The spleen is the most common primary site for hemangiosarcoma in dogs. This type of tumor is characterized by rapid growth and widespread metastasis, presumably owing to its tissue of origin, the vascular endothelium, and its resultant ready access to systemic circulation. Splenectomy provides palliation for splenic hemangiosarcoma, but median survival time is < 3 months; survival for > 1 year is uncommon.1–3
Clinical staging of dogs with hemangiosarcoma appears to be of prognostic benefit in some studies, particularly those in which adjuvant treatment is provided after surgery. Dogs with stage I disease have small (< 5-cm-diameter) tumors confined to the spleen with no evidence of metastasis, whereas dogs with stage II disease have tumors > 5 cm in diameter or have evidence of rupture, with or without regional lymph node involvement.4 Stage III disease is characterized by distant metastases.4 Among dogs with hemangiosarcoma treated by splenectomy and adjuvant chemotherapy, those with stage I disease have been found to live longer than those with stage II or III disease.2,3,5
Adjunctive chemotherapy with anthracyclines, alone or in combination with other drugs, appears to approximately double the survival time for dogs with stage II splenic hemangiosarcoma, compared with results for dogs undergoing surgery alone. Reported median survival times for dogs with stage II splenic hemangiosarcoma treated with surgery and adjuvant chemotherapy have ranged from 96 to 186 days,2,5–11 including dogs treated with epirubicin (median survival time, 98 days [n = 3]),2 doxorubicin (133 days [24]),6 and doxorubicin in combination with cyclophosphamide (96 days [9]5 and 186 days [4]8), ifosfamide (123 days [13]),9 deracoxib (120 days [7]),10 or vincristine and cyclophosphamide (133 days [17]).11 One study7 of 34 dogs treated with free or pegylated liposome–encapsulated doxorubicin after splenectomy did not separate dogs by disease stage for analysis; however, the information provided indicated that 30 dogs had stage II disease, 1 had stage III disease, and 3 had stage I or II disease, with an overall median survival time of 166 days. The 1-year survival rates for these dogs (when available) have ranged from 0%6 to 23%.7 Results of a more recent study3 suggested chemotherapy after splenectomy improved survival rates of dogs with this disease, but did not separately report findings for dogs with stage II disease.
Histologic grading as a prognostic factor for dogs with hemangiosarcoma (originating from any site) was first reported in 1996.12 The scheme took differentiation of the tumor cells as well as cellular pleomorphism, percentage of the tumor that was necrotic, and mitotic index (number/10 hpf) into account to arrive at a final grade. Tumor grade (and specifically nuclear pleomorphism and mitotic scores) appeared to predict survival time for these dogs, which were treated with surgery and doxorubicin. To the authors' knowledge, only 1 additional study2 has evaluated histologic features as a factor affecting outcome for this disease, and those investigators found that increasing mitotic rate (number per hpf) was associated with an increased risk of death (hazard ratio, 1.45), but further details were not provided.
As previously mentioned, anthracycline agents are the mainstay of adjuvant chemotherapy for hemangiosarcoma in dogs. Lomustine, or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, may have activity in treating sarcomas as determined by responses in cats and by responses in dogs with histiocytic sarcomas. The overall response rate to lomustine in 28 cats with measurable soft issue sarcomas was 25%.13 In addition, lomustine treatment resulted in a response rate of 27%14 to 46%15 in dogs with histocytic sarcomas, and dogs with these tumors that were treated with radiation and chemotherapy that included lomustine had a longer median survival time (208 days [n = 9]) than did dogs treated with surgery or radiation therapy alone or with a combination of radiation therapy and chemotherapy that did not include lomustine (68 days [28]).16 In the latter study,16 receiving > 3 doses of lomustine was associated with an even greater survival advantage. To the authors' knowledge, the effects of lomustine for the treatment of hemangiosarcoma in dogs have not been reported.
The purpose of the study reported here was to retrospectively evaluate histologic and clinical factors associated with survival times in a group of dogs with stage II splenic hemangiosarcoma that were treated with an alternating anthracycline and lomustine protocol after splenectomy.
Materials and Methods
Case selection criteria
Electronic medical records of 3 veterinary practices (Animal Referral Hospital, Homebush, NSW, Australia; Oncology Consultation Service, Veterinary Medical Center of Central New York, East Syracuse, NY; and Veterinary Oncology Consultants, Wauchope, NSW, Australia) were reviewed to identify dogs treated for stage II splenic hemangiosarcoma from June 15, 2011, to October 2, 2014. Criteria for entry in the study included histologic confirmation of hemangiosarcoma, staging information sufficient to exclude gross metastatic disease at the time of splenectomy (no thoracic structure involvement identified by 3-view thoracic radiography and no abdominal visceral involvement [with particular attention to the liver] evident by abdominal ultrasonography, exploratory laparotomy, or both), a histologic sample (tissue samples or slides) available for retrospective review, and postoperative treatment with a chemotherapy protocol of lomustine alternating with an anthracycline.
Medical records review
Information extracted from the medical records included patient breed, age, weight, sex, and reproductive status; results of diagnostic imaging and other methods used to assess for the presence of metastatic disease; presence or absence of anemia (Hct or PCV < 37%) prior to splenectomy; date of splenectomy; chemotherapy protocol, including drug dosages; any adverse effects noted; and date of death, if applicable. Follow-up was performed by telephone contact with primary care veterinarians for dogs that did not have follow-up information available in the records. The facility at which dogs were treated was also recorded.
Evaluation of histologic slides
All available histologic slides were retrospectively reviewed by the same veterinary pathologists who performed the original (diagnostic) histologic evaluations (12 individuals were invited to participate). The same histopathologic criteria12 were used in each case and applied to a representative slide if > 1 was available. These included the number of mitoses/10 hpf (400×), tumor differentiation as assessed by the presence and number of vascular channels, presence and degree of nuclear pleomorphism, and percentage of the tumor that was necrotic, each scored from 0 and 3 as previously described (Appendix).12 These values were summed, and the final score between 0 and 12 was used to determine overall histologic grade (grade 1 = 1 to 5; grade 2 = 6 to 9; and grade 3 = 9 to 12). The method used by all pathologists in the study for determination of mitotic counts was to scan for the area of the section with the highest mitotic activity and count mitoses in 10 adjacent fields in that area, omitting fields of necrosis and hemorrhage and instead using the next adjacent field for evaluation, as previously described.17,18
Chemotherapy
The chemotherapy protocol at each institution consisted of lomustine (50 to 90 mg/m2 [delivered to the nearest 5 mg], PO, q 6 wk), alternating every 3 weeks with an anthracycline (doxorubicin, 30 mg/m2 [1 mg/kg [0.45 mg/lb} for dogs weighing < 15 kg], IV; mitoxantrone, 5.5 to 6 mg/m2, IV; or epirubicin, 30 mg/m2, IV). Prophylactic antimicrobials (trimethoprim-sulfamethoxazole, trimethoprim-sulfadiazine, or enrofloxacin) and an antiemetic agent (maropitant) were administered with anthracyclines as well as with the initial dose of lomustine. Chemotherapy was started 11 to 28 days from the date of splenectomy. A total of 5 doses of lomustine and 5 doses of an anthracycline were planned; drug dosages were modified at the attending oncologist's discretion. At the end of the protocol, owners were offered the option of continuing treatment with metronomic administration of cyclophosphamide (15 mg/m2/d, PO, for durations deemed appropriate on the basis of clinical signs). When a drug dose or the number of treatments with a given drug was reduced, the reasons were recorded and a toxicosis grade, when applicable, was retrospectively assigned by application of the previously described Veterinary Cooperative Oncology Group's common terminology criteria for adverse events.19 Dogs that had drug dose reductions or schedule modifications because of toxicosis were retained in the analysis and were categorized according to the primary (original) treatment for treatment-based comparisons.
Statistical analysis
The main outcome evaluated was survival time, with the endpoint being death due to any cause; the day of splenectomy was considered day 1 for the analyses. Dogs were censored from survival analysis at the last day of contact if still alive or if lost to follow-up.
Variables evaluated for associations with overall survival time included the retrospectively assigned histologic scores, tumor grade, presence or absence of anemia prior to splenectomy, dose of lomustine (grouped as 80 to 90 mg/m2, 70 mg/m2, or 50 to 60 mg/m2), type of anthracycline administered, and whether metronomic cyclophosphamide treatment was provided in addition to the standard chemotherapy protocol. The Kaplan-Meier product limit method was used to estimate survival times for each potential risk factor. To evaluate the combined effects of potential risk factors on survival time, multivariate survival analysis was performed with forward conditional Cox regression analysis. Variables with values of P ≤ 0.1 in the univariate analysis were included in the multivariate analysis. All statistical analyses were performed by use of a software package.a For the final analysis, values of P < 0.05 were considered significant.
Results
Thirty dogs met the study inclusion criteria (6 were treated at Animal Referral Hospital, 7 at Oncology Consultation Service, and 17 at Veterinary Oncology Consultants). The median age was 10 years (range, 6 to 17 years), and median weight was 28 kg (61.6 lb; range, 8 to 47.6 kg [17.6 to 104.7 lb]). Three dogs weighed < 10 kg (22 lb). There were 15 females (14 spayed and 1 sexually intact) and 15 males (14 castrated and 1 sexually intact). Nine were crossbred dogs, and 6 were German Shepherd Dogs (the most commonly represented purebred). All 30 dogs were screened for evidence of gross thoracic metastases by 3-view thoracic radiography. The determination that there was no gross evidence of metastasis to the liver was made by pretreatment abdominal ultrasonography (n = 27), evaluation during splenectomy (30), or both; this was confirmed by biopsy and histologic examination of a liver sample in 4 dogs. Lymph node metastasis was not suspected or identified in any dog. Lack of evidence for a cardiac tumor was confirmed by cardiac ultrasonography in 7 dogs. Of 27 dogs that had Hct or PCV values recorded, 23 were anemic before splenectomy.
Histopathologic review of slides was performed for 28 of 30 patients (1 pathologist declined to participate). Of those 28 dogs, 9, 16, and 3 had overall histologic tumor grades of 1, 2, and 3, respectively. The mitotic score was 0 for 9 dogs, 1 for 7 dogs, 2 for 7 dogs, and 3 for 5 dogs.
In addition to lomustine, 25 of 30 dogs received doxorubicin, 4 received mitoxantrone, and 1 received epirubicin. Doses of lomustine were 80 to 90 mg/m2 for 10 dogs, 70 mg/m2 for 13 dogs, and 50 to 60 mg/m2 for 7 dogs. At the end of the protocol, 5 dogs had treatment continued with metronomic cyclophosphamide administration.
No dogs were lost to follow-up. Four dogs were still alive at 233, 358, 372, and 444 days after splenectomy. All other dogs had died; 1 dog had death attributed to suspected renal failure 292 days after splenectomy, with no clinical evidence of metastases. For the other 25 dogs, metastatic disease was believed to be the cause of death on the basis of clinical findings and diagnostic imaging (n = 15) or presence of hemoabdomen without additional imaging (10). No dog had metastasis confirmed by histopathologic evaluation.
The overall median survival time was 158 days (range, 55 to 560 days), with 4 of 30 dogs censored as alive at the last follow-up. The 1-year survival rate was 16%.
On univariate analysis, the presence of anemia, necrosis score, and nuclear pleomorphism score were not significantly associated with survival time (Table 1). Mitotic score (P = 0.002), tumor differentiation score (P = 0.014), and overall histologic tumor grade (P = 0.018) were each associated with survival times, as was the dose group for lomustine (P = 0.015) and whether a dog received metronomic cyclophosphamide (P = 0.017). This latter finding was biased, as cyclophosphamide was only given to dogs that completed the assigned chemotherapy protocol; when only dogs completing the protocol were considered, the association between cyclophosphamide and survival time was lost (P = 0.058).
Results of univariate regression analysis for associations between variables of interest and survival time in 30 dogs with stage II hemangiosarcoma that underwent splenectomy followed by a chemotherapeutic protocol in which anthracyclines and lomustine were administered alternately.
| Variable | Category | No. of dogs | MST (d) | P value |
|---|---|---|---|---|
| Tumor differentiation score | 1 | 7 | 336 | 0.014 |
| 2 | 16 | 158 | ||
| 3 | 5 | 116 | ||
| Nuclear pleomorphism score | 0 | 1 | 137 | 0.765 |
| 1 | 3 | 132 | ||
| 2 | 17 | 217 | ||
| 3 | 7 | 116 | ||
| Tumor necrosis score | 0 | 3 | — | 0.549 |
| 1 | 19 | 132 | ||
| 2 | 3 | 145 | ||
| 3 | 3 | 228 | ||
| Mitotic score | 0 | 9 | 292 | 0.002 |
| 1 | 7 | 212 | ||
| 2 | 7 | 118 | ||
| 3 | 5 | 116 | ||
| Overall histologic grade | 1 | 9 | 336 | 0.018 |
| 2 | 16 | 145 | ||
| 3 | 3 | 116 | ||
| Anemia prior to splenectomy | Absent | 4 | 217 | 0.289 |
| Present | 23 | 137 | ||
| Anthracycline administered | Doxorubicin | 25 | 172 | 0.335 |
| Mitoxantrone | 4 | 132 | ||
| Epirubicin | 1 | 137 | ||
| Lomustine dose (mg/m2) | 80–90 | 10 | 336 | 0.015 |
| 70 | 13 | 202 | ||
| 50–60 | 7 | 116 | ||
| Metronomic cyclophosphamide administration | Yes | 4 | 560 | 0.017 |
| No | 26 | 137 |
Dogs that had drug dose reductions or schedule modifications (n = 9) were retained in the analysis. Histologic slides were reviewed for 28 dogs, and information on presence or absence of anemia (Hct or PCV < 37%) prior to splenectomy was available for 27 dogs.
MST = Median survival time.
— = A median survival time was not calculated by the Kaplan-Meier method because 2 dogs were censored. Mean survival time of these dogs was ≥ 444 days.
On multivariate analysis, only mitotic score remained significantly (P = 0.002) associated with survival time. The median survival time for 9 dogs with a mitotic score of 0 (< 11 mitoses/10 hpf) was 292 days (range, 64 to 560 days [with 3 dogs still alive]); the 1-year survival rate for this group was 42%. For 7 dogs with a mitotic score of 1 (11 to 20 mitoses/10 hpf) and 7 dogs with a mitotic score of 2 (21 to 30 mitoses/10 hpf), median survival times were 212 days (range, 82 to 336 days) and 118 days (range, 55 to 291 days [with 1 dog still alive]), respectively; for 5 dogs with a mitotic score of 3 (> 30 mitoses/10 hpf), the median value was 116 days (range, 78 to 172 days). Survival times of dogs with mitotic scores of 2 and 3 did not differ significantly (P = 0.61); a combination of these 2 groups and comparison of results for dogs with mitotic scores of 0, 1, and ≥ 2 confirmed significant (P = 0.002) differences in survival times (Figure 1).
Results of Kaplan-Meier survival analysis for 28 of 30 dogs with stage II splenic hemangiosarcoma that underwent splenectomy followed by a chemotherapeutic protocol in which anthracyclines and lomustine were administered alternately. Dogs were grouped for analysis by tumor mitotic scores of 0 (solid line; 9 dogs; median survival time, 292 days), 1 (dashed line; 7 dogs; median survival time, 212 days), or ≥ 2 (dotted line; 12 dogs; median survival time, 116 days). Mitotic scores were determined on retrospective review of histologic slides by pathologists following a predetermined grading scheme, where grade 0 = < 11; grade 1 = 11 to 20; grade 2 = 21 to 30; and grade 3 = > 30 mitoses/10 hpf.12 Two dogs were excluded because the histologic slides were not reviewed. Survival time for dogs with scores of 2 and 3 did not differ significantly, and these were combined for the analysis shown. Vertical marks indicate censored data points.
Citation: Journal of the American Veterinary Medical Association 251, 5; 10.2460/javma.251.5.559
In most dogs, signs of toxicosis were insufficient to warrant a dose reduction or delay in administration of chemotherapeutic agents. Two dogs had a 25% reduction in the doxorubicin dose following signs of grade 3 gastrointestinal toxicosis19; an additional dog that developed grade 3 gastrointestinal toxicosis had doxorubicin discontinued after 2 doses and instead received mitoxantrone on the same schedule, at the previously described dose; and 1 other dog had grade 3 gastrointestinal toxicosis after epirubicin administration, but continued to receive that drug with maropitant and metoclopramide with no dose adjustment. Three dogs developed grade 3 neutropenia19 during treatment, which led to a 25% dose reduction in the drug being administered (doxorubicin [n = 1], mitoxantrone [1], and lomustine [after the first dose; 1]). Grade 3 increases in serum alanine aminotransferase activity19 were detected in 3 dogs following lomustine administration. One dog with this effect after the first dose of lomustine had resolution with S-adenosyl methionine treatment, and lomustine administration was continued without recurrence; 2 dogs had lomustine discontinued after 3 doses (replaced by additional mitoxantrone treatments for 1 dog; the other dog had progressive hepatic metastases, and all chemotherapy was discontinued). One other dog had lomustine treatment discontinued after the third dose when preexisting azotemia worsened during chemotherapy, but the anthracycline treatment was continued as planned.
Discussion
The contribution of chemotherapy to survival in dogs with stage II splenic hemangiosarcoma is difficult to elucidate from the literature, although a recent study3 suggested an overall (not defined by stage) benefit of such treatment. Some studies have grouped dogs with hemangiosarcoma from all anatomic sites together, and some have grouped dogs with stage I and II splenic disease together. One study12 that examined histologic findings and tumor grading as prognostic factors in dogs with metastatic hemangiosarcoma found that mitotic score, nuclear pleomorphism, and total histologic score were all associated with survival time and disease-free interval. However, we identified no studies that investigated histologic factors for dogs with stage II splenic hemangiosarcoma; 1 study11 that purported to evaluate grade only assessed tumor differentiation.
The combination of anthracyclines and lomustine in the protocol used in the present study was well tolerated by most dogs. Overall, dogs in our study had a median survival time of 158 days, and the 1-year survival rate was 16%, which was better than 1-year survival rates reported for dogs with splenic hemangiosarcoma treated by splenectomy alone (6%1 to 12%3), but not substantially different from rates described in the literature for dogs with splenic hemangiosarcoma treated by splenectomy and other chemotherapy protocols (ranging from 0% [all with stage II disease]6 to 23% [including ≥ 30/34 [88%] dogs with stage II disease]7). Our results suggested that in addition to tumor stage, histologic tumor grading is relevant when comparing outcomes for dogs with splenic hemangiosarcoma and might be a means to identify dogs that could potentially benefit most from chemotherapy, at least with the protocols used in the present study. The 1-year survival rate of dogs with a mitotic score of 0 (< 11 mitoses/10 hpf) was 42%, and 3 of these dogs were still alive at the end of the study. Other histologic factors did not have significant associations with this outcome on multivariate analysis, and this may have been attributable in part to the subjective nature of assessments for those variables (tumor differentiation, percentage of necrotic cells, and nuclear pleomorphism), compared with the relatively more objective mitotic index. The method used by all pathologists in the present study was to scan for the area of the section with the highest mitotic activity and to count mitoses in 10 adjacent fields in that area.
Although the present study had an advantage in the inclusion of dogs with the same stage of hemangiosarcoma affecting the same anatomic site (spleen), it was retrospective in nature and, as such, had limitations common to these types of studies. Not all dogs had a liver biopsy performed, but if the liver appeared normal on abdominal surgery, or both ultrasonography and surgery, gross metastatic disease was believed to be absent. This approach was supported by results of a recent study20 in which investigators found no metastases on biopsy of 21 of 21 (100%) grossly normal and 29 of 58 (50%) grossly abnormal livers of dogs with splenic hemangiosarcoma. Most dogs in the present study eventually developed hemoabdomen, and in many dogs, diagnostic imaging results supported the clinically suspected diagnosis of liver metastases that were presumably microscopic at the time of diagnosis. However, no evidence of metastatic disease was found prior to chemotherapy, and in this aspect, dogs in this study did constitute a reasonably homogeneous population. Toxicoses of grades I or II (signs that did not necessitate drug dosage alterations) were not consistently recorded, and this could have led to underestimation of the overall toxicity of drugs as used in the protocol. All dogs were analyzed according to the intent to treat, but some dogs had alterations in the intended chemotherapy protocol because of toxicosis; although in some dogs this was concurrent with the finding of metastatic disease (and therefore may not have influenced survival data), in other dogs, this may have led to inaccurate estimation of the associations of survival time with treatment. Disease-free interval was not considered to be a robust endpoint, as staging after chemotherapy concluded was inconsistent, and metastases were often only suspected or detected only when hemoabdomen was discovered, presumably after metastatic tumors had been present for some time. Survival time was therefore considered a more robust endpoint, as there was a high index of suspicion for disease progression or metastasis at the time of death for most dogs. The study did not include a control group of dogs with stage II hemangiosarcoma treated by splenectomy alone.
Counting the number of mitoses in a tumor where there is considerable necrosis or hemorrhage is difficult; however, all pathologists participating in the present study were asked to identify the areas of highest mitotic activity, within the most densely cellular areas of the hemangiosarcoma, and to omit areas of necrosis or hemorrhage, as has been suggested to allow for consistent results in counting mitoses.17,18 The present study also used several veterinary pathologists (11 individuals) to evaluate the histologic features of the hemangiosarcomas. This may be considered a weakness; however, these individuals all used a published grading scheme that was developed with the intent to allow consistent results among different pathologists. All pathologists participating in our study were certified specialists, and all were given consistent instructions and the grading scheme to follow. This would not allow for the same uniformity as would be expected had 1 pathologist read all slides. However, in clinical practice, histologic specimens will be read by various pathologists, and published grading schemes, when consistently used, should be considered an applicable method for producing reasonably consistent results among trained pathologists.
Although the administration of lomustine with an anthracycline as provided in the present study did not appear to improve survival times in dogs with stage II splenic hemangiosarcoma, compared with those reported for other adjunctive chemotherapy protocols for dogs, the investigation did identify a significant positive association between low mitotic index and survival time for dogs receiving this treatment protocol. This finding warrants further investigation and suggests future studies should take histologic factors, particularly mitotic rate, as well as tumor stage into account when assessing treatment effects on survival time in dogs with splenic hemangiosarcoma.
Acknowledgments
No third-party funding or support was received in connection with this study or the writing or publication of the manuscript. No conflicts of interest (financial or personal relationships) existed that could have influenced information in the manuscript.
The authors thank the following veterinary pathologists for review of histopathologic slides: David Taylor, Vetnostics, Australia; Peter Rowland, Histopath Consulting; Neill Sullivan, IDEXX Laboratories, Australia; Sean McDonough, Cornell University College of Veterinary Medicine; Judith Nimmo, Australian Specialized Animal Pathology, Australia; Malcolm A. Silkstone, Abbey Veterinary Services, England; Celia Hooper, Gribbles Pathology, Australia; Ann Crowley, Anapath, Australia; James Walberg, VetPath Services; Adrienne French, New Zealand Veterinary Pathology, New Zealand; and Ann Kincaid, Marshfield Clinic. The authors thank Dr. Catherine Chan, of the Animal Referral Hospital, NSW, Australia, for the use of clinical material.
Footnotes
SPSS, version 10, Statistical Analytical Software, Chicago, Ill.
References
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Appendix
Histologic grading scheme12 used for evaluation of hemangiosarcoma in dogs.
| Characteristic | Criterion | Score |
|---|---|---|
| Tumor differentiation | Well differentiated (well formed); numerous irregular vascular channels predominate in all fields | 1 |
| Moderately well differentiated; ≥ 50% of the tumor has well-defined vascular channels | 2 | |
| Poorly differentiated; most of the tumor is solid sheets of spindle cells with few vascular channels | 3 | |
| Nuclear pleomorphism | No difference in nuclear size and shape | 0 |
| Minimal variation | 1 | |
| Moderate variation (2× size difference) | 2 | |
| Marked variation (> 2× size difference) | 3 | |
| Tumor necrosis* | None | 0 |
| < 25% | 1 | |
| 25–50% | 2 | |
| > 50% | 3 | |
| Mitoses (No./10 hpf)† | < 11 | 0 |
| 11–20 | 1 | |
| 21–30 | 2 | |
| > 30 | 3 |
Areas of hematoma were excluded from these determinations.
Pathologists were instructed to scan for the area of the section with the highest mitotic activity and count 10 adjacent fields in that area, omitting regions of hemorrhage or necrosis.17,18
All histologic scores were summed to determine an overall histologic score for the tumor (grade 1 = 1 to 5; grade 2 = 6 to 9; and grade 3 = 9 to 12).12
