Abstract
Background
Anal sac adenocarcinoma (ASACA) in dogs is a malignant perianal tumour that often metastasizes to the iliosacral lymph nodes. Additionally, this tumour can be associated with hypercalcemia of malignancy. To date, no study has looked at the association between increased blood calcium levels and suspected or confirmed lymph node metastasis as a primary objective.
Objective
The objective of this study was to determine if increased total serum calcium level is associated with iliosacral lymph node metastasis in dogs diagnosed with ASACA.
Methods
Medical records of a single referral hospital were searched to identify dogs examined between 2011 and 2021 that had a diagnosis of ASACA via cytology or histopathology. Only dogs that had serum total calcium recorded and abdominal ultrasound were included in the study. All images were reviewed by a board‐certified radiologist blinded to any patient identifiers.
Results
Of the 58 dogs, 33% (19/58) had total hypercalcaemia, and of these, 68% had confirmed or suspected iliosacral lymph node metastasis. Total hypercalcaemia was significantly associated with confirmed or suspected iliosacral lymph node metastasis (p < 0.01). However, 46% (11/24) of dogs with confirmed or suspected iliosacral lymph node metastasis were normocalcaemic.
Conclusions
Based on these results, it is suggested that while the presence of total hypercalcaemia may increase the likelihood of concurrent lymph node metastasis, total hypercalcaemia alone cannot be used as a screening tool for lymph node metastasis. Dogs diagnosed with ASACA should undergo full staging regardless of total serum calcium values.
Keywords: anal gland neoplasms, lymph node metastasis, hypercalcaemia
Total hypercalcaemia is significantly associated with confirmed or suspected iliosacral lymph node metastasis (p < 0.01) in dogs with anal sac adenocarcinoma (ASACA). However, while the presence of total hypercalcaemia may increase the likelihood of concurrent lymph node metastasis, total hypercalcaemia alone cannot be used as a screening tool for lymph node metastasis. Dogs diagnosed with ASACA should undergo full staging regardless of total serum calcium values.
1. INTRODUCTION
Anal sac adenocarcinoma (ASACA) is an uncommon perianal tumour in dogs that arises from the apocrine glands in the wall of the anal sac and accounts for ∼17% of all perianal tumours (Anderson et al., 2015; Bennett et al., 2002; Berrocal et al., 1989). These tumours are malignant and have been described as both locally invasive and metastatic with a range of 36%–96% of animals having metastasis to the iliosacral lymphocentre (sacral, medial iliac, and internal iliac lymph nodes) (Bennett et al., 2002; Meuten et al., 1981; Polton & Brearley, 2007; Williams & Packer, 2003). Distant metastasis to the liver, spleen, lungs, and other sites can occur (Barnes & Demetriou, 2017; Bennett et al., 2002; Polton & Brearley, 2007). The presence of nodal metastasis has been associated with shorter survival times (Potanas et al., 2015; Williams & Packer, 2003).
Dogs with ASACA present with variable clinical signs which are generally related to the size of the primary tumour (e.g. visible swelling, pain, and discharge), metastatic lymph node enlargement (e.g. tenesmus), and hypercalcaemia (e.g. polyuria and polydipsia). Previous studies have reported between 27% and 51% of dogs have hypercalcaemia upon presentation, with some associating hypercalcaemia with shorter survival times (Hobsin, 2006; Ross et al., 1991; Williams & Packer, 2003), and others showing no difference in survival times (Bennett et al., 2002; Emms, 2005; Potanas et al., 2015). Several studies have also demonstrated that tumour size is associated with shorter survival times (Polton & Brearley, 2007; Williams & Packer, 2003; Wong et al., 2021). In a retrospective study evaluating risk factors associated with clinical outcomes in dogs diagnosed with ASACA, tumour size was weakly correlated with hypercaelcemia (Wong et al., 2021). This correlation suggests that the extent of disease burden (i.e. tumour size and/or lymph node metastasis) may have an association with hypercalcaemia. If this correlation is true, the presence of hypercalcaemia may be an effective screening tool for metastasis.
Staging of ASACA is routinely performed to determine prognosis and guide the management of the disease, and this commonly includes a minimum database (complete blood count, serum chemistry panel, and urinalysis), thoracic radiographs to identify pulmonary metastasis, and abdominal ultrasound (AUS) to identify potential metastatic disease. Abdominal and/or thoracic computed tomography (CT) may also be utilized and is considered the chosen imaging modality for identifying abnormal abdominal lymph nodes. Normal lymph nodes on AUS are generally small, fusiform in shape, homogenous in echotexture, and isoechoic to the surrounding fat (Mayer et al., 2010). Abnormal lymph nodes can have either benign (i.e. reactive) or malignant (i.e. metastatic) aetiologies (Anderson et al., 2015; Llabrés‐Diaz, 2004). The distinction between a reactive or metastatic lymph node can be difficult to determine on AUS; however, studies have indicated that lymph nodes that are heterogenous in echotexture (Kinns & Mai, 2007; Llabrés‐Díaz, 2015), rounded in shape (De Swarte et al., 2011; Llabrés‐Díaz, 2015) or increased in number (Llabrés‐Díaz, 2015) are more likely to be associated with neoplastic involvement. Cytology or histology of ultrasonographically abnormal lymph nodes is advised for the confirmation of metastasis, though this is not routinely performed at all institutions.
A diagnosis of ASACA is commonly made in the general practice setting, and in some cases, the affected anal gland is removed in general practice rather than at a specialty facility. However, the ability and expertise to perform an AUS to assess the iliosacral lymphocentre, as well as the liver/spleen for potential metastasis, is not always available in general practice. The approach to surgical treatment for a patient changes depending on the presence or absence of lymph node metastasis at the time of diagnosis, with metastectomy to remove the affected lymph nodes having the potential to increase survival times (Barnes & Demetriou, 2017; Repasy et al., 2022; Wong et al., 2021). Therefore, the ability to predict the presence or absence of metastasis at the time of diagnosis with an inexpensive, simple diagnostic test would be beneficial to general practitioners in determining which patients to refer to a specialty facility for complete staging and potential lymph node cytology if appropriate. The purpose of the current study was to determine if an association exists between increased total serum calcium concentrations and lymph node metastasis in dogs diagnosed with ASACA. Our null hypothesis was that increased total serum calcium concentrations are not associated with lymph node metastasis.
2. MATERIALS AND METHODS
2.1. Case selection criteria
The electronic and paper records database of a single veterinary hospital was searched to identify dogs examined between January 2011 and May 2021 that had a histologically or cytologically confirmed diagnosis of ASACA and had staging performed with a minimum of abdominal imaging and a serum chemistry panel (including total calcium, albumin, creatinine, and phosphorus) within 6 weeks of diagnosis of ASACA. Dogs that had incomplete records or staging, more than 21 days between total calcium analysis and abdominal imaging, a previously diagnosed condition that could lead to hypo‐ or hypercalcaemia (including elevated creatinine or blood urea nitrogen, hyper‐ or hypoalbuminaemia, hyper‐ or hypoadrenocorticism, hyper‐ or hypoparathyroidism, bone metastasis and concurrent neoplasia in addition to ASACA), or were presented on medications that could alter total serum calcium concentrations (including trilostane, toceranib phosphate, steroids, diuretics, gastroprotectants, and/or intratumoural immunotherapy) were excluded from this study. Dogs in which the primary tumour was removed prior to total calcium measurement were also excluded.
2.2. Medical records review
For each dog enrolled in the study, data extracted from the medical record included signalment, clinical signs, rectal examination findings, staging test results, and the presence or absence of hypercalcaemia. The phosphorus value at the time of staging was recorded; however, not all dogs had a phosphorus value available. Dogs were classified as hyper‐ or hypophosphataemic if the serum phosphorus value was above or below the reference range for the analyzer used, respectively. The primary tumour size was unable to be recorded due to inconsistent, or unreliable, reporting in the medical records.
2.3. Classification of hypercalcaemia
Blood serum samples were analyzed using one of two machines (Cobas C501; Roche and NOVA CCX; NOVA Biomedical), or on the chemistry analyzer from the referring veterinary practice. Dogs were classified as having hypercalcaemia if their total calcium concentration on a chemistry analyzer was above the reference range for the analyzer used for each sample. The time from initial diagnostic imaging and acquisition of a total serum calcium value was recorded for each dog.
2.4. Abdominal imaging review and lymph node classification
All ultrasound examinations were performed on one of three machines (Acuson Sequoia; Siemens Medical Solutions and Aplio i500 and Aplio i800; Canon Medical Systems) by either a boarded radiologist or a radiology resident supervised by a single boarded radiologist. Stored ultrasound images and original reports were retrospectively reviewed by a single boarded radiologist (R. Mackenzie Hallman). Lymph nodes of the iliosacral lymphocentrum were assessed for maximal thickness, maximal thickness‐to‐length (T:L) ratio, hypoechogenicity, heterogeneity, and perinodal fat hyperechogenicity (De Swarte et al., 2011; Mayer et al., 2010; Kinns & Mai, 2007; Llabrés‐Díaz, 2015). Additionally, the number of lymph nodes with abnormal characteristics was recorded. The T:L ratio was obtained using measurements 90o from one another, and the lymph nodes were considered rounded with a T:L ratio over 0.5 as previously described (Mayer et al., 2010). These visual characteristics were used to classify each subject as either ‘suspected metastasis‐free’ or ‘suspected metastatic’, based on the full available clinical picture, including comparison to the contralateral lymph node, and consensus of both the original ultrasonographic report and the reviewing radiologist. This classification did not include any results from cytology or histopathology as the reviewing radiologist was blinded to these findings. If there were no measurable lymph nodes identified on the original scan, but the area of the aortic bifurcation was well visualized, the subject was classified as ‘suspected metastasis‐free’ without additional lymph node parameters recorded. If subjects had multiple lymph nodes identified, the measurement of the largest lymph node was used. Any lymph node of abnormal size was classified as ‘suspected metastatic’ regardless of echogenicity and/or available image of the contralateral lymph node. If patients had both a CT and an ultrasound performed at the initial diagnostics, only the images from the ultrasound were included for analysis.
2.5. Lymph node cytology
Lymph node cytology and/or histopathology were performed on abdominal lymph nodes at the discretion of the attending clinician. When available, lymph node cytology and/or histopathology were used to confirm metastasis of the sampled lymph nodes. Based on the presence or absence of metastatic transformation in the sampled lymph nodes, the node was classified as either confirmed metastatic or confirmed metastasis‐free, respectively.
2.6. Lymph node classification
All lymph nodes were classified as suspected/confirmed metastasis‐free or suspected/confirmed metastatic based on the combination of abdominal ultrasound and lymph node cytology and/or histopathology. If abdominal ultrasound and lymph node cytology and/or histopathology revealed disparate results, the results of the cytology and/or histopathology were used to classify the lymph node.
2.7. Statistical analysis
Data were tested for normality with a Shapiro–Wilk test. A Fischer's exact test was used to determine if an association exists between hypercalcaemia and confirmed/suspected lymph node metastasis. The remaining data were reported using descriptive statistics. Normally distributed data were reported as mean ± standard deviation, and non‐normally distributed data were reported as median and range. All statistical analyses were performed using Microsoft Excel (Microsoft Excel 2010; Microsoft), and values of p < 0.05 were considered significant.
3. RESULTS
3.1. Animals
There were 81 dogs diagnosed with ASACA that were presented to a single institution during the study period. Of these patients, eight were excluded due to incomplete records or staging, nine were excluded for a concurrent disease that may affect serum calcium levels, two were excluded due to greater than 21 days between bloodwork analysis and abdominal imaging, and four were excluded for presenting on medications that could alter serum calcium levels. After these exclusions, there were 58 records available for evaluation.
The most common breeds reported were mixed breed dog (n = 13, 22%), Labrador retriever (10, 17%), cocker spaniel (3, 5%), German shepherd dog (3, 5%), Australian shepherd (2, 3%), boxer (2, 3%), English springer spaniel (2, 3%), Golden retriever (2, 3%), Irish setter (2, 3%), miniature poodle (2, 3%), Shih Tzu (2, 3%), and miniature Dachshund (2, 3%). The remainder included one each of Alaskan malamute, Belgian malinois, Brittany spaniel, bullmastiff, Cavalier King Charles spaniel, German shorthair pointer, miniature schnauzer, Parson Jack Russel terrier, Scottish terrier, shorthaired chihuahua, Siberian husky, standard poodle, and Yorkshire terrier.
The median age was 10 years (range, 5–15), and the median weight was 26.5 kg (range, 3.7–53.0). There was a sex predilection with 65% being male (37/58 castrated males, 1/58 sexually intact males) and 35% (20/58) being female (all spayed). Most dogs were asymptomatic (30/58, 52%), with the most common clinical signs being straining to defecate (13/58, 22%), weight loss (7/58, 12%), anal discharge (5/58, 9%), and polyuria and polydipsia (3/58, 5%). One dog presented with hyporexia and weight loss and was also hypercalcaemic. The ASACA was located in the right anal sac in 30/58 (52%) dogs, left anal sac in 21/58 (36%) dogs, bilaterally in 4/58 (7%) dogs, and was unknown in 3/58 (5%) dogs.
3.2. Serum calcium
Total serum calcium values were available for all dogs. The median time from the acquisition of a serum calcium value to abdominal imaging was 0 days (range, 0–21). Nineteen (33%) dogs were hypercalcaemic at the time of initial diagnosis. The median serum total calcium value for hypercalcaemic dogs was 14.45 mg/dL (range, 11.3–17.7 mg/dL). Of the 19 hypercalcaemic dogs, only three (16%) were reported to be polyuric and polydypsic.
3.3. Serum phosphorus
Serum phosphorus values were available for 51/58 (88%) of dogs. The mean value for serum phosphorus was 3.5 (range 2.0–5.1 mg/dL). Two (4%) dogs were hypophosphataemic (2.0 and 2.1 mg/dL), and no dogs were hyperphosphataemic. All dogs that were classified as having hypercalcaemia were normophosphataemic.
3.4. Classification of suspected metastatic lymph nodes
All included patients had AUS performed at the time of the initial diagnosis, by either a board‐certified radiologist or a radiology resident supervised by a board‐certified radiologist. The region of the iliosacral lymphocentre, surrounding the bifurcation of the aorta and cava, was well‐identified in all patients. Sixteen of 58 (28%) dogs had either lymph node cytology, histopathology, or both performed.
Of the 58 animals included in the study, 34 (59%) were classified as ‘suspected/confirmed metastasis‐free’, and 24 (41%) were classified as ‘suspected/confirmed metastatic’ based on abnormal lymph node characteristics on AUS and/or with histopathology/cytology. Figure 1A is representative of a normal lymph node on AUS, and Figure 1B is representative of an abnormal, or suspected metastatic, lymph node.
FIGURE 1.
Parasagittal/long‐axis images of the medial iliac lymph nodes in two dogs. (A) Medial iliac lymph node in a dog classified as ‘normal’. A‐plus signs denote the ventrodorsal thickness, and asterisks denote the craniocaudal length. This lymph node had a maximal thickness of 6 mm and a thickness‐to‐length ratio of 0.18. (B) Medial iliac lymph node in a dog classified as ‘abnormal’. Open calipers denote the ventrodorsal thickness, and asterisks denote the craniocaudal length. This lymph node had a maximal thickness of 22 mm and a thickness‐to‐length ratio of 1.0 and was classified as heterogenous.
In animals classified as ‘suspected/confirmed metastasis‐free’, 28/34 had visible and measurable lymph nodes on AUS. A total of 43 lymph nodes were evaluated. The median thickness of the nodes was 4 mm (range 2–8 mm), and the median length was 1.1 mm (range 0.4–5.3 mm). The T:L ratio was 0.29 (range 0.20–0.86). All of the ultrasonographically assessed lymph nodes except one (42/43) were described as homogenous with normal echogenicity. One patient in this category had a single lymph node with abnormal descriptors (hypoechoic). This node measured 6 mm in thickness and had a T:L ratio of 0.86. This node was thought to be most likely reactive based on the performing radiologist's clinical impression, as was retrospectively obtained from the original ultrasound report. Upon review, the reviewing radiologist (R. Mackenzie Hallman) categorized this lymph node as ‘suspected metastasis‐free' based on size and the absence of any other lymph nodes with abnormal characteristics or of abnormal size in this patient. This lymph node was subsequently biopsied and classified as reactive on histopathology.
In the 24 subjects classified as ‘suspected/confirmed metastatic’, a total of 65 lymph nodes were evaluated, with 63% having abnormal characteristics (41/65). The median thickness of the largest node on AUS was 28 mm (range 6–61 mm), and the median length was 5 mm (range 0.8–53 mm). The T:L ratio was 0.54 (range 0.27–1). Fifty‐five percent (36/65) of nodes were considered hypoechoic, and 45% (29/65) were described as heterogenous. Only five of 24 subjects had perinodal fat hyperechogenicity described. Fifty‐four percent (13/24) of subjects had multiple abnormal nodes, with the median number of abnormal nodes being 1.5 (range 1–3). Three subjects were classified as having ultrasonographically abnormal lymph nodes based on the echogenicity and other imaging characteristics, despite the lymph node size being within normal limits. The additional imaging characteristics that were used to determine the lymph nodes of normal size in these three subjects were suspected metastatic was the presence of perinodal hyperechoic fat and/or multiple lymph nodes with abnormal echogenicity within a single patient. All three of these lymph nodes were confirmed metastatic on histopathology.
Overall, among the 24 dogs in the ‘suspected/confirmed metastatic lymph node' group, 16 were sampled: 12 had histopathology alone, two had histopathology and cytology, and two had cytology alone. Metastases were confirmed in all cases sampled. In addition, two dogs with confirmed lymph node metastasis showed metastasis on histopathology of either the liver (1) or spleen (1). The eight cases that did not have cytology or histopathology performed were included in this category if they contained a single lymph node with more than one abnormal characteristic or more than one abnormal lymph node with a single abnormal characteristic. There were no cases of lymph nodes classified as ultrasonographically ‘suspected metastatic' in which cytology or histopathology diagnosed reactive or normal tissue samples.
3.5. Hypercalcaemia and lymph node metastasis
Of the 19 dogs with hypercalcaemia, 13/19 (68%) were classified as ‘suspected/confirmed metastatic' iliosacral lymph nodes, with 10/13 (77%) being confirmed with cytology and/or histopathology. Of the 39/58 (67%) animals whose total serum calcium was normal on initial presentation, 11/39 (28%) were classified as ‘suspected/confirmed metastatic’. Of the dogs with ’suspected/confirmed metastatic' lymph nodes, 11/24 (46%) were normocalcaemic. The number of hypercalcaemic dogs with ‘suspected/confirmed metastatic' and ‘suspected/confirmed metastasis‐free' lymph nodes and normocalcaemic dogs with ‘suspected/confirmed metastatic' and ‘suspect/confirmed metastasis‐free' lymph nodes are shown in Figure 2. Hypercalcaemia was significantly (p < 0.01) associated with the presence of ‘suspected/confirmed metastatic' lymph nodes.
FIGURE 2.
Graph demonstrating the number of hypercalcaemic dogs that were confirmed/suspected metastatic or confirmed/suspected metastasis‐free and the number of normocalcaemic dogs that were confirmed/suspected metastatic or suspected confirmed/suspected metastasis‐free.
The positive predictive value of hypercalcaemia on initial bloodwork was 68% for the presence of ‘suspected/confirmed metastatic' lymph nodes, and the negative predictive value was 72%. The presence of hypercalcaemia on initial bloodwork had a sensitivity of 54% and a specificity of 82% for the presence of ‘suspected/confirmed metastatic' lymph nodes.
4. DISCUSSION
The objective of this study was to determine if hypercalcaemia is associated with iliosacral lymph node metastasis, and the results suggest that dogs with increased total serum calcium levels at the time of diagnosis with ASACA are more likely to have suspected/confirmed lymph node metastasis than those with normal calcium levels. Approximately one third (33%) of the dogs included in this study were hypercalcaemic at the time of diagnosis. This is consistent with several prior studies demonstrating that 25% to 29% of dogs with ASACA had ionized hypercalcaemia (Barnes & Demetriou, 2017; Goldschmidt & Zoltowski, 1981; Williams & Packer, 2003; Wong et al., 2021); however, it is in contrast with another study that reported 51% (Bennett et al., 2002) of dogs were hypercalcaemic. In the Bennett study in 2002, 79% of the dogs had metastatic disease diagnosed on initial presentation, and therefore the greater percentage of hypercalcaemic dogs in that study may be due to the diagnosis of ASACA occurring later in the disease process for their sample. Of the dogs that were hypercalcaemic in the present study, 68% had suspected/confirmed lymph node metastasis, contrasted with 24% of dogs that were normocalcaemic. This finding rejects our hypothesis, since a positive association between hypercalcaemia and suspected/confirmed lymph node metastasis was identified.
Our current study found that 56% of dogs with suspected/confirmed metastatic disease had hypercalcaemia, and this contrasts with several previous studies that showed a prevalence ranging between 25% (Meier et al., 2016) and 29.8% (Tanis et al., 2021) using ionized calcium, and 31.6% (Huerta et al., 2022) using a combination of ionized and total calcium. This increase could potentially be due to the use solely of total calcium in our study rather than ionized calcium; however, in Tanis et al.'s study, nearly 20% of patients were reported as unknown in regard to hypercalcaemia, and Huerta et al.'s study used a combination of ionized and total calcium to define hypercalcaemia precluding the ability to directly compare our findings.
Additionally, our study utilized abdominal ultrasound to identify suspected metastatic lymph nodes. An abdominal CT is the gold standard for identifying abnormal abdominal lymph nodes for staging of ASACA as routine abdominal ultrasound scans do not fully evaluate all potential sites of metastasis such as the sacral and inguinal lymph nodes or pelvic bones, therefore leaving the possibility that some patients with metastatic disease in the current study were erroneously placed in the ‘confirmed/suspected metastasis‐free' group. The sacral lymph nodes are often the sentinel nodes (Linden et al., 2019; Majeski et al., 2017) and generally cannot be visualized on an AUS unless markedly enlarged. In addition, CT is more sensitive at identifying enlarged lymph nodes in the iliosacral lymphocentre than AUS (Palladino et al., 2016). Therefore, it is possible that dogs could have been incorrectly identified as having normal lymph nodes when abnormal sacral lymph nodes were present and not identified by the AUS. This underestimation of the number of dogs with metastatic disease may have artificially increased the prevalence of hypercalcemia in dogs with suspected/confirmed metastatic lymph nodes. That being said, it is nonetheless possible that in our study, some of the eight dogs with abnormal lymph nodes on ultrasound were erroneously classified as ‘metastatic' due to the lack of histopathological confirmation (Williams & Packer, 2003). In the future, a prospective study using abdominal CT to better detect abnormal lymph nodes in association with hypercalcaemia would be indicated.
It has also been shown that not all abnormal lymph nodes on ultrasound are truly metastatic, and therefore the number of metastatic lymph nodes may have been overestimated (Williams & Packer, 2003). In addition, this study showed that the presence of hypercalcaemia on initial bloodwork has a high specificity (82%) for suspected/confirmed lymph node metastasis; however, its sensitivity is low (54%). This suggests that hypercalcaemia cannot be used alone to predict the presence of lymph node metastasis, and while lymphadenopathy may be more likely in hypercalcemic dogs, further diagnostics such as abdominal imaging and lymph node cytology are still warranted in normocalcemic cases.
While hypercalcaemia is positively associated with suspected/confirmed lymph node metastasis, the absence of hypercalcaemia does not rule out its possibility. The current study found that 44% of dogs with suspected/confirmed lymph node metastasis were normocalcemic. The suspected primary factor in the development of hypercalcaemia in dogs with ASACA is circulating parathyroid hormone‐related protein (PTHrP) produced by the neoplastic cells (Gröne et al., 1998). This protein increases serum calcium through similar mechanisms as parathyroid hormone (PTH) by acting on osteoclasts to increase bone resorption and increase renal reabsorption of calcium (Mirrakhimov, 2015). This suggests that not all advanced staged ASACA tumours secrete PTHrP, or do not secrete enough PTHrP to lead to a clinically significant increase in serum calcium levels. Additionally, the secretion of PTHrP may not be the only factor that leads to hypercalcaemia in dogs with ASACA as there has been no study to document the association between PTHrP presence and hypercalcaemia in dogs diagnosed with ASACA. In the current study, no dogs were evaluated for PTHrP, and therefore further studies evaluating the presence of PTHrP in dogs with hypercalcaemia and lymph node metastasis would be warranted.
The current study agrees with prior studies (Bennett et al., 2002; Goldschmidt & Zoltowski, 1981; Ross et al., 1991; Williams & Packer, 2003) about the occult nature of ASACA, as 52% were diagnosed as an incidental finding. When present, the most common clinical signs of dogs diagnosed with ASACA were straining to defecate and weight loss. Only 16% of total hypercalcemic dogs in this study were reported to be polyuric. This is in contrast to two prior studies (Meuten et al., 1981; Tanis, 2022) that found a significantly higher percentage, 60%–64.7%, of dogs that were hypercalcaemic with polyuria and polydipsia. This could be due to total calcium overestimating the incidence of true hypercalcaemia in our study. It is also possible that owners were underreporting the incidence of polyuria and polydipsia in our study or that not all dogs with hypercalcaemia are also polyuric and polydipsic. Unfortunately, urinalyses were not performed routinely in the dogs in our study, and therefore detection of possible underreporting of polyuria was not possible. As such, concluding the incidence of polyuria and polydipsia in hypercalcaemic dogs from our study should be done cautiously.
The majority of the limitations of this study were associated with its retrospective nature. The clinical staging was at the discretion of the clinician in charge, and therefore not all dogs in this study received the same staging diagnostics. Therefore, it is possible that not all concurrent conditions that could affect serum calcium values were identified in each patient. The time between calcium assessment and AUS was up to 21 days, and therefore it is possible that hypercalcemia and/or lymph node metastasis could have occurred during that time frame. As previously discussed, the use of AUS may have underestimated the true prevalence of metastatic lymph node in our study. Additionally, we did not routinely perform cytology or remove lymph nodes in dogs that were considered normal at the time of staging. Only one lymph node that was categorized as suspected metastasis‐free was subsequently confirmed with histopathology and/or cytology. Therefore, we cannot definitively state that all dogs in this study classified as ‘suspected metastasis‐free’ were truly without metastasis.
In the current study, total serum calcium values were used to classify hypercalcaemia, as the majority of the patients in this study did not have an ionized calcium value. It has been previously reported that using total calcium to predict elevated ionized calcium may lead to an overestimation of normocalcaemia (Schenck & Chew, 2005). However, a more recent study demonstrated that total calcium may lead to an overestimation of elevated ionized calcium (Lebastard et al., 2021). In that study, the discordance between hypercalcaemia classified by total versus ionized calcium in non‐hyperphosphataemic patients was 34.1% (Lebastard et al., 2021). Therefore, the current study could have underestimated or overestimated the number of dogs that were truly hypercalcaemic. In addition, several different chemistry analyzers were used to obtain serum total calcium values. These analyzers had varying reference ranges and conditions for analysing samples, which precluded the ability to select a standard value for hypercalcaemia. This led to the inclusion of dogs with serum calcium values as low as 11.3 mg/dL (above the reference range of the specific analyzers), which could have contributed to an overestimation of true hypercalcaemia.
In one prior study (Meuten et al., 1981), 71% of patients were classified as hypophosphataemic, in contrast to 4% in the current study. As discussed above, the secretion of PTHrP is thought to be the main cause of hypercalcaemia in dogs with ASACA, and as PTHrP biologically acts like that of PTH, it may or may not cause concurrent hypophosphataemia. Therefore, the use of serum total calcium and therefore the potential for overestimation of true hypercalcaemia may explain this difference. However, the Meuten et al.'s study only contained 17 dogs with complete biochemical profiles available, and therefore the percentage of dogs with hypophosphataemia could have been falsely elevated due to a small sample size.
While the use of total calcium and multiple analyzers is a major limitation of the current study, many general practitioners do not have the ability to run in‐house ionized calcium and therefore will be making recommendations based on total calcium data from various analyzers. In conclusion, the current study establishes a positive association between increased total serum calcium and the presence of lymph node metastasis. While this association exists, using the presence of hypercalcaemia on initial bloodwork is not a sensitive test for the presence of lymphadenopathy, as 24% of normocalcaemic dogs were categorized as ‘suspected/confirmed metastatic’. Based on these findings, hypercalcaemia cannot be used as a screening tool for predicting metastatic lymph nodes, but when present, should raise concern for a higher likelihood of metastasis at the time of initial diagnosis. It is therefore recommended that complete staging with abdominal imaging and lymph node cytology is pursued in both hypercalcemic and normocalcemic dogs diagnosed with ASACA.
5. CONCLUSION
Based on these results, it is suggested that while the presence of hypercalcaemia may increase the likelihood of lymph node metastasis, hypercalcaemia alone cannot be used as a screening tool. Dogs diagnosed with ASACA should undergo full staging regardless of total serum calcium values.
AUTHOR CONTRIBUTIONS
Darby Toth drafted, revised, and approved the submitted version of the manuscript. David Upchurch revised the manuscript drafts critically and approved the submitted version of the manuscript. R. Mackenzie Hallman revised the manuscript drafts critically and approved the submitted version of the manuscript.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflicts of interest.
The authors have no funding information to disclose.
ETHICS STATEMENT
No ethical approval was required as this is a retrospective article with no original research data.
PEER REVIEW
The peer review history for this article is available at https://publons.com/publon/10.1002/vms3.1324.
Toth, D. , Upchurch, D. , & Hallman, R. M. (2024). Association between total hypercalcaemia and iliosacral lymph node metastasis in dogs diagnosed with anal sac adenocarcinoma using abdominal ultrasonography. Veterinary Medicine and Science, 10, e1324. 10.1002/vms3.1324
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions
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Associated Data
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Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions