity of the main protein tumour biomarkers studied to date. However, great hope is currently placed in biomarkers grouped under the name of liquid biopsy, which could prove to be effective tools for common clinical use in the near future. This review gives an update on blood cancer biomarkers studied in dogs, such as ions, proteins, nucleic acids and also circulating cells, of which some might become more prominent in the coming years to help improve the management of animal care.

KEYWORDS

biomarker, cancer, liquid biopsy, veterinary

# 1 | INTRODUCTION

The use of biomarkers represents a major challenge in the field of oncology in both human and veterinary medicine. The growing knowledge of biomarkers over the last two decades has enabled better management of patients with tumours. Ideally a tumour biomarker would be easy to measure, should have perfect sensitivity and specificity, with the aim of perfectly differentiating individuals suffering from a neoplastic process from healthy individuals, and at the earliest possible stages. It should also be able to identify a subject affected by any tumour process, allow monitoring of the evolution of cancer over time and forecasting of relapses or recurrences.<fnref n="1" />

The vascular compartment is easily accessible, so blood is a sample of choice to identify and measure tumour biomarkers. Indeed, the

sampling procedure is minimally invasive and the measurement of one (or more) component is often quick. In contrast, tumour biopsies are more invasive and often take longer to analyse and interpret. Moreover, in veterinary medicine, tumour biopsies, by requiring sedation or general anaesthesia, increase the owner's expenses as well as exposure of the animal to additional risks such as dissemination of tumour cells, enlargement of the surgical site, and possible post-anaesthetic complications. Furthermore, unlike the majority of tumour biopsies, blood sampling can be repeated at close intervals, which allows monitoring of parameters over time and therefore assessment of potential changes in cancer behaviour, with or without treatment. There are many types of blood biomarkers: ions, proteins, carbohydrates, lipids, nucleic acids and also cells. However, none of them is yet commonly used for diagnostic purposes in veterinary medicine. Instead of

This is an open access article under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2022 The Authors. *Veterinary and Comparative Oncology* published by John Wiley & Sons Ltd.

<footnote n="1"><sup>1</sup>Ecole Nationale Vétérinaire d'Alfort, BioPôle Alfort, Maisons-Alfort, France</footnote>

<footnote n="2"><sup>2</sup>Ecole Nationale Vétérinaire d'Alfort, CHUVA, Service de Médecine Interne, Maisons-Alfort, France</footnote>

<footnote n="3"><sup>3</sup>Anses, INRAE, Ecole Nationale Vétérinaire d'Alfort, UMR VIROLOGIE, Laboratoire de Santé Animale, Maisons-Alfort, France</footnote>

<footnote n="4"><sup>4</sup>Ecole nationale Vétérinaire d'Alfort, Univ Paris Est Créteil, INSERM, IMRB, Maisons-Alfort, France</footnote>

<footnote n="5"><sup>5</sup>Anses, INRAE, Ecole Nationale Vétérinaire d'Alfort, UMR BIPAR, Laboratoire de Santé Animale, Maisons-Alfort, France</footnote>
focusing on one single category of biomarker or all biomarkers in one
single form of cancer, this review provides an overview of the scien-
tific advances concerning selected blood parameters of various type,
that have shown potential interest as biomarkers for different canine
cancers, inspired from extensive studies in human oncology, the latter
being excluded from this manuscript as reviewed elsewhere.²

2 | IONS AS BIOMARKERS

2.1 | The copper ion and copper isotopes

Copper ion ($Cu^{2+}$) is an essential micronutrient, with involvement in many fundamental physiological processes. In humans, copper has also been involved in the processes of tumorigenesis, angiogenesis, tumour migration and metastasis development³⁻⁶ but only a few animal studies have focused on this topic. However, one study demonstrated a two-fold increase in $Cu^{2+}$ concentrations in bitches with mammary tumours of various types compared with healthy bitches.⁷ Unfortunately, the small size of this study and the lack of comparison of nutrient intake, precludes for the moment, the possibility to use serum $Cu^{2+}$ as a diagnostic biomarker for mammary tumours in dogs.

New mass spectrometry methods such as multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS), allowed the measurement of copper isotopes ⁶⁵Cu/⁶³Cu ratio (δCu) in dogs' blood. Blood δCu measured in dogs with neoplastic or non-neoplastic disease was significantly lower compared with healthy dogs. However, δCu levels between neoplastic and non-neoplastic dogs were not significantly different. In addition, following a chemotherapy protocol for lymphoma, an increase of the δCu was seen in five out of six dogs in clinical remission.⁸ Interestingly, to measure these copper ions, whole blood can be frozen at -20°C and processed once defrosted, to purify copper elements before performing MC-ICP-MS, which is therefore a technical advantage to investigate larger cohorts. Indeed, to show a possible interest of the δCu in monitoring the response to chemotherapy treatment in dogs, these larger studies are now required, with a focus on specific neoplastic conditions to identify those that may present significant isotope variation compared with other non-neoplastic conditions.

2.2 | The zinc ion

Only two published studies have focused on measuring Zinc ion ($Zn^{2+}$) serum concentration in neoplastic and healthy dogs. A significant decrease in $Zn^{2+}$ serum concentrations was observed in 50 dogs diagnosed with Illa or IVa lymphoma (classification according to the World Health Organisation's) compared with 50 healthy dogs. This decrease was also seen in dogs suffering from osteosarcoma but the difference with the healthy dogs was not significant.⁹ A significant decrease in serum $Zn^{2+}$ concentration was also observed in bitches with mammary tumours compared with healthy bitches.⁷ Since these two studies from the past decades, $Zn^{2+}$ serum levels have not been

further studied in other tumour types or with bigger dog cohorts. This
could be attributed to the fact that dietary intake may influence $Zn^{2+}$
serum levels, which could be hard to monitor in animals. Yet, a lot of
studies in human oncology have shown a significant decrease in
serum $Zn^{2+}$ concentration in many cancers and some also suggest the
potential benefit of $Zn^{2+}$ supplementation during therapy.¹⁰⁻¹⁴ This
should justify and motivate more research on $Zn^{2+}$ blood levels in the
veterinary oncology field since it is now easily measurable in serum
with a colorimetric assay.¹²

3 | PROTEINS AS BIOMARKERS

3.1 | The carcino-embryonic antigen

The carcino-embryogenic antigen (CEA) is a membrane glycoprotein, mainly synthesised in certain portions of the digestive tract and plays a role in cell adhesion. CEA blood concentration is very low under physiological conditions but some tumour cells can produce this protein in large quantities and express it over the entire membrane surface when losing their polarity. As these cells no longer rest on a basal lamina, CEA can be found in high concentrations in the blood of human patients with certain tumours.¹⁵ In healthy dogs, serum CEA concentrations reference values were initially set using radio-immuno assays, ranging from 0.12 to 0.23 ng/ml,¹⁶,¹⁷ and interestingly, bitches with mammary tumours presented CEA serum concentration above the reference values. At the 0.23 ng/ml cut-off concentration, the sensibility and specificity of this assay was of 60% et 95% respectively for tumour diagnosis in dogs.¹⁷ Other studies evaluated the difference in CEA expression between female dogs with mammary tumours and healthy dogs using ELISA kits available for human CEA. Two studies confirmed a significant increase in serum CEA concentration in bitches with mammary tumours compared with healthy bitches¹⁸,¹⁹ whereas a previous one showed no correlation of the CEA serum levels with the staging of the tumour.²⁰ Interestingly, Senhorello et al., observed a significant increase in CEA levels in bitches with tumours greater than 3 cm in size compared with tumours less than 3 cm, and a significant increase in CEA was noticed for grade III tumours compared with grade I and II tumours. Based on the Receiver Operating Characteristic (ROC) curve performed using 1.08 ng/ml as the cut-off value, they established the sensitivity and specificity of serum CEA, measured by ELISA, to detect the presence of mammary tumour at 82.14% and 95.24%, respectively. Sensitivity for this threshold value was improved to 100% for tumours larger than 3 cm and metastasised tumours but down to 70% for tumours smaller than 3 cm.¹⁹ Taken together, these studies confirmed that CEA is a relatively sensitive and specific diagnostic biomarker for canine mammary tumours, especially in more advanced stages, which, however, might not allow CEA to be a biomarker for early diagnostic of breast tumour. The use of human ELISA kits, commercially available to measure CEA in dog blood, made these studies possible but more recently, canine specific kits are also available, which is a great advantage to acknowledge CEA as an easily accessible canine cancer biomarker. These more recent
studies are in agreement with what had been shown previously.<sup>21,22</sup> Using canine specific CEA ELISA kits, lower CEA levels were observed 15 days after surgery on 41 dogs but this difference was not significant.<sup>21</sup> Interestingly, with human CEA ELISA kits, the reduced CEA serum levels were significantly different 15 and 45 days post-masectomy as compared with before surgery in 11 female dogs.<sup>19</sup> In terms of prognostic biomarkers, they also show that the higher the initial serum CEA levels in bitches, the shorter the survival time is,<sup>21</sup> but longer-term studies involving a larger number of bitches are required to assert this.

## 3.2 | The carbohydrate antigen 15-3

The Carbohydrate Antigen 15-3 (CA15-3) is transmembrane glycoprotein belonging to the mucin family produced by the MUC-1 gene. The products of the MUC-1 gene are involved in carcinogenesis as they participate to immunosuppression,<sup>23</sup> promote the proliferation and survival of tumour cells,<sup>24</sup> and also contribute to their dissemination.<sup>25</sup> CA15-3 concentration is increased in blood when tissues are altered, however, this is not specific to a particular neoplastic disease and can be found in different human cancers<sup>26-31</sup> as well as in hepatitis<sup>32</sup> and arthritis.<sup>33</sup>

In veterinary oncology, several studies have found a significant increase in serum CA15-3 concentration in female dogs suffering from canine mammary tumours compared with healthy ones.<sup>17,18,20-22,34-36</sup> The sensitivity and specificity of CA15-3 to detect mammary neoplastic damage at a threshold value of 7 IU/ml, was 100% and 95% respectively.<sup>17</sup> However, a more recent study exhibited a sensitivity of CA15-3 to 51.8% in the malignant group but with a specificity close to previously described (93.9%). More interestingly, combining CA15-3 to CEA for instance, increased sensitivity to 64.2%, but combining these two biomarkers reduced specificity down to 81.7%.<sup>22</sup> In addition, a significant positive correlation between increasing serum CA15-3 concentrations and advancing stages of tumour has been observed.<sup>20</sup> However, the preliminary study from 2007 by Marchesi et al., showed no significant difference in serum CA15-3 concentrations between dogs with different types of neoplastic processes and healthy dogs.<sup>34</sup> This lack of significance could be explained, as in humans, by the fact that not all tumour types are associated with an elevated CA15-3 production. Finally, as for CEA, low serum CA15-3 concentrations before surgery are associated with longer survival times, while higher concentrations are associated with shorter survival times.<sup>21</sup> Thus, the value of CA15-3 may provide prognostic information in bitches with mammary tumours. Now, studies showing the independence of this parameter from those used in practice for prognostic purposes are required to recommend serum CA15-3 in prognostic procedures. Combining CA15-3 with other biomarkers, as suggested above,<sup>22,37</sup> could also be a lead to follow for canine cancer prognosis.

## 3.3 | The alpha-fetoprotein

The alpha-fetoprotein (AFP) is synthesised in large quantities mainly by the liver, during embryonic development. It is indeed one of the

major proteins in foetal circulation, however, its expression is transcriptionally repressed at birth. AFP has been shown to have an immunosuppressive activity and also pro-angiogenic properties that promote neovascularisation in foetal and tumour tissues.<sup>38-40</sup> Considering that AFP is mainly produced by liver, veterinary studies have focused on this protein as a biomarker for canine liver cancer.

At birth, serum AFP concentration in dogs is high: 14080 ± 5944 µg/ml, then a strong decrease during growth is observed until reaching 0.014 to 0.069 µg/ml in adult dogs.<sup>41</sup> In vitro, AFP can be produced by canine hepatocellular carcinoma cells, suggesting that this production could be linked to the neoplastic process in dogs.<sup>42</sup> This observation had been confirmed by Marchesi et al. 2007, in which, no significant differences were noted in the serum AFP concentrations between healthy dogs and those with non-neoplastic diseases but dogs with lymphomas and mastocytomas showed an increase in serum AFP concentrations. However, AFP concentrations in serum of dogs with sarcomas and carcinomas were not significantly different from healthy dogs.<sup>34</sup> A high AFP serum concentration cut-off value could indicate a high probability of neoplastic process.<sup>43,44</sup> but to date, no studies have proposed a cut-off value in dogs defining a sensitivity and specificity value for this biomarker. Moreover, no studies have been conducted on hepatocellular carcinomas in dogs to establish a correlation between serum AFP concentration and histological characteristics, tumour size, or tumour stage. Despite a significant decrease in serum AFP concentrations observed following surgical removal of affected liver lobes,<sup>43,44</sup> the available evidence is not strong enough yet to consider AFP serum concentrations as a good follow-up biomarker of individuals with hepatocellular carcinoma.

## 3.4 | Lactate dehydrogenase

Rapid cancer cell proliferation and high metabolic demands lead to an increase in lactate dehydrogenase (LDH), which catalyses the reversible transformation of pyruvate into lactate and is well established in human medicine as a prognosis and follow-up biomarker in many cancers.<sup>45</sup> It has been shown in veterinary medicine that LDH is significantly increased in blood of dogs with malignancies compared with healthy dogs or dogs with non-tumour disease and the highest LDH concentration is observed among dogs with lymphoma.<sup>46</sup> This enzyme is easily measurable in freshly collected serum using commercially available kits and reference values in dog serum are set from 45 to 233 U/L.<sup>20</sup> Interestingly, a significant increase of serum LDH in dogs with malignant mammary tumours has been measured compared with healthy dogs and a positive correlation was shown between LDH serum concentrations and tumour stage.<sup>20</sup> Therefore, serum LDH concentration shows an interesting diagnostic value for canine tumours, despite an earlier study showing that dogs affected with malignant lymphoma kept LDH levels in the reference values.<sup>47</sup> In cats, a study shows a poor accuracy for serum LDH to discriminate oral lymphoma and inflammatory bowel disease (IBD)<sup>48</sup> and LDH levels were also high in serum of dogs infected with canine
parvovirus.⁴⁹ However, interestingly, an increased LDH activity (> 280 U/L) anticipated clinical stages of lymphomas in dogs and was also often seen at completion of chemotherapy and at 1 month after chemotherapy, in dogs with recurrence during the successive 45 days, with a sensitivity of 77.8% and 96.2% of specificity.⁵⁰ Thereby, rather than a diagnostic biomarker, LDH could be an interesting prognostic tool to predict recurrence and a follow-up biomarker in veterinary medicine, but further studies are needed to assess this hypothesis.

## 3.5 | The anti-mullerian hormone

The anti-mullerian hormone (AMH) is a glycoprotein secreted by Sertoli cells in males and by the granulosa cells of small growing follicles in females. A decrease of its secretion has been observed after puberty in men, bulls and stallions.⁵¹ Thus, this hormone is mainly studied and used in relation to its potential as a biomarker in granulosa cell tumours (GCT) and Sertoli cell tumours (SCT) in humans.⁵² Given the importance of reproduction in veterinary breeding, blood AMH concentrations in the case of ovarian pathologies were first investigated in cows and mares. It was shown in both animals that an increase in the serum AMH concentration above the threshold limit diagnostics the presence of GCT, with the cut-off set at 0.36 ng/ml for cows and 4 ng/ml for mares, with sensitivity respectively evaluated at 100% and 98%.⁵³,⁵⁴ AMH serum concentrations were significantly higher in female dogs with GCT compared with healthy bitches as well as bitches with other ovarian neoplasia or non-neoplastic ovarian disease such as follicular or luteal cysts. The cut-off value was set at 0.99 ng/ml, which gives to AMH a sensitivity of 100% and specificity at 94.44% to detect GCT.⁵⁵ These values suggest accurate diagnostic performance for AMH, but further studies would be required in bitches, including a larger number of animals, to confirm these results. Interestingly, in male dogs, several articles have also shown a three-fold or more increase in serum AMH concentrations in SCT dogs compared with healthy dogs.⁵⁶,⁵⁷ These results strengthened AMH as a potential biomarker for SCT in male dogs as well. However, the diagnostic interest of this marker is limited by the fact that any pathology concerning the ovaries or testicles is mostly treated by surgical excision in veterinary medicine. Therefore, a precise diagnostic test to detect a GCT or SCT before surgical removal is rarely necessary. Further studies are now needed to establish the potential clinical utility

of this marker in the follow-up of animals with GCT or SCT, including its ability to detect metastatic processes after surgical excision.

## 3.6 | The thymidine kinase 1

The thymidine kinase 1 (TK1) is one isoform of the thymidine kinase (TK) which phosphorylates thymidine, an essential nucleotide for DNA replication. TK1 is located in the cytosol where its quantity and activity are low when the cell is quiescent and increase during cell division.⁵⁸ Many studies in dogs have focused on TK1 as a biomarker in oncology. In most of these studies, it has been shown that the activity of TK1, measured by radioenzymatic assays or ELISA, is increased particularly in the blood of dogs suffering from lymphomas and myeloid leukaemia compared with healthy dogs.⁴⁷,⁵⁹⁻⁶⁶ The diagnostic performances of TK1 activity, outlined by some of these authors, to differentiate dogs with haematopoietic neoplastic processes from healthy dogs is shown in Table 1. Thus, the activity of TK1, significantly increased in haematopoietic tumours, may be interesting in terms of diagnostic performances mainly for these specific tumours. In terms of prognostic interest, it has been shown that the activity of TK1 increased with tumour stage.⁶⁰,⁶⁵ In addition, an increase in TK1 activity above 30 U/L is significantly associated with a decrease in overall survival time, in dogs with lymphomas⁶⁰ and more recent studies showed that a combination of TK1 with the inflammation marker C Reactive Protein (CRP) gave higher diagnosis accuracy and prognostic values than either of both alone, in dogs with haematological malignancies.⁶⁵,⁶⁷ Finally, several studies have investigated the interest of TK1 as a pharmacodynamic biomarker in dogs with haematopoietic tumours (lymphomas and leukaemias) during treatment. All these studies consistently showed that dogs undergoing chemotherapy had a significant decrease in TK1 activity, with partial or complete response, and up to physiological values.⁶⁰,⁶¹,⁶⁴,⁶⁶,⁶⁸,⁶⁹ According to these same studies, these values remained significantly higher in cases of relapse or no response to treatment. Thus, TK1 shows good values in terms of diagnostic, prognostic and follow-up performance in canine haematopoietic tumours, and therefore seems to be a good candidate as a biomarker in this field, despite a certain lack of specificity of this enzyme for other neoplastic pathologies. Interestingly, the stability of TK1 enzyme activity is not affected by storage at -20°C for up to 3 months⁶⁰ and has also been correlated with ELISA values in

<table><caption>TABLE 1 Diagnostic performances of TK1 in dogs with haematopoietic cancers (lymphomas and leukaemias) compared with healthy dogsª</caption><thead><tr><td>Study</td><td>Dogs with neoplasia (n)</td><td>Healthy dogs (n)</td><td>Used technology</td><td>Sensitivity</td><td>Specificity</td></tr></thead><tbody><tr><td>Nakamura et al., 1997<sup>60</sup></td><td>20</td><td>13</td><td>Radioenzyme assay</td><td>100%</td><td>100%</td></tr><tr><td>von Euler et al., 2004<sup>61</sup></td><td>52</td><td>21</td><td>Radioenzyme assay</td><td>92%</td><td>98.1%</td></tr><tr><td>Thamm et al., 2012<sup>64</sup></td><td>14</td><td>24</td><td>ELISA</td><td>80%</td><td>n.d.</td></tr><tr><td>Sharif et al., 2012<sup>65</sup></td><td>34</td><td>35</td><td>Radiochemical assay</td><td>94%</td><td>68%</td></tr><tr><td>Jagarlamudi et al., 2015<sup>67</sup></td><td>43</td><td>42</td><td>ELISA</td><td>79%</td><td>97%</td></tr></tbody></table>

Abbreviation: n.d., non-determined.

<footnote>ªCalculated by authors from data in the articles.</footnote>
dogs with malignant lymphomas,⁴⁷ therefore TK1 measurement could
easily become a routine procedure in veterinary oncology.

4 | NUCLEIC ACID BIOMARKERS

4.1 | Circulating deoxyribonucleic acids

Increased concentrations of cell-free circulating Deoxyribonucleic Acids (cell-free DNA or cfDNA) have been observed in blood of human cancer patients as compared with healthy individuals. However, this increase has been shown later not to be specific of tumours since cfCDNA is also increased in other human conditions such as pregnancy or after transplantation and in some inflammatory conditions.⁷⁰ Whereas cfDNA is the total pool of circulating cell-free DNA, circulating-tumour DNA (ctDNA) is tumour-specific, released from circulating tumour cells (CTCs) and from cells within the tumour. Several studies in humans suggest that cells within the tumour are the main source of ctDNA compared with CTCs since ctDNA has been detected even in the absence of CTCs in the blood.⁷¹,⁷² The ctDNA forms a very small part of the total circulating cfDNA, so it is particularly complex to detect. Somatic mutations (point mutations, insertions, rearrangements), variability in the number of copies of a gene, size of the fragment, aneuploidy and degree of methylation are used to identify ctDNA among cfDNA, using many different methods.⁷³,⁷⁴ Furthermore, it was shown that, thanks to different properties of the ctDNA, such as its methylation profile, it was possible to determine the tissue of origin of the identified ctDNA.⁷⁵⁻⁷⁷ Thus, a combination of somatic and epigenetic changes could detect a tumour process and determine its location.

A limited number of studies have been carried out on cfDNA in
veterinary oncology rather than on ctDNA. Most of the studies

investigating the variation in the amount of cfDNA in dogs with neo-
plastic processes have shown a significant increase in the amount of
cfDNA in patients compared with healthy dogs as shown in
Table 2.⁷⁸⁻⁸⁰ Interestingly, Tagawa et al., show that the amount of
plasma cfDNA is correlated with clinical stage, with a significant
increase in plasma cfDNA in metastatic versus non-metastatic
dogs.⁷⁸ In 2013, a first study carried out the complete sequencing of
the tumour genome of four dogs with mammary tumours. Based on
the genetic rearrangements observed within the tumours, the
authors were able to detect in the four dogs, circulating DNA with
these same rearrangements, that is, tumour circulating DNA.⁸¹ The
specific BRAF gene mutation was also investigated in dogs with
urothelial carcinomas since it had been shown that nearly 80% of
these tumours in dogs have a mutation in this gene.⁸² The BRAF
V595E mutation was found in almost 73% of the plasma of dogs
with tumour and the concentration increased with disease progres-
sion. Thus, the authors suggest that the amount of ctDNA with BRAF
mutations may represent a useful non-invasive diagnostic biomarker
in canine urothelial carcinoma.⁸³ Interestingly the BRAF V595E
mutation can also be monitored in urine samples using the new
droplet digital Polymerase Chain Reaction (dPCR) molecular tech-
nique which allowed to identify the mutation in 83% of dogs with
urothelial carcinoma (19 positive out of 23 patients) and 100% of
dogs with protastic carcinoma (3 dogs only) when it was completely
absent in healthy controls.⁸⁴

Another specific element of ctDNA is the hypomethylation of the Long Interspersed Nuclear Element-1 (LINE-1) sequences which are associated with oncogenesis and metastases formations.⁸⁵ In a study involving healthy dogs, dogs with malignant or benign cancers, the index of the relative level of methylation of the LINE-1 sequences of the cfDNA was significantly reduced in dogs with benign and malignant mammary tumours (0.29 ± 0.061 and 0.39 ± 0.066, respectively)

<table><caption>TABLE 2 Studies comparing cfDNA quantities in neoplastic and healthy dogs</caption>
  <thead>
    <tr>
      <th rowspan="2">Study</th>
      <th colspan="2">Tumour group</th>
      <th colspan="2">Healthy group</th>
      <th rowspan="2">p-value<sup>a</sup></th>
    </tr>
    <tr>
      <th>Type and number of tumour bearing dogs</th>
      <th>[cfDNA]<sup>b</sup> in blood</th>
      <th>Number of healthy dogs</th>
      <th>[cfDNA]<sup>b</sup> in blood</th>
    </tr>
  </thead>
  <tbody>
    <tr>
      <td>Letendre and Goggs, 2017<sup>81</sup></td>
      <td>Sarcomas n = 15</td>
      <td>8.1 µg/ml<sup>c</sup> (5.1-15.8)</td>
      <td>n = 15</td>
      <td>4.7 µg/ml<sup>c</sup> (0.6-8.5)</td>
      <td>p = .003</td>
    </tr>
    <tr>
      <td>Beffagna et al., 2017<sup>82</sup></td>
      <td>Mammary tumours n = 44</td>
      <td>Short fragments: 128.5 ng/ml (56.8-200.2)<br/>Long fragments: 87.5 ng/ml (33-142)</td>
      <td>n = 15</td>
      <td>Short fragments: 28.8 ng/ml (6.5-51.1)<br/>Long fragments: 18.2 ng/ml (3.9-32.6)</td>
      <td>Short fragments: p = .018<br/>Long fragments: p = .009</td>
    </tr>
    <tr>
      <td>Tagawa et al., 2019<sup>80</sup></td>
      <td>Various tumours<sup>d</sup> n = 50</td>
      <td>6290 ng/ml (51-115 000)</td>
      <td>n = 11</td>
      <td>481 ng/ml (205-802)</td>
      <td>p = .011</td>
    </tr>
  </tbody>
</table>

<footnote><sup>a</sup>In all studies, *p* < .05 is considered significant.</footnote>

<footnote><sup>b</sup>[cfDNA]: concentration of cell-free DNA.</footnote>

<footnote><sup>c</sup>Median values and when non-specified mean values.</footnote>

<footnote><sup>d</sup>Lymphoma & leukaemia (n = 11), hemangiocarcinoma (n = 8), urothelial carcinoma (n = 5), mammary carcinoma (n = 4), orar squamous cell carcinoma (n = 3) apocrine gland anal sac carcinoma (n = 3) hepatic tumour (n = 3), soft tissue carcinoma (n = 2), malignant melanoma (n = 2), gastrointestinal stroma tumour (n = 2), mast cell tumour (n = 1), osteosarcoma (n = 1), ceruminous adenocarcinoma (n = 1), salivary adenocarcinoma (n = 1), apocrine adenocarcinoma (n = 1), pheocromocytoma (n = 1), seminoma (n = 1).</footnote>
compared with healthy dogs (0.92 ± 0.067). However, this decrease
was less significant for other tumour types studied in this article.⁸⁶

cfDNA and ctDNA appear to be of interest as well as prognostic
and follow-up biomarkers in dogs with neoplastic processes. Indeed,
dogs with high cfDNA concentrations had a significantly shorter dura-
tion of remission/lower overall survival rate than dogs with low
cfDNA concentration with a threshold set at 25 ng/ml when the ref-
erence value for cfDNA in normal canine plasma is 1–15 ng/ml.⁸⁷
Looking more specifically at ctDNA, two other studies came to the
same conclusions.⁸³,⁸⁸

Despite the low number of studies in dogs, cfDNA and ctDNA as
diagnostic, prognostic and monitoring biomarkers in patients suffering
from neoplastic processes should be considered. The advantages of
measuring cfDNA and/or ctDNA are numerous: it can be detected in
blood as well as in non-invasive samples such as urine, appears reli-
able in detecting a tumour process in a sensitive and specific manner,
and allows the entire tumour process of an individual to be properly
characterised. However, studies involving a larger number of dogs are
needed to establish reliable results and to be integrated into clinical
practice in veterinary oncology.

## 4.2 | Circulating ribonucleic acids

As for DNA, RNA can also be found in the bloodstream. The main cir-
culating RNAs are microRNAs (miRNAs). Highly conserved in evolu-
tion, they act as post-transcriptional regulators, based on the principle
of partial or complete correspondence with the complementary
mRNA. To date, several thousand miRNAs have been identified in
humans.⁸⁹ Several studies have been conducted on miRNAs as a bio-
marker in dogs, but only a few have focused on their expression in the
blood of dogs with neoplastic processes. *In vitro*, the miRNA profiles
contained in exosomes released from normal canine mammary epithe-
lial cells obtained from dogs without mammary tumours were com-
pared with exosomes released by tumour breast cells, derived from
canine mammary carcinoma. A total of 145 miRNAs were differen-
tially expressed by tumour cells compared with healthy cells.⁹⁰ How-
ever, two other studies in dogs concluded that there was no
correlation between the miRNA profiles recorded within the tumour
and in the blood of the same patient.⁹¹,⁹² Thus, the increases or
decreases in blood concentrations recorded for some miRNAs, may
not be due solely to differential expression of the miRNAs by the
tumour, but may be the result of a general response of the body to
the presence of the tumour. Nevertheless, some studies focused on
specific miRNA, such as miRNA-126 and -214, which are known to
play a role in angiogenesis and tumorigenesis.⁹³ Two studies showed
a significant increase in blood expression of both biomarkers in dogs
with hemangiosarcoma, compared with dogs with benign processes
and healthy dogs. It should also be noted that the values of these two
markers did not show a strong correlation with other clinical parame-
ters (haematocrit, platelet count, prothrombin time and activated par-
tial thromboplastin time, blood urea nitrogen, creatinine, alanine
aminotransferase, aspartate aminotransferase, alkaline phosphatase),

thus attesting to the independent nature of these biomarkers.⁹³,⁹⁴
Interestingly, as in human medicine where a panel of several miRNA
showed very high values of diagnosis accuracy to detect people with
early stage of non-small-cell-lung-tumours compared with either
miRNA alone,⁹⁵ a combination of miRNA-214 and miRNA-126 has
showed better diagnosis accuracy to detect different types of epithe-
lial and non-epithelial tumours than both independently.⁹³,⁹⁴ Several
additional studies have looked at other mi-RNAs, particularly with
regard to specific tumour conditions. Serum miRNA-122 concentra-
tions have been shown to be increased in dogs with neoplastic liver
damage compared with healthy dogs.⁹⁶ RNA deep-sequencing
(RNASeq) and dPCR techniques identified differential expression of
65 miRNAs in the blood of healthy female dogs and of dogs with
mammary carcinomas, with two of them significantly over-expressed
in the plasma of dogs with tumours: miRNA-19b and miRNA-125a. In
addition, the plasma concentration of miRNA-18a was significantly
increased in dogs with Stage III versus Stage I–II tumours. A significant
increase in plasma miRNA-18a concentration was also observed when
lymphatic invasion was noticed on histological analysis, and could
therefore be a predictive biomarker of metastasis.⁹⁷

Regarding the prognostic performance of blood miRNAs in dogs,
an increase in serum miRNA-125a concentration showed a correla-
tion, although not significant, with a decrease in survival time in
bitches with mammary carcinoma.⁹⁷ Moreover, combined miRNA-126
and -214 concentrations did not show a significant impact on survival
times of dogs affected by various type of neoplasia.⁹³ A study focus-
ing on the prognostic impact of miRNA-126 and -214 concentrations
in dogs with osteosarcoma treated by amputation and adjuvant che-
motherapy, revealed that an increase in serum miRNA-214 concentra-
tion was significantly associated with poorer duration of remission
and shorter overall survival time. Higher miRNA-126 concentrations
were on the contrary, significantly associated with longer durations of
remission and overall survival. It should be noted that the values of
these markers were independent of different histopathological char-
acteristics (mitotic index, histological subtype), but an association was
noted with tumour grade.⁹⁸ For dogs with multicentric lymphomas,
elevated plasma miRNA-21, -31, -45, -150, -155 and -222 concentra-
tions were significantly correlated with lower progression-free sur-
vival rates, and elevated plasma miRNA-181c and -222
concentrations significantly associated with a lower median overall
survival time but in the B-cell lymphoma group only.⁹² Thus, several
circulating miRNAs appear to have shown potential as prognostic bio-
markers in dogs with different tumour types, although few studies
have been performed on this subject.

Interestingly, serum miRNA-126 and -214 concentrations were
decreased in dogs after surgical removal of a hemangiosarcoma,
attesting the dynamic nature of these two miRNAs.⁹⁴ Some other
miRNAs also showed significant variation in their serum concentra-
tions in dogs with lymphomas during relapses (increased miRNA-125b
but miRNA-30b, -34a and -182 decreased).⁹² These observations
could trigger an interest to use these miRNAs for monitoring tumour
processes in dogs and early detection of relapse. However, it is impor-
tant to underline that a lack of specificity of miRNAs in neoplastic
condition could be a main limitation for these as diagnostic bio-
markers. Indeed, for example, there were no significant differences in
plasma concentrations of miRNA-122 between dogs with hepatic
neoplasia, hepatic fibrosis or inflammatory liver disease.⁹⁶ Moreover,
some miRNA detected in the blood such as miR-144 or miR-32, are
not able to discriminate dogs with metastasis from dogs without,⁹¹
adding one more limitation to their value as prognostic biomarkers.

In conclusion, some specific circulating miRNAs have been shown
in humans and dogs to be of interest for diagnosis, prognosis or pre-
diction of responses to treatment and for monitoring individuals with
neoplastic processes. For all these reasons, miRNAs are presented as
the future major biomarkers in the field of oncology by many authors
and more studies on these in veterinary oncology can be expected in
the future.

5 | CIRCULATING CELLS AS BIOMARKERS

5.1 | Circulating tumour cells

Circulating Tumour Cells (CTCs) are responsible for the formation of metastases. CTCs originate from a solid tumour from which they have lost their ability to adhere, allowing them to migrate into the blood. As these cells are very rare, about 1 CTC among $10^6$ to $10^7$ leukocytes, the tools used for their detection must be highly sensitive and techniques for enrichment and isolation of these cells, are necessary.⁹⁹,¹⁰⁰ They are based on the recognition of CTCs' cell surface markers, on their physical or biological characteristics (density, size, invasiveness), or on molecular techniques such as RT-PCR for the detection of CTCs-specific RNA. However, only one technique for identifying, isolating and enumerating CTCs was approved in 2004 by the Food and Drug Agency (FDA) which includes an enrichment step followed by a more specific identification, both using specific antibodies and high resolution imaging systems.¹⁰⁰ The cells considered as CTCs express the Epithelial cell adhesion molecule (EpCAM), Cytokeratins (CK), and are positive for 4',6-diamidino-2-phenylindole (DAPI) but do not express the Cluster of Differentiation 45 (CD45). Their phenotype is therefore EpCAM+/CK+/DAPI+/CD45-.¹⁰¹ Due to the fact that this is the only FDA-approved technology, the majority of studies in human oncology have been conducted using this system, but in dogs, studies have been published using RT-PCR as it is possible to detect the presence of mRNAs specifically expressed in CTCs in the blood.¹⁰² Out of 16 human breast cancer CTCs markers mRNAs, six were overexpressed in the blood of bitches with mammary carcinoma but none were in the blood of healthy dogs.¹⁰³,¹⁰⁴ Moreover, products of the CLDN7, CRYAB, ATP8B1 and EGFR genes can differentiate with varying degrees of accuracy between bitches with metastatic breast carcinoma and bitches with no evidence of metastasis. In particular, mRNAs from the CRYAB and ATP8B1 genes could be biomarkers for CTCs in female dogs with mammary carcinoma, since they both exhibit respectively the best sensitivity and specificity values (35% and 100% for CRYAB and 32.5% and 90% for AT8B1) among all the other genes tested.¹⁰⁵

EpCAM+/CK+/DAPI+/CD45- CTCs have also been identified in the blood of bitches with metastatic mammary carcinoma using the FDA approved method for human samples. This new technique showed a sensitivity of 44.4% for the detection of metastasized mammary carcinoma, and revealed that the presence of CTCs in the blood has a negative prognostic impact.¹⁰⁶ Recently, flow cytometry was also used to detect CTCs in the blood of three dogs with osteosarcoma with labelling of Collagen I and Osteocalcin. Interestingly, CTC frequencies in blood were greatly reduced following amputation in all three dogs, were variable during chemotherapy and an increase has been seen in all three dogs within the 4 weeks prior to apparent metastasis or death.¹⁰⁷ This first study showed a dynamic evolution of CTCs during chemotherapy treatment in dogs, suggesting a potential follow-up biomarker role for CTCs in dogs with osteosarcoma during treatment.

In conclusion, CTCs have shown their potential for diagnosis of
tumour processes despite limited and variable sensitivity. They have
also proven to be interesting in terms of prognosis, monitoring and
predictive tools. However, given that flow cytometry and CTCs
research is a relatively recent field in veterinary oncology, it is likely
that the increasing number of studies will lead in the near future to a
wider use of these cellular biomarkers.

5.2 | Myeloid derived suppressor cells

Myeloid derived suppressor cells (MDSCs) form a group of heteroge-
neous cells mainly present in the bone marrow where they represent
the immature stages of granulocytes and macrophages. They have a
strong immunomodulatory activity by mainly inhibiting the prolifera-
tion of T lymphocytes, reducing their cytotoxic power and inducing
their apoptosis. They also reduce the proliferation and activity of B
lymphocytes, as well as natural killer (NK) cells and dendritic cells.
Acute or chronic inflammatory phenomena favour the accumulation
of MDSCs within the sites of inflammation, prevent their differentia-
tion and induce their activation.¹⁰⁸ It has been shown that many fac-
tors allow the recruitment and expansion of MDSCs into the tumour
environment from the bone marrow and secondary lymphoid organs.
Due to their immunosuppressive role and their angiogenic capacities,
human MDSCs are key players in tumour escape and metastasis for-
mation.¹⁰⁹ A significant increase in the proportion of MDSCs among
peripheral blood mononuclear cells (%MDSCs) has been shown in a
wide range of tumour types compared with the %MDSCs in healthy
human subjects.¹¹⁰ Thus, due to the large number of tumour types
with a significant increase in %MDSCs, this endpoint cannot represent
a diagnostic biomarker of a particular tumour but may indicate the
possibility of a tumour involvement. MDSCs were first identified in
dog blood by flow cytometry as immature myeloid cells expressing
CD11b, but lacking the expression of CD14 and the major histocom-
patiblity complex class II (MHCII).¹¹¹ The immunosuppressive charac-
ter of these cells was confirmed by co-culture experiments in the
presence of autologous or heterologous T lymphocytes, whose activ-
ity was reduced in the presence of MDSCs. Blood of dogs with
advanced neoplastic processes showed a significant increase in %
MDSCs (36.04%) compared with healthy dogs (10.24%) and early-
stage tumours (9.40%).<sup>111</sup> These results were confirmed by other
studies on canine MDSCs in the context of sarcoma, carcinomas and
oral melanomas<sup>112</sup> and mammary tumours.<sup>113,114</sup> It was proposed
later, that canine MDSCs could be distinguished, as in humans and
mice, in two different phenotypes: monocyte-derived MDSCs (M-
MDSCs) and polymorphonuclear-MDSCs (PMN-MDSCs) by flow
cytometry, microscopy, and co-culture experiments. Both cell popula-
tions are CD5<sup>-</sup>/CD21<sup>-</sup>/MHCII<sup>-</sup> and CD11b<sup>+</sup> and they differ from
each other by the expression of CD14 and CADO48A, a canine spe-
cific neutrophil marker. M-MDSCs are CD14<sup>+</sup>/CADO48A<sup>-</sup>, whereas
PMN-MDSCs are CD14<sup>-</sup>/CADO48A<sup>+</sup>. The percentage of PMN-
MDSCs among blood mononuclear cells (%PMN-MDSC) was
increased in dogs with tumour processes compared with healthy
dogs, and this difference was significant in some tumour types.<sup>115</sup> In
dogs with melanomas, a significant increase in %M-MDSC and %
PMN-MDSC compared with healthy dogs was also observed.<sup>116</sup>
In humans, MDSCs have shown a moderate diagnostic capacity, with
modest sensitivities and specificities for the detection of tumour
processes.<sup>117-119</sup> However, the prognostic capacities of blood %
MDSCs have been widely demonstrated, as well as their ability to
predict the response to treatments based on their initial levels.
However, although their increase is more pronounced for advanced
neoplastic processes, their prognostic impact has not been clearly
established in dogs with tumours. Finally, their usefulness in terms of
follow-up during treatment could not yet be clearly established in both
humans and dogs. Therefore, additional studies are necessary to con-
firm the results obtained, to study the prognostic impact of MDSCs and
their usefulness in the follow-up of animals under treatment.

6 | CONCLUSION

In this review, we focused on blood biomarkers studied in veterinary oncology, and that shows a certain degree of specificity and sensitivity for neoplastic processes. Most of the biomarkers highlighted to date are protein-based, including CEA, CA15-3, AFP, LDH, AMH and TK1. They all show varying performances in terms of sensitivity and specificity to tumour, and each has shown some evidence of their potential as diagnostic, prognostic, predictive and monitoring biomarkers. However, none of them has perfect specificity for distinguishing individuals with tumour processes from healthy individuals or those with non-neoplastic pathologies. The next generation of biomarkers from liquid biopsy, seems to be particularly encouraging such as circulating miRNAs, ctDNA, cfDNA, CTCs and MDSCs. Some of these biomarkers have shown high performances in terms of sensitivity and specificity for identifying individuals presenting a tumour condition. It also appears that they have the capacity to describe tumour characteristics and monitor their evolution. More specifically, they allow to predict precisely sensitivities of the tumours studied to the various treatment protocols, both in humans and dogs. According to the One Health concept, most of the biomarkers studied in humans

are also accurate to follow in dogs, and conversely dogs could also be
a good model to identify or better characterise them, leading to a true
personalised medicine, for both humans and dogs.

AUTHOR CONTRIBUTIONS

**Philippe Colombe:** Acquisition of data, analysis, and interpretation of data, drafting and revising the manuscript. **Jérémy Béguin:** revising the manuscript. **Ghita Benchekroun:** revising the manuscript. **Delphine Le Roux:** conception and design, analysis and interpretation of data, drafting and revising the manuscript.

ACKNOWLEDGMENT

The authors thank Dr Darragh Duffy for critical reading of the
manuscript.

FUNDING INFORMATION

The authors received no funding for this work.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

ORCID

Delphine Le Roux https://orcid.org/0000-0003-3252-9155

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**How to cite this article:** Colombe P, Béguin J, Benchekroun G, Le Roux D. Blood biomarkers for canine cancer, from human to veterinary oncology. *Vet Comp Oncol*. 2022;20(4):767-777. doi:10.1111/vco.12848