SEARCH

SEARCH BY CITATION

Keywords:

  • Arthritis;
  • CD18;
  • Dendritic cells;
  • Pancytokeratin;
  • Synovium

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Background

Peri-articular histiocytic sarcoma (PAHS) occurs in dogs, including Bernese Mountain Dogs (BMD). An etiologic relationship with previous joint disease has not been documented.

Hypothesis

Peri-articular histiocytic sarcoma in BMD will be more frequently encountered around previously diseased joints compared with normal joints.

Animals

920 European BMD.

Methods

A retrospective study, in which data were obtained through an Internet questionnaire and from 2 veterinary pathology laboratories. Archived samples of hematoxylin-eosin (H&E) staining diagnosed PAHS and synovial cell sarcoma (SCS) were immunolabeled with CD18 and pancytokeratin. Descriptive, comparative, and actuarial statistics comprise the data analysis.

Results

All primary synovial tumors were identified as PAHS based on their morphology, positive CD18, and negative pancytokeratin labeling. Joint disease was diagnosed in 226 BMD, of which 15 developed PAHS in a previously diseased joint and 3 in a nondiseased joint. Of the remaining 694 BMD without joint disease, 9 developed PAHS. The odds ratio for a dog with previous joint disease developing PAHS is calculated as 5.4 (95% CI: 2.3–12.5; P < .0001) compared with no previous joint problem. A significant association between previous joint disease and PAHS in the same joint was demonstrated for the left elbow (P = .016), right elbow (P = .006), right shoulder (P = .047), left and right stifle (P < .001), and left carpal joint (P = .010).

Conclusions and Clinical Importance

The results of this study suggest a relation between previous joint disease and the development of PAHS in the same joint of European BMD. Owners of BMD should monitor dogs for peri-articular swellings, particularly around previously diseased joints.

Abbreviations
BMD

Bernese Mountain Dog

FMCP

fragmented medial coronoid process

H&E

hematoxylin-eosin

PAHS

peri-articular histiocytic sarcoma

SCS

synovial cell sarcoma

Histiocytic sarcoma complex is more frequently recognized in Bernese Mountain Dogs (BMD), Rottweilers, and Retrievers, compared with other breeds of dog.[1, 2] In BMD, a genetic predisposition is suspected.[2-5] The histiocytic sarcoma complex includes hemophagocytic, disseminated, and localized histiocytic sarcoma.[1, 6, 7] Localized histiocytic sarcoma most often occurs as a primary lesion involving the skin and subcutis of the extremities, though it can also be found in peri-articular tissues of large appendicular joints, in the spleen, lymph nodes, lung, or bone marrow.[1, 8] In a group of 50 dogs, of different breeds with histiocytic sarcoma, an incidence of peri-articular histiocytic sarcoma (PAHS) of 38% (19 cases) is reported. Of these 19 dogs, there was only 1 BMD.[8] It has been suggested that PAHS has a more indolent clinical course compared with other localized histiocytic sarcomas, and has the best prognosis if treated early by definitive surgical excision, radiation therapy, or both, in combination with adjuvant chemotherapy.[8]

Identification of the cell origin in synovial tumors is not always possible based on routine histological examination.[9-11] Ancillary techniques, such as immunohistochemistry, have proven to be of use in determination of this cell of origin. In synovial tumors, immunolabeling for CD18, cytokeratin, and smooth muscle actin is recommended for differentiation between PAHS and synovial cell sarcoma (SCS).[9]

The hypothesis of this study was that PAHS in BMD will be more frequently encountered around previously diseased joints compared with normal joints. The objective was to investigate this hypothesis in the European BMD population through an Internet questionnaire and data from 2 veterinary diagnostic pathology laboratories.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Data Collection

Data from European BMD were obtained through an Internet survey, which has been opened from September 1, 2008 to February 1, 2010. For this web-based questionnaire, the online Check Market software was used. The questionnaire was available in 10 different languages and contained in total 29 questions (Table 1).

Table 1. Overview of the questions in the internet survey
QuestionsSub Questions
  1. a

    Osteochondritis dissecans.

  2. b

    Fragmented medial coronoid process.

  3. c

    Ununited anconeal process.

  4. d

    Cranial cruciate ligament rupture.

  5. e

    Hip dysplasia.

  6. f

    Luxated patella.

  7. g

    Histiocytic sarcoma.

  8. h

    Synovial cell sarcoma.

1. Survey number of the owner 
2. Dog's name
3. Chip registration number
4. Pedigree number of the dog
5. Dog's date of birthDayMonthYear 
6. SexMaleFemale  
7. Is the dog still alive?YesNo  
8. If no, when did the dog die?DayMonthYear 
9. Is the dog neutered?YesNo  
10. If neutered, at what age? 
11. Has your dog ever been lame?YesNo  
12. Which leg was affected?Forelimb leftForelimb rightHind limb leftHind limb right
13. Localization of lamenessJointOther  
14. Which joint? Shoulder leftShoulder rightElbow leftElbow right
Carpus leftCarpus rightHip leftHip right
Stifle leftStifle rightTarsus leftTarsus right
15. Cause of lamenessOCDaFMCPbUAPcCCLRd
HDeLPfOther 
16. Date of onset (approximately)DayMonthYear 
17. TreatmentSurgeryArthroscopyMedicationPhysiotherapy
18. More info about surgery/arthroscopyWhenLocalizationWhy 
19. Has your dog ever had a tumor?YesNo  
20. If yes: what type of tumor?HSgSCShOther, specify if known 
21. Localization of the tumorLimbJointOther 
22. If joint, which joint?Shoulder leftShoulder rightElbow leftElbow right
Carpus leftCarpus rightHip leftHip right
Stifle leftStifle rightTarsus leftTarsus right
23. More info about tumorDate of diagnoseRemovalSize
24. Owner's name   
25. Owner's address   
26. Owner's email address   
27. Name & address veterinary practice   
28. Do you allow us to contact your veterinarian?YesNo  
29. Any other comment? 

The national European BMD societies were contacted and the study was announced in their newsletters and on their websites. Owners were asked to complete the questionnaire, also whether their dog was no longer alive. Introductory information to the owners and on the homepage of the website was carefully designed to avoid mentioning the goal of the study. Inevitably, the board members of the European BMD societies were aware of the intention of the study. An audit was performed to ensure that dogs were not more than once included. Questionnaires that were not completed were excluded. Diagnosis of joint disease, mentioned by the owner on the web form, was accepted in first instance, and made the dog eligible to enter the study, on the condition that the questionnaire was completed.

Additionally, the BMD data files from 2 veterinary diagnostic pathology laboratories were obtained. Of one, clinicopathologic data were collected from 2006 to 2008 and of the second one from 2000 to 2008. These data were compared with the Internet questionnaire to avoid duplications in the study. To overcome possible selection bias by adding data from both pathology laboratories to the online obtained data, all veterinarians of the BMD originating from both pathology labs were either contacted by phone or by a written letter. They were invited to provide information on the health status of the joints of the specific dog. Without this explicit information, the dog was not eligible to enter the study. In doing so, possible threats to the internal validity have been minimized.

All veterinarians of the BMD initially diagnosed with PAHS or SCS, independent through which arm they entered the study, were contacted to obtain the relevant history of the dog.

Histologic Confirmation and Identification

At first, dogs with PAHS and SCS based on archived histopathologic diagnosis of hematoxylin-eosin (H&E) stained samples were included in the study. All archival cases were reviewed by 1 pathologist of each laboratory. The diagnosis of a primary synovial neoplasm was confirmed based on the pleomorphic morphology of the cells: spindle cells, round cells, and multinucleated cells.

All histologic samples initially classified as SCS and 4 randomly chosen samples initially classified as PAHS were immunolabeled for cytokeratin (AE1/AE3) and CD18, as described by Craig et al.[9]

Statistical Analysis

Chi-square test was used to compare the number of dogs developing PAHS with previous joint disease versus no previous joint disease. The odds ratio between these 2 groups was also calculated. In order to analyze possible associations between previous disease in specific joints and PAHS, Fisher Exact tests were used.[12] To test the difference in age between the 2 groups of dogs developing PAHS, with previous joint disease versus no previous joint disease, a 2 sample (unpaired) t-test was performed.

Although not a primary aim of this study, survival analysis was performed for dogs after being diagnosed with PAHS. Survival time (days) was defined as date of death minus date of tumor diagnosis. Kaplan–Meier product-limit analysis was performed. Dogs dead from disease or related causes were classified as events; dogs dead from unrelated causes or lost to follow-up at the time of study closure were censored. Logrank test for equality was used to compare survival times between pairs of variables (previous joint disease versus no previous joint disease; treatment versus no treatment). Median survival times and 95% confidence intervals (95% CI) were derived from the Kaplan–Meier data. P-values of <.05 were considered significant for all tests. All calculations were performed using commercial statistical software packages.1

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Data were obtained from 1554 BMD, submitted from 12 different European countries and both pathology laboratories. After removal of duplicates, 1513 BMD remained, of which 103 had histiocytic sarcoma on different locations in the body, including PAHS. After deletion of incomplete files, 920 dogs were included in the study, of which 471 were male and 449 female.

Nine BMD with PAHS originated from the questionnaire. This diagnosis was confirmed by the original pathology report, additionally submitted by the owner. Ten dogs with PAHS, all eligible for the study, originated from the pathology clinicopathologic data-files. From those dogs, mentioned to have histiocytic sarcoma on a non peri-articular location, only pathology reports were available when the data originated from the 2 pathology labs. Because histiocytic sarcoma on a nonperi-articular location in the body was beyond the scope of the study, pathology reports of these dogs originating from the Internet questionnaire were not recruited. Other tumor types were diagnosed in 85 BMD, however, none of them in a peri-articular localization.

Diagnosis of PAHS and SCS Based on H&E Staining

In total, 19 dogs had 20 PAHS, based on histological examination of H&E stained samples. One dog developed synchronously 2 histiocytic sarcomas in both stifle joints.

Ten dogs were initially diagnosed with SCS, 9 through H&E staining on histological biopsies and 1 on cytology. Eight had an appendicular SCS, 1 was located in a temporomandibular joint, and 1 in the vertebral column. The latter 2 were excluded from the study, because the questionnaire was directed to appendicular tumors.

Reclassification of SCS Based on Immuonohistochemistry

One of the 8 appendicular SCS was diagnosed on cytology and so excluded for immunohistochemistry. Immunolabeling with CD18 was positive on all 7 primary synovial tumors initially diagnosed as SCS on H&E staining and 4 randomly chosen PAHS, whereas none of them reacted with the pancytokeratin marker (Fig 1). Based on these findings, the tumors initially diagnosed as SCS were considered as PAHS for the study, including the SCS diagnosed on cytology.

image

Figure 1. Positive CD18 (A) and negative pancytokeratin (B) immunolabeling in PAHS.

Download figure to PowerPoint

Statistical Results

A diagnosis of joint disease was made in 226 BMD, of which 15 developed PAHS in a previously diseased joint. Three dogs with previous joint disease developed PAHS in a nondiseased joint, including the dog with the initially cytology diagnosed SCS. Of the remaining 694 BMD without joint disease, 9 developed PAHS. From these numbers an odds ratio of 5.4 (95% CI: 2.3–12.5) can be calculated as an estimation of the risk for a dog of developing PAHS in a previously diseased joint compared to a dog with no diseased joints. The corresponding Chi-square test yielded a value of 19.14 (df = 1; P < .0001).

Of the 15 dogs that developed a PAHS in a previously diseased joint, 5 had a ruptured cranial cruciate ligament, 1 had a chronic patella luxation, 3 had a fragmented medial coronoid process (FMCP), 1 suffered from elbow incongruence resulting in an arthritic joint, 1 had a ruptured biceps tendon, and 4 dogs had joint trauma because of an accident. In those joints of these 15 dogs treated surgically, no tumor was macroscopically visible at the time of surgery for the original disease.

There was a significant association between previous joint disease and the development of PAHS in 6 out of 12 joints (Table 2).

Table 2. Overview of the numbers of diseased joints and the numbers of PAHS per diseased and nondiseased joint. Of the 226 BMD with joint disease, 130 had one joint affected, 70 two joints, 12 three joints, 9 four joints, 2 five joints, 1 six joints, and in 2 dogs eight joints were diseased. Statistical results for the individual joints, depicting the association of previous joint disease and the development of PAHS in the same joint, are shown as Fisher Exact values. The association was significant for 6 out of 12 joints
 226 BMD with Joint Disease694 BMD without Joint DiseaseFisher Exact
Diseased JointsDiseased Joints with PAHSNondiseased Joints with PAHSPAHS
  1. a

    One dog developed a PAHS in both stifle joints.

Shoulder left20000n.a.
Shoulder right22101P = .047
Elbow left69201= .016
Elbow right70200= .006
Carpus left9100= .010
Carpus right10000n.a.
Hip left35000n.a.
Hip right40001= 1.000
Stifle left39513a< .001
Stifle right45413a< .001
Tarsus left7001= 1.000
Tarsus right8010= 1.000
Total37415310 

Peri-articular histiocytic sarcoma developed in dogs with previous joint disease in the same joint at a median and mean age of 6.7 and 6.5 years (SD = 1.28; n = 15), respectively. In dogs without previous joint disease in the same joint, the median and mean age being diagnosed with PAHS was 7.6 and 7.2 years (SD = 2.66; n = 12), respectively. However, this difference in age of onset was not statistically significant (t(25) = 0.98; P = .34).

The median and mean time elapsed between previous joint trauma and development of PAHS in the same joint was 1.5 and 1.9 years (SD = 2.17; n = 13), respectively. Two dogs with FMCP, in which PAHS was diagnosed at an age of 5.5 and 5 years respectively, were censored for this calculation, because the cause of the long-lasting arthritis of the elbow joint was diagnosed at time of tumor diagnosis, and the onset of lameness caused by FMCP was unknown.

A ruptured cranial cruciate ligament was the only cause of joint injury that showed a significant association with the development of PAHS by means of a Fisher's Exact test (P = .026).

Sex was not found to be associated with the development of PAHS (Fisher's Exact test, P = .44).

At the time of analysis, follow-up time data were unknown for 3 of the 27 dogs diagnosed with PAHS and these were not included in the survival analysis. Of the remaining 24 dogs, 1 died of immune mediated hemolytic disease (considered unrelated death, therefore censored). The other 23 dogs all died of PAHS or a related problem. From the Kaplan–Meier product limit data, the median survival time after diagnosis for the cohort of 24 dogs with PAHS was 61 days (95% CI: 50.4–81.8 days). For those dogs (n = 13) with previous joint disease, the median survival time was 49 days (95% CI: 31.6–83.1); for those dogs without known previous joint disease (n = 11) the median survival was 173 days (95% CI: 12.8–487.0) (Fig 2). By logrank test, this difference was statistically significant (P = .035).

image

Figure 2. Survival probabilities estimated by Kaplan–Meier method for dogs with PAHS, with and without previous joint disease. Arrow represents censored case.

Download figure to PowerPoint

One dog was treated with surgery alone, 1 with surgery and lomustine, and 2 dogs were treated with lomustine only. The use of prednisolone was not considered as a treatment. Assessing the effect of treatment, the logrank test showed no statistical difference (P = .73) between the survival times of those dogs (n = 4) receiving treatment (146 days; 95% CI: 82.1–271.3) and those (n = 20) not receiving treatment (61 days; 95% CI: 51.6–79.5).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

The overall odds ratio of 5.4 suggests a statistically significant association between previous joint disease and the development of a PAHS in that joint. Despite the small number of BMD with PAHS and previous joint disease in the same joint, the significance of the associations clearly point in the direction of a relation between previous joint disease and the development of PAHS within the European BMD population. Because data were not available for all specific appendicular joints, this association cannot be generalized yet for random previously diseased joints, but a strong indication of such a relation is shown in 6 out of 12 joints (Table 2). These results are in line with the hypothesis of a previous study, that rupture of the cranial cruciate ligament, resulting in degenerative joint disease, is associated with a higher incidence of PAHS.[9]

The statistically significant difference in survival time between BMD with PAHS with and without previous joint disease is not easy to explain. Development of a tumor in an already chronically diseased joint could have more clinical impact on mobility and lameness, compared with PAHS development in a previously healthy joint, leading to an early decision of euthanasia. Demotivation of the owners to continue treatment after diagnosis of PAHS in BMD already suffering from chronic degenerative joint disease for a longer period, also could influence survival time.

In our retrospective study, identification of histiocytic sarcomas and SCS was initially based on routine examination of H&E slides. However, differentiation of both tumors cannot easily be done on H&E staining, and immunohistochemistry for differentiation at least CD18 and pancytokeratin should be performed.[9] Craig et al described that only a minority (14%) of on H&E diagnosed SCS, stained positive for pancytokeratin, whereas 50% were positively labeled with CD18, and therefore were reclassified as PAHS.[9] Based on this information, CD18 and pancytokeratin immunolabeling was performed on all appendicular SCS in our study, diagnosed on histology. All of them were pancytokeratin negative and CD18 positive histiocytic sarcomas, confirming the results of Craig et al[9] and demonstrating that H&E staining alone is not sufficient to make a diagnosis of SCS in veterinary medicine.

The authors are aware of the fact that the results of this study do not point to a causal effect of joint disease in the carcinogenesis of PAHS, but only show an association between these 2 phenomena. However, one could hypothesize that tumor-promoting inflammation, one of the newly proposed enabling characteristics within the hallmarks of cancer, in general could help to understand the pathogenesis of PAHS in previously diseased joints.[13-15]

The normal synovial membrane in dogs consists of a mixture of fibroblasts and bone-marrow derived macrophages.[11, 16, 17] Several studies showed that in the inflamed synovial membranes of dogs with degenerative joint disease, next to T- and B-lymphocytes and plasma cells, increased numbers of CD18 expressing dendritic cells are present.[18, 19] Also one of the coauthors of our study did find a considerable number of CD18 expressing cells in synovial membranes of joints with chronic hypertrophic synovitis, which may link to histiocytic cells and their precursors (unpublished data, E. van Garderen; Fig 3).

image

Figure 3. Chronic hypertrophic synovitis demonstrating CD18 positive immunolabeling (A) and negative pancytokeratin staining (B).

Download figure to PowerPoint

BMD are considered genetically predisposed to develop histiocytic tumors through one or more germ line mutations.[2, 5] We hypothesize that in BMD, a combination of tumor-promoting inflammation in synovial membranes of joints with chronic degenerative disease and genetically predisposed cells from the histiocytic lineage, present in these synovial membranes, may lead to PAHS formation. However, prospective studies on PAHS development need to be designed to investigate this hypothesis. The moment the causative genes for HS in BMD are discovered, a follow-up of genetically predisposed and nonpredisposed BMD with joint disease could lead to a better understanding of the tumorigenesis of PAHS in this breed.

The nature of self-selection sampling does not imply high external validity (generalizability), especially because the selected cases for this study need to meet the aforementioned inclusion criteria.[20, 21] It is recommended that in future studies methods to minimize this limitation are taken into consideration. However, we feel that there is enough evidence to recommend owners of BMD to be extra aware of any peri-articular mass around previously diseased joints.

On the other hand, there is also a possibility that BMD are misdiagnosed as having degenerative joint disease when they actually have PAHS. Veterinarians must be extra aware of this differential diagnosis in BMD, presenting with lameness originating from a clinical abnormal joint.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Study was supported by the Società Italiana Bovaro del Bernese, 2007 Alberto Vittone Award.

Conflict of Interest : Authors disclose no conflict of interest.

Footnote
  1. 1

    SPSS for Windows Version 15 and 18; Statviews for Windows v4.57, Abacus Concepts Inc for Kaplan–Meier analysis, Piscataway, NJ

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References