Musculoskeletal sarcomas are rare and aggressive human malignancies affecting bones and soft tissues with severe consequences, in terms of both morbidity and mortality. An innovative technique that combines photodynamic surgery (PDS) and therapy (PDT) with acridine orange has been recently suggested, showing promising results. However, due to the low incidence of sarcoma in humans, this procedure has been attempted only in pilot studies and stronger evidence is needed. Naturally occurring tumors in cats are well-established and advantageous models for human cancers. Feline injection-site sarcoma (FISS) shares with human musculoskeletal sarcomas a mesenchymal origin and an aggressive behavior with a high relapse rate. Furthermore, wide surgical excision is not always possible due to the size and site of development. We assessed the feasibility and the effectiveness of PDS and PDT with acridine orange to prevent FISS recurrence by treating a short case series of cats. For PDS, the surgical field was irrigated with an acridine orange solution and exposed to UV light to enlighten the residual tumor tissue, and the resultant fluorescent areas were trimmed. For PDT, before wound closure, the field was again irrigated with acridine orange solution and exposed to visible light to get the antitumoral cytocidal effect. The procedure was easy to perform and well tolerated, we did not observe any major complications, and all the surgical resection margins were free of disease. Finally, at follow-up, all treated patients did not show evidence of tumor recurrence and had a significantly higher event-free survival rate in respect to a control group treated only by surgery. In conclusion, by this study we demonstrated that, in FISS, PDS and PDT with acridine orange may improve local tumor control, granting a better outcome, and we laid the foundation to validate its effectiveness for the treatment of human musculoskeletal sarcomas.
Musculoskeletal sarcomas are relatively rare human malignancies that often occur in children and young adults, leading to devastating consequences in terms of both morbidity and mortality [
Limb salvage is a well-established technique for the treatment of musculoskeletal sarcomas and the most common approach includes wide resection followed by limb reconstruction [
In this context, we have recently described a novel limb salvage technique based on the use of acridine orange for photodynamic surgery (PDS) that is coupled with photodynamic therapy (PDT) and radiodynamic therapy (RDT) [
Despite the presence of numerous pilot studies concerning the use of acridine orange for the treatment of musculoskeletal sarcomas, the low incidence of these tumors in humans is limiting the development of clinical trials demonstrating the efficacy of this procedure compared to standard treatments. To extend the clinical database and increase the statistical significance of these results, in this study we involved comparative oncology. Naturally occurring tumors in animals, such as cats or dogs, are well-established models for human cancers [
Feline Injection Site Sarcoma (FISS) is a malignant tumor of mesenchymal origin that develops in 1–10 of 10,000 injected cats. The pathogenesis of this disease is unknown, although the most accepted hypothesis is a hyatrogenic origin due to local postinjection inflammatory response that leads to neoplastic transformation [
In this study, through comparative oncology, we aimed to establish FISS as an optimum model to consolidate the effectiveness of the combined approach PDS-PDT using acridine orange in reducing postsurgical recurrence rate of human musculoskeletal sarcoma.
Starting from March 2014 and up to April 2015, we enrolled a short case series that presented for examination at the Veterinary Teaching Hospital of the University of Turin (Grugliasco, Italy) with a growing mass at injection sites. Before any procedure, the Institutional Animal Care and Use Committee approval (11 September 2011) was obtained, and the informed consent clearly describing the technique was signed by the animal owner, clearly describing the technique. After a thorough physical examination, each patient underwent a complete blood count, a biochemical profile, and a blood test for Feline Immunodeficiency and Feline Leukaemia Viruses (FIV, FeLV) and was diagnosed with FISS, as confirmed by fine needle biopsy and histological examination. The history always reported the occurrence of a rapidly growing mass at sites of injections of vaccines or other drugs. All cats underwent total body computed tomography (CT) scan under general anesthesia to estimate the size of the tumor, to determine the area of surgical excision, and to detect lung metastases.
A standard oncological resection was performed in the sternal decubitus position, with the exception of one case for which the patient was put in lateral decubitus due to the location of the tumor on the lateral aspect of the thorax. The surgical excision was performed according to the FISS treatment guidelines [
Soon after the surgical procedure, the field was irrigated with a 50 mL syringe filled with acridine orange sterile solution (1
Immediately after the excision, the tumor pH was assessed (three replicates for each lesion) using a portable pH Level Monitor (Hanna Instruments, Padova, Italy) and recorded.
After the procedure, animals were hospitalized for 2 days and reassessed after 7 and 15 days and every 3 months thereafter for the first year and every 6 months the second year. At each control, starting at 3 months, chest radiographs were taken to evaluate the development of metastases and a thorough physical examination was performed. After the second year the follow-up was updated by phone call to the owners or the referring veterinarian. As a control, we considered both a retrospective and a prospective group of 30 FISS treated by en-bloc surgery alone, recruited from January 2009 to September 2015. In order to parallel the maximum follow-up time of the treated group, the maximum follow-up considered for both groups was 1444 days from the date of the surgery. The decision to retrospectively recruit cases for the control group was due to the decrease in the number of cats affected by the disease visited in the last years, at least at the institution where the study was conducted. Anyway, the diagnosis and staging of each animal were accomplished in the same way, and the surgical procedure was performed by the same surgeons in both groups. The only variable in the procedure was the PDS-PDT added in the study group. To evaluate the rate of success for the PDS and PDT-treatment, the event-free survival was calculated from the date of surgery to the date of detection of recurrence or metastasis. The average follow-up was 902 ± 84 days for the control group and 961 ± 223 days for the treated group.
Statistical analysis was performed by GraphPad Prism 7 software (GraphPad Software, Inc). Quantitative results were expressed as mean ± standard error (SEM). Due to the low number of events considered in the study, we used nonparametric test. Survival analysis was performed by using Kaplan-Meier survival plot (Log-rank Mantel-Cox test). Only
In this study we enrolled seven client-owned cats, while 30 cats were used as control (Table
Patients series.
Case# | Age | Sex | Race | Recurrence | Metastasis | Status at follow-up |
Length of follow-up | |
---|---|---|---|---|---|---|---|---|
treated | 1 | 11 | M | Domestic short hair | No | No | censored | 1197 |
treated | 2 | 10 | MC | Domestic short hair | No | No | censored | 1340 |
treated | 3 | 12 | F | Domestic short hair | No | No | censored | 173 |
treated | 4 | 12 | FS | Siamese | No | No | censored | 1444 |
treated | 5 | 10 | MC | Domestic short hair | No | No | censored | 1424 |
treated | 6 | 11 | FS | Maine Coon | No | No | censored | 60 |
treated | 7 | 12 | MC | Domestic short hair | No | No | censored | 1088 |
control | 1 | 6 | FS | Domestic short hair | No | No | censored | 1444 |
control | 2 | 10 | MC | Domestic short hair | No | No | censored | 1444 |
control | 3 | 9 | MC | Domestic short hair | No | No | censored | 1444 |
control | 4 | 8 | MC | Domestic short hair | Yes | Yes | uncensored | 317 |
control | 5 | 6 | FS | Domestic short hair | No | No | censored | 1163 |
control | 6 | 9 | FS | Domestic short hair | Yes | No | uncensored | 718 |
control | 7 | 7 | FS | Domestic short hair | Yes | No | uncensored | 1302 |
control | 8 | 8 | FS | Domestic short hair | No | yes | uncensored | 243 |
control | 9 | 13 | FS | Domestic short hair | Yes | No | uncensored | 558 |
control | 10 | 11 | FS | Domestic short hair | No | No | censored | 1444 |
control | 11 | 12 | FS | Domestic short hair | No | Yes | uncensored | 354 |
control | 12 | 3 | FS | Domestic short hair | No | No | censored | 1444 |
control | 13 | 10 | MC | Domestic short hair | Yes | No | uncensored | 1236 |
control | 14 | 12 | FS | Domestic short hair | No | Yes | uncensored | 339 |
control | 15 | 11 | MC | Domestic short hair | No | No | censored | 1444 |
control | 16 | 10 | FS | Domestic short hair | Yes | No | uncensored | 350 |
control | 17 | 13 | MC | Domestic short hair | No | No | censored | 663 |
control | 18 | 13 | FS | Domestic short hair | No | No | censored | 1379 |
control | 19 | 13 | MC | Domestic short hair | Yes | No | uncensored | 986 |
control | 20 | 14 | MC | Domestic short hair | Yes | No | uncensored | 1256 |
control | 21 | 15 | FC | Domestic short hair | No | No | censored | 577 |
control | 22 | 13 | MC | Birmano | No | Yes | uncensored | 483 |
control | 23 | 7 | FC | Domestic short hair | No | Yes | uncensored | 806 |
control | 24 | 9 | FC | Domestic short hair | Yes | No | uncensored | 503 |
control | 25 | 11 | FC | Domestic short hair | Yes | No | uncensored | 1409 |
control | 26 | 11 | MC | Domestic short hair | Yes | No | uncensored | 238 |
control | 27 | 11 | FC | Domestic short hair | Yes | No | uncensored | 289 |
control | 28 | 13 | FC | Domestic short hair | No | No | censored | 1310 |
control | 29 | 13 | MC | Domestic short hair | No | No | censored | 1139 |
control | 30 | 14 | MC | Domestic short hair | No | No | censored | 765 |
M, male; MC, male castrated; F, female; FS, female spayed; status at follow-up
The median age of the treated cats was 11 years (min 10 – max 12); for the control group it was 11 years (min 3 – max 15). As revealed by CT scan, the maximum tumor volume, that was calculated according to the formula: tumor volume [cm3] = (length [cm] x width2 [cm2])/2 [
Preoperative CT scan of cats # 2 and 3 of the treated group. The extent of the tumor after contrast medium administration was considered including the lateral laciniae; infiltration into surrounding tissues and the vicinity with spinal processes were assessed.
Histopathological image of FISS (Courtesy of dr. S. Iussich). Upper panel, multiple nodules of fusiform cells arranged in sheets in abundant hyaline stroma infiltrating the subcutaneous tissue (H&E, 2x objective); lower panel, multiple nodules of fusiform cells arranged in sheets in moderate myxoid stroma; the cells (20-30
After tumor resection, the median pH value was 6.14 (min 6.08, max 6.84, Table
Intratumoral pH.
Case | Tumour size | pH |
---|---|---|
# | width x length [cm] | |
1 | 5.0x8.8 | n.d. |
2 | 8.5x9.0 | 6.14 |
3 | 9.0x17.4 | 6.26 |
4 | 5.5x5.6 | 6.08 |
5 | 7.3x10.7 | 6.10 |
6 | 6.0x7.0 | 6.84 |
7 | 10.0x5.0 | n.d. |
n.d., not determined.
PDS and PDT are summarized in Figure
Graphical representation of acridine orange PDS and PDT technique. Surgical excision (a), pH measurement (b), acridine orange irrigation (c), PDS (d), PDT (e). On the bottom line, representative photographs of the procedure are shown.
Resection of spinous processes of adjacent vertebrae was needed in three cases (#2, 3, and 7). Overall, the surgery time was 30 minutes longer as compared to the standard surgical procedure. No major complications were observed. The procedure was well tolerated by the animals. Tumor-free excision margins were obtained in all cases.
Two patients (#3, 6) were euthanized for unrelated causes after 60 and 173 days from surgery (infectious viral peritonitis and chronic bowel disease, respectively), and they did not present evidence of local or systemic relapse of the tumor at the time of death. All remaining cats were alive and did not develop recurrence and/or metastasis at the time of last follow-up. The Kaplan-Meier analysis was applied to estimate the survival rate. Although no significant association was found with local recurrence rate (data not shown), the PDS and PDT treatment was associated with a significantly higher event-free survival rate in comparison with the control group (Figure
Effect of PDS and PDT with acridine orange on FISS disease-free survival time at follow-up, as analyzed by Kaplan-Meier. Both local recurrence and metastasis are considered as event (n. of uncensored events was 17 for control and 0 for treated, out of 30 and 7 patients, respectively).
Intratumoral acidosis is a hallmark of cancer [
Despite the preclinical evidence of the potential of acridine orange combined to PDS and PDT to treat sarcomas, the low incidence of these malignancies in humans has not allowed a wide application of such technique. In this context, comparative oncology may greatly help in the evaluation and interpretation of the response to treatment, especially when human and animal diseases are biologically and clinically similar [
In this pilot study, we enrolled cats with FISS, a soft tissue sarcoma with clinical futures very similar to those of human tumors, including an aggressive local behavior. Up to one-quarter of cats with this condition have metastatic lung involvement, but the major concern is related to the high recurrence rate of the disease. The mainstay of treatment is aggressive surgery, but even in cases of wide excision with clean margins, tumor recurrence occurs in a high proportion of cases [
After the execution of PDS and PDT, we did not observe any adverse event, neither during nor in the immediate follow-up. The procedure was very similar to that previously used in human patients with sarcomas [
The recurrence rate observed in the control group was higher compared to what is reported by the most recent veterinary literature [
Our data suggest that PDS-PDT based on the use of acridine orange has the potential to exert a similar effect in comparison to the gold standard treatment of FISS and that, according to the results obtained by analyzing a pilot series of animal patients with FISS, this technique may be considered a useful adjuvant therapeutic modality.
Our data lay the foundation for additional research in this spontaneous animal condition that may foster the development of novel treatment options to be translated for human musculoskeletal sarcomas.
All the data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there is no conflict of interest regarding the publication of this paper.
The work was supported by the Italian Association for Cancer Research (to Nicola Baldini, n. 15608) and by the financial support for Scientific Research “5 per mille 2016” to IRCCS Istituto Ortopedico Rizzoli (to Nicola Baldini)