- Email: [email protected]
Long-term clinical outcome in patients with high-grade soft tissue sarcoma who were treated with surgical adjuvant therapy using acridine orange after intra-lesional or marginal resection
Accepted Manuscript Title: Long-term clinical outcome in patients with high-grade soft tissue sarcoma who were treated with surgical adjuvant therapy using acridine orange after intra-lesional or marginal resection Authors: Tomoki Nakamura, Katsuyuki Kusuzaki, Takao Matsubara, Hiroaki Murata, Tomohito Hagi, Kunihiro Asanuma, Akihiro Sudo PII: DOI: Reference:
S1572-1000(18)30082-6 https://doi.org/10.1016/j.pdpdt.2018.06.001 PDPDT 1177
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
15-3-2018 1-6-2018 1-6-2018
Please cite this article as: Nakamura T, Kusuzaki K, Matsubara T, Murata H, Hagi T, Asanuma K, Sudo A, Long-term clinical outcome in patients with high-grade soft tissue sarcoma who were treated with surgical adjuvant therapy using acridine orange after intra-lesional or marginal resection, Photodiagnosis and Photodynamic Therapy (2018), https://doi.org/10.1016/j.pdpdt.2018.06.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Long-term clinical outcome in patients with high-grade soft tissue sarcoma who were treated with surgical adjuvant therapy using acridine orange after intra-lesional or marginal resection.
IP T
Head line title: Long-term results of acridine orange therapy Tomoki Nakamura1, Katsuyuki Kusuzaki2, Takao Matsubara3, Hiroaki Murata4, Tomohito Hagi1, Kunihiro Asanuma1, Akihiro Sudo1 1Department
of Orthopaedic Surgery, Mie University Postgraduate School of Medicine.
2Department
SC R
Edobashi 2-174, Tsu-city, 514-8507, Japan
of Musculoskeletal Oncology, Takai Hospital. Kuranosho 470-8, Tenri, 632-
0006, Japan.
Orthopaedic Clinic. Shiroyama 3-4-25, Tsu-city, 514-0818, Japan.
4Department
U
3Matsubara
of Orthopaedic Surgery, Matsushita Memorial Hospital. Moriguchi-city,
A
N
570-8540, Japan. Corresponding author: Tomoki Nakamura
M
Department of Orthopaedic Surgery, Mie University Postgraduate School of Medicine 2-174 Edobashi, Tsu-city, Mie, 514-8507, Japan
ED
Tel: +81592315022, FAX; +81592315211
Highlights
PT
E-mail: [email protected]
CC E
・ We have established acridine orange (AO) therapy, including photodynamic surgery, photodynamic therapy, and radiodynamic therapy. ・ AO therapy was applied for soft tissue sarcomas to reduce local recurrence and maintaining limb function.
A
・ The 5- and 10-year local recurrence-free rates in 48 patients were 78.9% and 73.3%, respectively. ・Tumor size is an important factor for the indication of AO therapy.
1
Abstract Background: We investigated the long-term clinical efficacy of acridine orange (AO) therapy on the inhibition of local recurrence after marginal or intra-lesional tumor resection in highgrade soft tissue sarcomas (STSs).
IP T
Methods: Our study consisted of 48 patients with STSs who received AO therapy after
marginal or intra-lesional resection. The median and mean follow-up durations were 76 and
surgery, photodynamic therapy, and radiodynamic therapy.
SC R
78 months, respectively. Our AO therapy procedure was combined with photodynamic
U
Results: There were 25 men and 23 women, with a mean age of 46 years. The average tumor
N
size at surgery was 8.5 cm. At the last follow-up, 11 patients developed local recurrence. The
A
5- and 10-year local recurrence-free rates were 78.9% and 73.3%, respectively. In multivariate
M
analysis, tumor size remained significant for local control. None of the patients developed systemic or local complications. All patients recovered activities of daily life before AO
ED
therapy.
PT
Conclusion: AO therapy can be performed in safety and may be a useful therapy for acquiring long-term local control in patients with high-grade STSs. Tumor size is an
CC E
important factor for the indication of AO therapy.
Key Words: Soft tissue sarcoma; Acridine orange; Photodynamic therapy; Radiodymanic
A
therapy; Local control
2
Introduction Adequate surgical margins inhibit local tumor recurrence in high-grade soft tissue sarcomas (STSs) [1-3]. However, if tumors are in close contact with major nerves, vessels,
IP T
bones, or joints, patients can develop serious dysfunction of the affected limb after wide
resection, which might involve the sacrifice of some or all those structures. Adjuvant
SC R
radiotherapy (RT) may be performed when surgeons perform dissection between tumors
and these structures with marginal or intra-lesional resections to maintain limb function [1,4-
U
6]. Although adjuvant RT may be effective to reduce local recurrence, the long-term effects
N
of RT can include fibrosis, edema, fractures, and contractures, all of which can substantially
A
impair limb function [7-9]. Patients who undergo adjuvant RT are also likely to develop
M
major complications, such as pathological fractures and second malignancies [4,5,10]. Therefore, it is imperative to develop a new adjuvant therapy as soon as possible to reduce
ED
the local recurrence rate after marginal or intra-lesional tumor resection, but without causing
PT
damage to adjacent normal tissues for patients with high-grade STSs, in order to maintain excellent postoperative limb function.
CC E
Based on basic research studies [11-14], we have established acridine orange (AO) therapy, including photodynamic surgery (PDS), photodynamic therapy (PDT), and radiodynamic therapy (RDT) after marginal or intra-lesional tumor resection, with the aim of reducing local
A
recurrence and maintaining limb function in patients with STSs under the approval of the institutional review board [7,15-17]. Although the clinical outcome reportedly showed excellent limb function with a local control similar to that following a wide resection with a mean follow-up time of 3 or 4 years, long-term results should be evaluated for this procedure.
3
Therefore, we investigated the long-term clinical efficacy of this new AO therapy modality on the inhibition of local recurrence after marginal or intra-lesional tumor resection in high-grade STSs.
IP T
Materials and Methods Between July 1999 and October 2014, a total of 58 patients was treated with AO therapy
SC R
after marginal or intra-lesional resection. The study was approved by the institutional review board of Kyoto Prefectural University of Medicine and Mie University Postgraduate School
U
of Medicine. We defined a marginal surgical margin with the surgical plane running through
N
the pseudo-capsule (reactive zone) and an intra-lesional margin with the margin running
A
through the tumor and, therefore, the tumor remains histologically according to the
M
Enneking criteria of surgical margins (1,18). Briefly, intralesional resection means that tumor has been removed from within the capsule, while marginal resection means removed from
ED
outside the capsule. Both methods can leave residual tumor cells under some circumstances. Seven patients were excluded from the current study because of the difficulty of evaluation
PT
of local tumor control due to the short follow-up duration. We also excluded 3 patients who
CC E
received adjuvant RT after AO therapy. A total of 48 patients was therefore available for evaluation. We retrospectively studied the clinical data of the patients as contained on a prospectively maintained database program, clinical notes, and radiological imaging of the
A
patients. This clinical study was performed after obtaining informed consent from both the patients and their families following a full explanation of the study. The survival rate of all patients and local recurrence-free rates after AO therapy were analyzed using the Kaplan– Meier method. Local recurrence of tumors was evaluated every 3 to 6 months using magnetic
4
resonance imaging (MRI). The median and mean follow-up durations were 76 and 78 months (range, 3-167), respectively. Procedure of AO therapy combined with PDS, PDT, and RDT Initially, we performed intra-lesional or marginal tumor resection to avoid any damage
IP T
to intact muscles, bones, joints, major nerves, and vessels in close contact with the tumor, resulting in good limb function after surgery. In the next procedure involving PDS,
SC R
microscopic curettage with an ultrasonic surgical knife (Olympus Co. Ltd., Tokyo, Japan) was immediately followed with a fluorescence surgical microscope equipped with a high-
U
power xenon lamp (500 mW, >10,0000 lx) and special interference and resorption filters for
N
AO. We sprayed a 1-μg/ml solution of AO (Sigma Aldrich Co, St Louis, MO, USA) into the
A
surgical field. Repeated fluorescence-guided resection using the ultrasonic knife was then
M
performed by excitation with blue light from a xenon lamp using an interference filter (450– 490 nm), and the remaining tumor fragments, which had selectively taken up AO, emitted a
ED
green fluorescence that was visible under a fluorescence microscope fitted with a yellow resorption filter (>520 nm) [7,15-17]. This PDS procedure was repeated until AO fluorescence
PT
could not be elicited [7,15-17] (Figure 1).
CC E
PDT sequentially followed PDS. For AO-PDT, an unfiltered full-light beam from a xenon lamp was applied to the tumor curettage area for 10 minutes using a surgical microscope after administration of AO solution [7,15-17].
A
The final step of RDT was performed as follows. After closure of the surgical wound,
without washing out the AO solution, single-session radiotherapy with 5 Gy was immediately administered to the resected area in the RT room in 25 patients who agreed to undergo RDT [7,15-17].
5
We evaluated the staining of AO using biopsy sample when we started this pilot study because STSs had variety of histology and those tumors had a good affinity of AO (unpublished data). Statistical analysis
IP T
Survival time was measured from the date of surgery using AO to the date of sarcomarelated death or last follow-up. Local recurrence time was measured from the date of surgery
SC R
using AO to the date of local recurrence. Even if the AO therapy was performed in patients who developed local recurrence or metastasis, local recurrence time was also measured from the date of surgery using AO to the date of local recurrence. Survival curves were created
U
using the Kaplan–Meier method. Univariate and multivariate analyses were performed
N
using Cox proportional hazard regression models. All statistical analyses were conducted
A
using Stat View for Windows, version 5.0 (SAS Institute Inc., Cary, NC, USA). P values <0.05
M
were considered significant.
ED
Results
PT
Our study consisted of 48 patients with STSs who received AO therapy (Table 1). We included 12 patients who had local recurrence. There were 25 men and 23 women, with a
CC E
mean age of 46 years (range, 11 months–85 years). Primary tumor sites included the thigh (n=13), forearm (n=7), buttock (n=5), popliteal lesion (n=4), foot (n=3), lower leg (n=3), inguinal region (n=3), upper arm (n=2), and other regions (n=8). The tumors were
A
histologically classified as synovial sarcomas (n=9), undifferentiated pleomorphic sarcomas (UPSs) (n=8), rhabdomyosarcomas (n=6), liposarcomas (n=5; myxoid type n=4, pleomorphic type n=1), leiomyosarcomas (n=4), myxofibrosarcomas (n=4), malignant peripheral nerve sheath tumors (n=4), and others (n=8). All tumors were high-grade sarcomas categorized as
6
grade 2 (n=16) or 3 (n=32) according to the criteria of the French Federation of Cancer Centers Sarcoma Group (FNCLCC) grading system. We included 11 patients who developed lung metastasis at initial presentation. The average tumor size at surgery was 8.5 cm (range, 3–27). Neo- and adjuvant chemotherapy was performed in 26 patients. Chemotherapy was
IP T
administered in younger patients (p<0.0001, Mann–Whitney U test). Surgeons performed
dissection between tumors and preserved structures with marginal (in 14 patients) or intra-
SC R
lesional (in 34 patients) resection to maintain limb function. The distribution of AO therapy for each patient is shown in Table 1.
U
At the last follow-up, local recurrence occurred in 11 patients: 4/10 patients treated with
N
PDT after intra-lesional excision; 1/6 patients treated with PDT after marginal resection; 5/24
A
patients treated with PDT and RDT after intra-lesional excision; and 1/8 patients treated with
M
PDT and RDT after marginal resection. Although there was a high rate of local recurrence (40%, 4 of 10 patients) in patients treated with PDT after intra-lesional excision, there was no
ED
significant difference between other methods of AO therapy (Table 2). The 5- and 10-year
PT
local recurrence-free rates were 78.9% [95% confidence interval (CI): 67.3–90.6] and 73.3% [95% CI: 58.1–88.5], respectively (Figure 2). In Cox hazard univariate analysis, the patients
CC E
with large tumor sizes had worse local control (hazard ratio (HR): 1.2, 95% CI: 1.068–1.349, p=0.002). Female patients had significantly better local control (HR: 0.212: 0.045–0.986, p=0.048). Age, administration of chemotherapy, status of tumors (primary vs. local
A
recurrence), FNCLCC grade (grade 2 vs. 3), and type of AO therapy were not significant predictive factors for local control (Table 2). In multivariate analysis, tumor size remained significant. Of 11 patients with local recurrence after AO therapy, three patients underwent amputation.
7
Two of three patients died of stomach cancer and pancreatic cancer, respectively. The remaining one survived with no disease after undergoing amputation. Two of eight patients received palliative radiation because of their progressive lung metastasis; they died of their disease. Another 2 patients died from multiple distant metastases. The remaining 4 patients
IP T
underwent tumor re-excision and survived with no disease. Three of four patients underwent tumor excision with adjuvant RT.
SC R
None of the patients developed systemic or local complications following local
administration of AO, light, and X-ray irradiation. All patients recovered activities of daily
U
life before AO therapy at the last follow-up, except for the patients who underwent
A
N
amputation or additional tumor resection because of local recurrence.
M
Case presentation
Case 1: The patient was a 21-year-old woman with synovial sarcoma arising at the right
ED
shoulder. Neo- and adjuvant chemotherapy comprising doxorubicin and ifosfamide were
PT
administered. The tumor was in close contact with the axillary nerve (Figure 3). If the tumor was resected with a strictly wide margin, the patient could develop serious dysfunction of
CC E
the shoulder. Therefore, we planned PDS followed by AO-PDT and RDT to preserve the axillary nerve. After informed consent for the study was provided, AO therapy was performed. After therapy, the motor and sensory functions of the right shoulder were normal
A
and the patient is now able to elevate her shoulder without any obvious handicap. There is no evidence of local tumor recurrence even after a follow-up of 136 months (Figure 4). Limb function evaluated based on the International Society of Limb Salvage (ISOLS) criteria was 100%.
8
Case 2: The patient was a 67-year-old woman with a myxofibrosarcoma arising at an inguinal region. MRI revealed that the tumor was in close contact with the femoral artery and vein, as well as the femoral nerve (Figure 5). Since wide resection of the tumor could cause serious
IP T
dysfunction of the lower extremities even after reconstruction of vessels by transplantation
of the femoral artery and vein, we proposed AO therapy to the patient and her family. After
SC R
their agreement with the study, we planned PDS followed by AO-PDT and RDT to preserve
the vessels and nerve. There is no evidence of local tumor recurrence even after a follow-up
N
U
of 73 months (Figure 6). Limb function evaluated by the ISOLS criteria was 100%.
A
Discussion
M
In the present study, local recurrence after AO therapy occurred in 11 patients (23%). The 5and 10-year local recurrence-free rates were 78.9% and 73.3%, respectively. In multivariate
ED
analysis, the patients with larger tumor sizes had significantly poorer local control. We firstly reported the long-term results of AO therapy and suggest that AO therapy is safe procedure
PT
and may be useful for acquiring long-term local control after marginal or intra-lesional tumor
CC E
resection and for preserving excellent limb function in selected patients with high-grade STSs.
We established AO therapy based on the characteristics of AO. AO excited by blue light
A
produces an excited singlet state which can lose energy by fluorescence or undergo intersystem crossing to the triplet state which can then undergo an energy transfer mechanism to triplet oxygen producing singlet oxygen (photochemical reaction II) (19,20). AO is accumulated into acidic environments, sarcoma cells having a lot of large acidic
9
vesicles that readily take up AO easily, whereas normal cells with non-acidic environments and weak acidic lysosomes can quickly excluded AO. It has been clarified that the inhibition of V-ATPase activity by bafilomycin causes a decrease in AO accumulation into the lysosome, suggesting that AO accumulates into the lysosome in an acidity-dependent manner (16,21).
IP T
Wide tumor resection for high-grade STSs in contact with critical muscles, vessels, nerves,
bones, or joints causes a serious deficit of limb function [7,22,23]. Especially at the forearm,
SC R
hand, lower leg, foot, popliteus, axilla, inguinal region, and buttock, it is not easy to reserve
such structures by surgical tumor resection with a strict wide margin. However, marginal
U
excision includes peritumoral reactive tissue, but insufficient margins and is associated with
N
high local recurrence rates [1-3]. Adjuvant RT is the standard approach for high-grade STSs
A
[1,4-6]. Two randomized clinical trials demonstrated a 20–25% reduction in the risk of local
M
recurrence with radiation in patients with extremity STSs [4,24,25]. Trovik et al. also reported the affection of surgical margins with or without adjuvant RT for local recurrence of STSs in
ED
a Scandinavian group [26]. They showed that the rate was 25 of 64 patients (39%) after an
PT
intra-lesional or marginal resection without postoperative RT, versus 28 of 119 patients (24%) when RT was administered. However, Kneisl et al. reported that the patients who underwent
CC E
adjuvant RT (median dose of 50–60 Gy) had a major complication rate of 16.2%, including complications such as pathological fractures and secondary malignancies, compared to 3.9% in those who did not receive RT [4]. Postoperative wound complications should also be
A
considered in patients who undergo RT [4,5]. In the present study, local recurrence after AO therapy occurred in 11 patients (23%), which is similar to the results in patients who received adjuvant RT in a study by Trovik et al [24]. When we performed RDT, only single-session radiotherapy of 5 Gy was administered to the resected area. We had no experience of
10
complications after AO therapy. Therefore, we believe that AO therapy was safe and effective for local control. We also suggest that if the tumor can be resected without serious dysfunction of the affected limb after wide resection, AO therapy should not be indicated because tumor resection with wide surgical margin is standard treatment for good local
IP T
control.
Interestingly, when we divided the patients into 4 groups according to the AO therapy,
SC R
intra-lesional tumor resection with PDT alone conferred a high rate of local recurrence (of 40%), although there were no significant differences in the rates between the types of AO
U
therapy. Furthermore, there was no significant difference between the patients who
N
underwent PDT alone and those who underwent PDT and RDT. RDT may be necessary to
M
future studies are necessary to validate this.
A
prevent local recurrence in addition to intra-lesional tumor resection with PDT, although
Concerning large tumors, AO therapy may be insufficient because it is so difficult to expose
ED
all the remaining cells to AO and illuminate such wide areas with the surgical fluorescence
PT
microscope. Therefore, we consider that tumors with a diameter larger than 10 cm are not a suitable indication for AO therapy. Furthermore, this procedure is indicated as local therapy.
CC E
A clinical trial of RT after intravenous injection of AO for patients with advanced cancer is ongoing [25]. Although a more intensive clinical trial is necessary, PDT and RDT involving systemic administration of AO may be applied for unresectable or metastatic tumors.
A
There are some limitations in this study. First, there is a small number of patients. We should
validate our findings, especially for efficacy, in a future study. Second, this study is not a randomized clinical trial, but a pilot study. Therefore, local control cannot be directly compared in the patients with or without AO therapy.
11
In conclusion, AO therapy may be useful for acquiring long-term local control in patients with high-grade STSs. Tumor size is an important factor for the indication of AO therapy.
SC R
There was no funding for this study. There is no conflict of interest.
IP T
Acknowledgement
References
U
[1] Grimer R, Judson I, Peake D, Seddon B, Guidelines for the management of soft tissue
N
sarcomas, Sarcoma 2010; 2010: 5061182.
A
[2] Herbert SH, Corn BW, Solin LJ, et al, Limb-preserving treatment for soft tissue sarcomas of the extremities, The significance of surgical margins. Cancer 72 (1993) 1230-1238.
M
[3] Weiss SW, goldblum JR, Local recurrence In Enzinger and Weiss’s soft tissue tumors,5th edition, St Louis, MO, USA; Mosby (2008) 15-31.
ED
[4] Kneisl JS, Ferguson C, Robinson M et al, The effect of radiation therapy in the treatment of adult soft tissue sarcomas of the extremities: a long-term community-based cancer center
PT
experience, Cancer Medicine 6 (2016) 516-525. [5] Haas RL, Delaney TF, O’Sullivialn B et al, Radiationtherapy for management of extremity
CC E
soft tissue sarcomas: why, when, and where? Int J Radiat Oncol Biol Phys. 83 (2012) 572-580. [6] Abouarab MH, Salem IL, Degheidy MM et al, Therapeutic options and postoperative wound complications after extremity soft tissue sarcoma resection and postoperative external beam radiotherapy, Int Wound J 15 (2018) 148-158.
A
[7] Matsubara T, Kusuzaki K, Matsumine A, Nakamura T, Sudo A, Can a less radical surgery using photodynamic therapy with acridine orange be equal to a wide-margin resection? Clin Orthop Relat Res 471 (2013) 792-802. [8] Fuchs B, Davis AM, Griffin AM, et al, Sciatic nerve resection in the thigh: a functional evaluation, Clin Orthop Relat Res 382 (2001) 34-41.
12
[9] Ghert MA, Davis AM, Griffin AM, et al, The surgical and functional outcome of limbsalvage surgery with vascular reconstruction for soft tissue sarcoma of the extremity, Ann Surg Oncol 12 (2005) 1102-1110. [10] Sholl LM, Barletta JA, Hornick JL, Radiation-associated neoplasia: clinical, pathological and genomic correlates, Histopathology 70 (2017) 70-80.
IP T
[11] Kusuzaki K, Takeshita H, Murata H et al, Photodynamic inactivation with acridine orange on a multidrug-resistant mouse osteosarcoma cell line, Jpn J Cancer Res 91 (2000) 439445.
on mouse osteosarcoma, Anticancer Res 20 (2000) 3019-3024.
SC R
[12] Kusuzaki K, Suginoshita T, Minami G et al, Fluorovisualization effect of acridine orange
[13] Kusuzaki K, Aomori K, Suginoshita et al, Total tumor cell elimination with minimum
U
damage to normal tissues in muscloskeltal sarcomas by photodynamic reaction with acridine
N
orange, Oncology 59 (2000) 174-180.
[14] Hashiguchi S, Kusuzaki K, Hirasawa Y et al, Acridine orange excited by low-dose
A
radiation has a strong cytcidal effect on mouse osteosarcoma, Oncology 62 (2002) 85-93.
M
[15] Kusuzaki K, Murata H, Uchida A et al, Clinical trial of photodynamic therapy using acridine orange with/without low dose radiation as new limb salvage modality in
ED
muscloskeltal sarcomas, Anticancer Res 25 (2005) 1225-1235. [16] Kusuzaki K, Murata H, Uchida A et al, Clinical outcome of a new phodynamic therapy
PT
with acridine orange for synovial sarcomas, Rhotochem Photobil 83 (2005) 705-709. [17] Nakamura T, Kusuzaki K, Matsubara T, Matsumine A, Murata H, Uchida A, A new limb salvage surgery in cases of high-grade soft tissue sarcoma using photodynamic surgery,
CC E
followed by photo- and radiodynamic therapy with acridine orange, J Surg Oncol 97 (2008) 523-528.
[18] Enneking WF, Spanier SS, Goodman M, A system for the surgical staging of
A
musculoskeletal sarcoma, Clin Orthop Relat Res 153 (1980) 106-120. [19] Zdolsek JM, Acridine orange-mediated photodamage to cultured cells. Apmis 101 (1993) 127-132. [20] Miccoli L, Beurdeley-Thomas A, De Pinieux G, Sureau F, Oudard S, Dutrillaux B, Poupon MF, Light-induced photoactivation of hypericin affects the energy metabolism of human glioma cells by inhibited hexokinase bound to mitochondria. Cancer Res 17 (1998) 5777-5786.
13
[21] Hiruma H, Katakura, T, Igawa S, Kanoh M, Fujimura T, Kawakami T, vesicle disruption, plasma membrane bleb formation, and acute cell death caused by illumination with blue light in acridine-loaded malignant melanoma cells. J Photochem Photobiol 86 (2007) 1-8. [22] Brooks AD, Gold JS, Graham D et al, Resection of the sciatic, peroneal, or tibial nerves: assessment of fuctional status, Ann Surg Oncol. 9 (2002) 41-47.
IP T
[23] Stinson SF, DeLaney TF, Greenberg J et al, Acute and long-term effect on limb function of combined modality limb sparing therapy for extremity soft tissue sarcoma, Int J Radiat Oncol Biol Phys 21 (1991) 1493-1499.
SC R
[24] Pisters PW, Harrison LB, Leung DH, Woodruff JM, Casper ES, Brennan MF, Long-term
results of a prospective randomized trial of adjuvant brachytherapy in soft tissue sarcoma, J Clin Oncol 14 (1996) 859-868.
U
[25] Yang JC, Chang AE, Baker AR, et al, Randomized prospective study of the benefit of
N
adjuvant radiotherapy in the treatment of soft tissue sarcomas of the extremity, J Clin Oncol 16 (1998) 197-203.
A
[26] Trovik CS, Scandinavian Sarcoma Group Project. Local recurrence of soft tissue sarcoma.
M
A Scandinavian Sarcoma Group Project. Acta Orthop Scand Suppl 72 (2001) 1-31. [27] Kusuzaki K, Takai T, Yoshimura H, Inoue K, Takai S, Baldini N, Clinical trial of radiation
ED
after intravenous injection of acridine for patients with cancer, Anticancer Res 38 (2018)
PT
481-489.
CC E
Legends for figures
Figure.1: Photodynamic surgery. (a) surgical field after tumor resection (b-d) We sprayed
a 1-μg/ml solution of AO into the surgical field (b). Repeated fluorescence-guided resection
A
using the ultrasonic knife was then performed by excitation with blue light from a xenon lamp using an interference filter (450–490 nm), and the remaining tumor fragments, which had selectively taken up AO, emitted a green fluorescence that was visible under a fluorescence microscope fitted with a yellow resorption filter (c,d). Figure 2: Kaplan–Meier curve showing local recurrence-free rate for all patients Figure 3: Magnetic resonance imaging findings in case 1 with synovial sarcoma arising at
14
the right shoulder Left: axial T1-weighted image (arrow; axillary nerve) Middle: axial T2-weighted image, Right: axial T1-weighted image with fat suppression enhanced with gadolinium
IP T
Figure 4: Magnetic resonance imaging findings 5 years after surgery Left: axial T1-weighted image Right: axial T2-weighted image,
SC R
Figure 5: Computed tomography findings in case 2 with myxofibrosarcoma arising at the inguinal region.
Figure 6: Magnetic resonance imaging findings 5 years after surgery
U
Left: axial T1-weighted image
A
CC E
PT
ED
M
A
N
Right: axial T2-weighted image
15
CC E
IP T SC R
PT
ED
M
A
N
U
Table.1 Patients and tumor background Age Years Mean (Range) 46 (0-85) Sex N Male 25 Female 23 Patients’ status at surgery N Primary 36 Local recurrence 12 Tumor size cm Mean (Range) 8.5 (3-27) Neo- and adjuvant chemotherapy N Yes 26 No 22 Surgery and acridine orange therapy Intra-lesional + PDT 10 Intra-lesional+ PDT, RDT 24 Marginal + PDT 6 Marginal + PDT+ RDT 8 AJCC: American Joint Committee on Cancer PDT: Photodynamic therapy, RDT: Radiodynamic therapy
Table.2 prognostic factors for local control Variables
A
Age (years) Gender
Male Female
Tumor size (cm) Chemotherapy
No Yes
Univariate analysis HR (95% CI) p value 1.024 0.09 (0.996-1.052) 1 0.212 0.048 (0.045-0.986) 1.2 0.002 (1.068-1.349) 1 2.228 0.2 (0.647-7.675)
16
Multivariate analysis HR (95%CI) p value
1 0.258 (0.054-1.23) 1.176 (1.0441.324)
0.09 0.008
primary Recurrence
Tumor grade
Grade3 Grade2
Acridine orange therapy
MPDT-RDT
1 2.036 (0.593-6.987) 1 0.391 (0.084-1.814)
0.26
0.23
1
M-PDT I-PDT-RDT I-PDT
1.383 (0.086-22.174) 1.853 (0.216-15.873) 3.665 (0.409-32.848)
0.82 0.57 0.25
A
CC E
PT
ED
M
A
N
U
SC R
HR; Hazard ratio 95% CI; 95% confidence interval
IP T
Patient’s status
17
S1572-1000(18)30082-6 https://doi.org/10.1016/j.pdpdt.2018.06.001 PDPDT 1177
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Revised date: Accepted date:
15-3-2018 1-6-2018 1-6-2018
Please cite this article as: Nakamura T, Kusuzaki K, Matsubara T, Murata H, Hagi T, Asanuma K, Sudo A, Long-term clinical outcome in patients with high-grade soft tissue sarcoma who were treated with surgical adjuvant therapy using acridine orange after intra-lesional or marginal resection, Photodiagnosis and Photodynamic Therapy (2018), https://doi.org/10.1016/j.pdpdt.2018.06.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Long-term clinical outcome in patients with high-grade soft tissue sarcoma who were treated with surgical adjuvant therapy using acridine orange after intra-lesional or marginal resection.
IP T
Head line title: Long-term results of acridine orange therapy Tomoki Nakamura1, Katsuyuki Kusuzaki2, Takao Matsubara3, Hiroaki Murata4, Tomohito Hagi1, Kunihiro Asanuma1, Akihiro Sudo1 1Department
of Orthopaedic Surgery, Mie University Postgraduate School of Medicine.
2Department
SC R
Edobashi 2-174, Tsu-city, 514-8507, Japan
of Musculoskeletal Oncology, Takai Hospital. Kuranosho 470-8, Tenri, 632-
0006, Japan.
Orthopaedic Clinic. Shiroyama 3-4-25, Tsu-city, 514-0818, Japan.
4Department
U
3Matsubara
of Orthopaedic Surgery, Matsushita Memorial Hospital. Moriguchi-city,
A
N
570-8540, Japan. Corresponding author: Tomoki Nakamura
M
Department of Orthopaedic Surgery, Mie University Postgraduate School of Medicine 2-174 Edobashi, Tsu-city, Mie, 514-8507, Japan
ED
Tel: +81592315022, FAX; +81592315211
Highlights
PT
E-mail: [email protected]
CC E
・ We have established acridine orange (AO) therapy, including photodynamic surgery, photodynamic therapy, and radiodynamic therapy. ・ AO therapy was applied for soft tissue sarcomas to reduce local recurrence and maintaining limb function.
A
・ The 5- and 10-year local recurrence-free rates in 48 patients were 78.9% and 73.3%, respectively. ・Tumor size is an important factor for the indication of AO therapy.
1
Abstract Background: We investigated the long-term clinical efficacy of acridine orange (AO) therapy on the inhibition of local recurrence after marginal or intra-lesional tumor resection in highgrade soft tissue sarcomas (STSs).
IP T
Methods: Our study consisted of 48 patients with STSs who received AO therapy after
marginal or intra-lesional resection. The median and mean follow-up durations were 76 and
surgery, photodynamic therapy, and radiodynamic therapy.
SC R
78 months, respectively. Our AO therapy procedure was combined with photodynamic
U
Results: There were 25 men and 23 women, with a mean age of 46 years. The average tumor
N
size at surgery was 8.5 cm. At the last follow-up, 11 patients developed local recurrence. The
A
5- and 10-year local recurrence-free rates were 78.9% and 73.3%, respectively. In multivariate
M
analysis, tumor size remained significant for local control. None of the patients developed systemic or local complications. All patients recovered activities of daily life before AO
ED
therapy.
PT
Conclusion: AO therapy can be performed in safety and may be a useful therapy for acquiring long-term local control in patients with high-grade STSs. Tumor size is an
CC E
important factor for the indication of AO therapy.
Key Words: Soft tissue sarcoma; Acridine orange; Photodynamic therapy; Radiodymanic
A
therapy; Local control
2
Introduction Adequate surgical margins inhibit local tumor recurrence in high-grade soft tissue sarcomas (STSs) [1-3]. However, if tumors are in close contact with major nerves, vessels,
IP T
bones, or joints, patients can develop serious dysfunction of the affected limb after wide
resection, which might involve the sacrifice of some or all those structures. Adjuvant
SC R
radiotherapy (RT) may be performed when surgeons perform dissection between tumors
and these structures with marginal or intra-lesional resections to maintain limb function [1,4-
U
6]. Although adjuvant RT may be effective to reduce local recurrence, the long-term effects
N
of RT can include fibrosis, edema, fractures, and contractures, all of which can substantially
A
impair limb function [7-9]. Patients who undergo adjuvant RT are also likely to develop
M
major complications, such as pathological fractures and second malignancies [4,5,10]. Therefore, it is imperative to develop a new adjuvant therapy as soon as possible to reduce
ED
the local recurrence rate after marginal or intra-lesional tumor resection, but without causing
PT
damage to adjacent normal tissues for patients with high-grade STSs, in order to maintain excellent postoperative limb function.
CC E
Based on basic research studies [11-14], we have established acridine orange (AO) therapy, including photodynamic surgery (PDS), photodynamic therapy (PDT), and radiodynamic therapy (RDT) after marginal or intra-lesional tumor resection, with the aim of reducing local
A
recurrence and maintaining limb function in patients with STSs under the approval of the institutional review board [7,15-17]. Although the clinical outcome reportedly showed excellent limb function with a local control similar to that following a wide resection with a mean follow-up time of 3 or 4 years, long-term results should be evaluated for this procedure.
3
Therefore, we investigated the long-term clinical efficacy of this new AO therapy modality on the inhibition of local recurrence after marginal or intra-lesional tumor resection in high-grade STSs.
IP T
Materials and Methods Between July 1999 and October 2014, a total of 58 patients was treated with AO therapy
SC R
after marginal or intra-lesional resection. The study was approved by the institutional review board of Kyoto Prefectural University of Medicine and Mie University Postgraduate School
U
of Medicine. We defined a marginal surgical margin with the surgical plane running through
N
the pseudo-capsule (reactive zone) and an intra-lesional margin with the margin running
A
through the tumor and, therefore, the tumor remains histologically according to the
M
Enneking criteria of surgical margins (1,18). Briefly, intralesional resection means that tumor has been removed from within the capsule, while marginal resection means removed from
ED
outside the capsule. Both methods can leave residual tumor cells under some circumstances. Seven patients were excluded from the current study because of the difficulty of evaluation
PT
of local tumor control due to the short follow-up duration. We also excluded 3 patients who
CC E
received adjuvant RT after AO therapy. A total of 48 patients was therefore available for evaluation. We retrospectively studied the clinical data of the patients as contained on a prospectively maintained database program, clinical notes, and radiological imaging of the
A
patients. This clinical study was performed after obtaining informed consent from both the patients and their families following a full explanation of the study. The survival rate of all patients and local recurrence-free rates after AO therapy were analyzed using the Kaplan– Meier method. Local recurrence of tumors was evaluated every 3 to 6 months using magnetic
4
resonance imaging (MRI). The median and mean follow-up durations were 76 and 78 months (range, 3-167), respectively. Procedure of AO therapy combined with PDS, PDT, and RDT Initially, we performed intra-lesional or marginal tumor resection to avoid any damage
IP T
to intact muscles, bones, joints, major nerves, and vessels in close contact with the tumor, resulting in good limb function after surgery. In the next procedure involving PDS,
SC R
microscopic curettage with an ultrasonic surgical knife (Olympus Co. Ltd., Tokyo, Japan) was immediately followed with a fluorescence surgical microscope equipped with a high-
U
power xenon lamp (500 mW, >10,0000 lx) and special interference and resorption filters for
N
AO. We sprayed a 1-μg/ml solution of AO (Sigma Aldrich Co, St Louis, MO, USA) into the
A
surgical field. Repeated fluorescence-guided resection using the ultrasonic knife was then
M
performed by excitation with blue light from a xenon lamp using an interference filter (450– 490 nm), and the remaining tumor fragments, which had selectively taken up AO, emitted a
ED
green fluorescence that was visible under a fluorescence microscope fitted with a yellow resorption filter (>520 nm) [7,15-17]. This PDS procedure was repeated until AO fluorescence
PT
could not be elicited [7,15-17] (Figure 1).
CC E
PDT sequentially followed PDS. For AO-PDT, an unfiltered full-light beam from a xenon lamp was applied to the tumor curettage area for 10 minutes using a surgical microscope after administration of AO solution [7,15-17].
A
The final step of RDT was performed as follows. After closure of the surgical wound,
without washing out the AO solution, single-session radiotherapy with 5 Gy was immediately administered to the resected area in the RT room in 25 patients who agreed to undergo RDT [7,15-17].
5
We evaluated the staining of AO using biopsy sample when we started this pilot study because STSs had variety of histology and those tumors had a good affinity of AO (unpublished data). Statistical analysis
IP T
Survival time was measured from the date of surgery using AO to the date of sarcomarelated death or last follow-up. Local recurrence time was measured from the date of surgery
SC R
using AO to the date of local recurrence. Even if the AO therapy was performed in patients who developed local recurrence or metastasis, local recurrence time was also measured from the date of surgery using AO to the date of local recurrence. Survival curves were created
U
using the Kaplan–Meier method. Univariate and multivariate analyses were performed
N
using Cox proportional hazard regression models. All statistical analyses were conducted
A
using Stat View for Windows, version 5.0 (SAS Institute Inc., Cary, NC, USA). P values <0.05
M
were considered significant.
ED
Results
PT
Our study consisted of 48 patients with STSs who received AO therapy (Table 1). We included 12 patients who had local recurrence. There were 25 men and 23 women, with a
CC E
mean age of 46 years (range, 11 months–85 years). Primary tumor sites included the thigh (n=13), forearm (n=7), buttock (n=5), popliteal lesion (n=4), foot (n=3), lower leg (n=3), inguinal region (n=3), upper arm (n=2), and other regions (n=8). The tumors were
A
histologically classified as synovial sarcomas (n=9), undifferentiated pleomorphic sarcomas (UPSs) (n=8), rhabdomyosarcomas (n=6), liposarcomas (n=5; myxoid type n=4, pleomorphic type n=1), leiomyosarcomas (n=4), myxofibrosarcomas (n=4), malignant peripheral nerve sheath tumors (n=4), and others (n=8). All tumors were high-grade sarcomas categorized as
6
grade 2 (n=16) or 3 (n=32) according to the criteria of the French Federation of Cancer Centers Sarcoma Group (FNCLCC) grading system. We included 11 patients who developed lung metastasis at initial presentation. The average tumor size at surgery was 8.5 cm (range, 3–27). Neo- and adjuvant chemotherapy was performed in 26 patients. Chemotherapy was
IP T
administered in younger patients (p<0.0001, Mann–Whitney U test). Surgeons performed
dissection between tumors and preserved structures with marginal (in 14 patients) or intra-
SC R
lesional (in 34 patients) resection to maintain limb function. The distribution of AO therapy for each patient is shown in Table 1.
U
At the last follow-up, local recurrence occurred in 11 patients: 4/10 patients treated with
N
PDT after intra-lesional excision; 1/6 patients treated with PDT after marginal resection; 5/24
A
patients treated with PDT and RDT after intra-lesional excision; and 1/8 patients treated with
M
PDT and RDT after marginal resection. Although there was a high rate of local recurrence (40%, 4 of 10 patients) in patients treated with PDT after intra-lesional excision, there was no
ED
significant difference between other methods of AO therapy (Table 2). The 5- and 10-year
PT
local recurrence-free rates were 78.9% [95% confidence interval (CI): 67.3–90.6] and 73.3% [95% CI: 58.1–88.5], respectively (Figure 2). In Cox hazard univariate analysis, the patients
CC E
with large tumor sizes had worse local control (hazard ratio (HR): 1.2, 95% CI: 1.068–1.349, p=0.002). Female patients had significantly better local control (HR: 0.212: 0.045–0.986, p=0.048). Age, administration of chemotherapy, status of tumors (primary vs. local
A
recurrence), FNCLCC grade (grade 2 vs. 3), and type of AO therapy were not significant predictive factors for local control (Table 2). In multivariate analysis, tumor size remained significant. Of 11 patients with local recurrence after AO therapy, three patients underwent amputation.
7
Two of three patients died of stomach cancer and pancreatic cancer, respectively. The remaining one survived with no disease after undergoing amputation. Two of eight patients received palliative radiation because of their progressive lung metastasis; they died of their disease. Another 2 patients died from multiple distant metastases. The remaining 4 patients
IP T
underwent tumor re-excision and survived with no disease. Three of four patients underwent tumor excision with adjuvant RT.
SC R
None of the patients developed systemic or local complications following local
administration of AO, light, and X-ray irradiation. All patients recovered activities of daily
U
life before AO therapy at the last follow-up, except for the patients who underwent
A
N
amputation or additional tumor resection because of local recurrence.
M
Case presentation
Case 1: The patient was a 21-year-old woman with synovial sarcoma arising at the right
ED
shoulder. Neo- and adjuvant chemotherapy comprising doxorubicin and ifosfamide were
PT
administered. The tumor was in close contact with the axillary nerve (Figure 3). If the tumor was resected with a strictly wide margin, the patient could develop serious dysfunction of
CC E
the shoulder. Therefore, we planned PDS followed by AO-PDT and RDT to preserve the axillary nerve. After informed consent for the study was provided, AO therapy was performed. After therapy, the motor and sensory functions of the right shoulder were normal
A
and the patient is now able to elevate her shoulder without any obvious handicap. There is no evidence of local tumor recurrence even after a follow-up of 136 months (Figure 4). Limb function evaluated based on the International Society of Limb Salvage (ISOLS) criteria was 100%.
8
Case 2: The patient was a 67-year-old woman with a myxofibrosarcoma arising at an inguinal region. MRI revealed that the tumor was in close contact with the femoral artery and vein, as well as the femoral nerve (Figure 5). Since wide resection of the tumor could cause serious
IP T
dysfunction of the lower extremities even after reconstruction of vessels by transplantation
of the femoral artery and vein, we proposed AO therapy to the patient and her family. After
SC R
their agreement with the study, we planned PDS followed by AO-PDT and RDT to preserve
the vessels and nerve. There is no evidence of local tumor recurrence even after a follow-up
N
U
of 73 months (Figure 6). Limb function evaluated by the ISOLS criteria was 100%.
A
Discussion
M
In the present study, local recurrence after AO therapy occurred in 11 patients (23%). The 5and 10-year local recurrence-free rates were 78.9% and 73.3%, respectively. In multivariate
ED
analysis, the patients with larger tumor sizes had significantly poorer local control. We firstly reported the long-term results of AO therapy and suggest that AO therapy is safe procedure
PT
and may be useful for acquiring long-term local control after marginal or intra-lesional tumor
CC E
resection and for preserving excellent limb function in selected patients with high-grade STSs.
We established AO therapy based on the characteristics of AO. AO excited by blue light
A
produces an excited singlet state which can lose energy by fluorescence or undergo intersystem crossing to the triplet state which can then undergo an energy transfer mechanism to triplet oxygen producing singlet oxygen (photochemical reaction II) (19,20). AO is accumulated into acidic environments, sarcoma cells having a lot of large acidic
9
vesicles that readily take up AO easily, whereas normal cells with non-acidic environments and weak acidic lysosomes can quickly excluded AO. It has been clarified that the inhibition of V-ATPase activity by bafilomycin causes a decrease in AO accumulation into the lysosome, suggesting that AO accumulates into the lysosome in an acidity-dependent manner (16,21).
IP T
Wide tumor resection for high-grade STSs in contact with critical muscles, vessels, nerves,
bones, or joints causes a serious deficit of limb function [7,22,23]. Especially at the forearm,
SC R
hand, lower leg, foot, popliteus, axilla, inguinal region, and buttock, it is not easy to reserve
such structures by surgical tumor resection with a strict wide margin. However, marginal
U
excision includes peritumoral reactive tissue, but insufficient margins and is associated with
N
high local recurrence rates [1-3]. Adjuvant RT is the standard approach for high-grade STSs
A
[1,4-6]. Two randomized clinical trials demonstrated a 20–25% reduction in the risk of local
M
recurrence with radiation in patients with extremity STSs [4,24,25]. Trovik et al. also reported the affection of surgical margins with or without adjuvant RT for local recurrence of STSs in
ED
a Scandinavian group [26]. They showed that the rate was 25 of 64 patients (39%) after an
PT
intra-lesional or marginal resection without postoperative RT, versus 28 of 119 patients (24%) when RT was administered. However, Kneisl et al. reported that the patients who underwent
CC E
adjuvant RT (median dose of 50–60 Gy) had a major complication rate of 16.2%, including complications such as pathological fractures and secondary malignancies, compared to 3.9% in those who did not receive RT [4]. Postoperative wound complications should also be
A
considered in patients who undergo RT [4,5]. In the present study, local recurrence after AO therapy occurred in 11 patients (23%), which is similar to the results in patients who received adjuvant RT in a study by Trovik et al [24]. When we performed RDT, only single-session radiotherapy of 5 Gy was administered to the resected area. We had no experience of
10
complications after AO therapy. Therefore, we believe that AO therapy was safe and effective for local control. We also suggest that if the tumor can be resected without serious dysfunction of the affected limb after wide resection, AO therapy should not be indicated because tumor resection with wide surgical margin is standard treatment for good local
IP T
control.
Interestingly, when we divided the patients into 4 groups according to the AO therapy,
SC R
intra-lesional tumor resection with PDT alone conferred a high rate of local recurrence (of 40%), although there were no significant differences in the rates between the types of AO
U
therapy. Furthermore, there was no significant difference between the patients who
N
underwent PDT alone and those who underwent PDT and RDT. RDT may be necessary to
M
future studies are necessary to validate this.
A
prevent local recurrence in addition to intra-lesional tumor resection with PDT, although
Concerning large tumors, AO therapy may be insufficient because it is so difficult to expose
ED
all the remaining cells to AO and illuminate such wide areas with the surgical fluorescence
PT
microscope. Therefore, we consider that tumors with a diameter larger than 10 cm are not a suitable indication for AO therapy. Furthermore, this procedure is indicated as local therapy.
CC E
A clinical trial of RT after intravenous injection of AO for patients with advanced cancer is ongoing [25]. Although a more intensive clinical trial is necessary, PDT and RDT involving systemic administration of AO may be applied for unresectable or metastatic tumors.
A
There are some limitations in this study. First, there is a small number of patients. We should
validate our findings, especially for efficacy, in a future study. Second, this study is not a randomized clinical trial, but a pilot study. Therefore, local control cannot be directly compared in the patients with or without AO therapy.
11
In conclusion, AO therapy may be useful for acquiring long-term local control in patients with high-grade STSs. Tumor size is an important factor for the indication of AO therapy.
SC R
There was no funding for this study. There is no conflict of interest.
IP T
Acknowledgement
References
U
[1] Grimer R, Judson I, Peake D, Seddon B, Guidelines for the management of soft tissue
N
sarcomas, Sarcoma 2010; 2010: 5061182.
A
[2] Herbert SH, Corn BW, Solin LJ, et al, Limb-preserving treatment for soft tissue sarcomas of the extremities, The significance of surgical margins. Cancer 72 (1993) 1230-1238.
M
[3] Weiss SW, goldblum JR, Local recurrence In Enzinger and Weiss’s soft tissue tumors,5th edition, St Louis, MO, USA; Mosby (2008) 15-31.
ED
[4] Kneisl JS, Ferguson C, Robinson M et al, The effect of radiation therapy in the treatment of adult soft tissue sarcomas of the extremities: a long-term community-based cancer center
PT
experience, Cancer Medicine 6 (2016) 516-525. [5] Haas RL, Delaney TF, O’Sullivialn B et al, Radiationtherapy for management of extremity
CC E
soft tissue sarcomas: why, when, and where? Int J Radiat Oncol Biol Phys. 83 (2012) 572-580. [6] Abouarab MH, Salem IL, Degheidy MM et al, Therapeutic options and postoperative wound complications after extremity soft tissue sarcoma resection and postoperative external beam radiotherapy, Int Wound J 15 (2018) 148-158.
A
[7] Matsubara T, Kusuzaki K, Matsumine A, Nakamura T, Sudo A, Can a less radical surgery using photodynamic therapy with acridine orange be equal to a wide-margin resection? Clin Orthop Relat Res 471 (2013) 792-802. [8] Fuchs B, Davis AM, Griffin AM, et al, Sciatic nerve resection in the thigh: a functional evaluation, Clin Orthop Relat Res 382 (2001) 34-41.
12
[9] Ghert MA, Davis AM, Griffin AM, et al, The surgical and functional outcome of limbsalvage surgery with vascular reconstruction for soft tissue sarcoma of the extremity, Ann Surg Oncol 12 (2005) 1102-1110. [10] Sholl LM, Barletta JA, Hornick JL, Radiation-associated neoplasia: clinical, pathological and genomic correlates, Histopathology 70 (2017) 70-80.
IP T
[11] Kusuzaki K, Takeshita H, Murata H et al, Photodynamic inactivation with acridine orange on a multidrug-resistant mouse osteosarcoma cell line, Jpn J Cancer Res 91 (2000) 439445.
on mouse osteosarcoma, Anticancer Res 20 (2000) 3019-3024.
SC R
[12] Kusuzaki K, Suginoshita T, Minami G et al, Fluorovisualization effect of acridine orange
[13] Kusuzaki K, Aomori K, Suginoshita et al, Total tumor cell elimination with minimum
U
damage to normal tissues in muscloskeltal sarcomas by photodynamic reaction with acridine
N
orange, Oncology 59 (2000) 174-180.
[14] Hashiguchi S, Kusuzaki K, Hirasawa Y et al, Acridine orange excited by low-dose
A
radiation has a strong cytcidal effect on mouse osteosarcoma, Oncology 62 (2002) 85-93.
M
[15] Kusuzaki K, Murata H, Uchida A et al, Clinical trial of photodynamic therapy using acridine orange with/without low dose radiation as new limb salvage modality in
ED
muscloskeltal sarcomas, Anticancer Res 25 (2005) 1225-1235. [16] Kusuzaki K, Murata H, Uchida A et al, Clinical outcome of a new phodynamic therapy
PT
with acridine orange for synovial sarcomas, Rhotochem Photobil 83 (2005) 705-709. [17] Nakamura T, Kusuzaki K, Matsubara T, Matsumine A, Murata H, Uchida A, A new limb salvage surgery in cases of high-grade soft tissue sarcoma using photodynamic surgery,
CC E
followed by photo- and radiodynamic therapy with acridine orange, J Surg Oncol 97 (2008) 523-528.
[18] Enneking WF, Spanier SS, Goodman M, A system for the surgical staging of
A
musculoskeletal sarcoma, Clin Orthop Relat Res 153 (1980) 106-120. [19] Zdolsek JM, Acridine orange-mediated photodamage to cultured cells. Apmis 101 (1993) 127-132. [20] Miccoli L, Beurdeley-Thomas A, De Pinieux G, Sureau F, Oudard S, Dutrillaux B, Poupon MF, Light-induced photoactivation of hypericin affects the energy metabolism of human glioma cells by inhibited hexokinase bound to mitochondria. Cancer Res 17 (1998) 5777-5786.
13
[21] Hiruma H, Katakura, T, Igawa S, Kanoh M, Fujimura T, Kawakami T, vesicle disruption, plasma membrane bleb formation, and acute cell death caused by illumination with blue light in acridine-loaded malignant melanoma cells. J Photochem Photobiol 86 (2007) 1-8. [22] Brooks AD, Gold JS, Graham D et al, Resection of the sciatic, peroneal, or tibial nerves: assessment of fuctional status, Ann Surg Oncol. 9 (2002) 41-47.
IP T
[23] Stinson SF, DeLaney TF, Greenberg J et al, Acute and long-term effect on limb function of combined modality limb sparing therapy for extremity soft tissue sarcoma, Int J Radiat Oncol Biol Phys 21 (1991) 1493-1499.
SC R
[24] Pisters PW, Harrison LB, Leung DH, Woodruff JM, Casper ES, Brennan MF, Long-term
results of a prospective randomized trial of adjuvant brachytherapy in soft tissue sarcoma, J Clin Oncol 14 (1996) 859-868.
U
[25] Yang JC, Chang AE, Baker AR, et al, Randomized prospective study of the benefit of
N
adjuvant radiotherapy in the treatment of soft tissue sarcomas of the extremity, J Clin Oncol 16 (1998) 197-203.
A
[26] Trovik CS, Scandinavian Sarcoma Group Project. Local recurrence of soft tissue sarcoma.
M
A Scandinavian Sarcoma Group Project. Acta Orthop Scand Suppl 72 (2001) 1-31. [27] Kusuzaki K, Takai T, Yoshimura H, Inoue K, Takai S, Baldini N, Clinical trial of radiation
ED
after intravenous injection of acridine for patients with cancer, Anticancer Res 38 (2018)
PT
481-489.
CC E
Legends for figures
Figure.1: Photodynamic surgery. (a) surgical field after tumor resection (b-d) We sprayed
a 1-μg/ml solution of AO into the surgical field (b). Repeated fluorescence-guided resection
A
using the ultrasonic knife was then performed by excitation with blue light from a xenon lamp using an interference filter (450–490 nm), and the remaining tumor fragments, which had selectively taken up AO, emitted a green fluorescence that was visible under a fluorescence microscope fitted with a yellow resorption filter (c,d). Figure 2: Kaplan–Meier curve showing local recurrence-free rate for all patients Figure 3: Magnetic resonance imaging findings in case 1 with synovial sarcoma arising at
14
the right shoulder Left: axial T1-weighted image (arrow; axillary nerve) Middle: axial T2-weighted image, Right: axial T1-weighted image with fat suppression enhanced with gadolinium
IP T
Figure 4: Magnetic resonance imaging findings 5 years after surgery Left: axial T1-weighted image Right: axial T2-weighted image,
SC R
Figure 5: Computed tomography findings in case 2 with myxofibrosarcoma arising at the inguinal region.
Figure 6: Magnetic resonance imaging findings 5 years after surgery
U
Left: axial T1-weighted image
A
CC E
PT
ED
M
A
N
Right: axial T2-weighted image
15
CC E
IP T SC R
PT
ED
M
A
N
U
Table.1 Patients and tumor background Age Years Mean (Range) 46 (0-85) Sex N Male 25 Female 23 Patients’ status at surgery N Primary 36 Local recurrence 12 Tumor size cm Mean (Range) 8.5 (3-27) Neo- and adjuvant chemotherapy N Yes 26 No 22 Surgery and acridine orange therapy Intra-lesional + PDT 10 Intra-lesional+ PDT, RDT 24 Marginal + PDT 6 Marginal + PDT+ RDT 8 AJCC: American Joint Committee on Cancer PDT: Photodynamic therapy, RDT: Radiodynamic therapy
Table.2 prognostic factors for local control Variables
A
Age (years) Gender
Male Female
Tumor size (cm) Chemotherapy
No Yes
Univariate analysis HR (95% CI) p value 1.024 0.09 (0.996-1.052) 1 0.212 0.048 (0.045-0.986) 1.2 0.002 (1.068-1.349) 1 2.228 0.2 (0.647-7.675)
16
Multivariate analysis HR (95%CI) p value
1 0.258 (0.054-1.23) 1.176 (1.0441.324)
0.09 0.008
primary Recurrence
Tumor grade
Grade3 Grade2
Acridine orange therapy
MPDT-RDT
1 2.036 (0.593-6.987) 1 0.391 (0.084-1.814)
0.26
0.23
1
M-PDT I-PDT-RDT I-PDT
1.383 (0.086-22.174) 1.853 (0.216-15.873) 3.665 (0.409-32.848)
0.82 0.57 0.25
A
CC E
PT
ED
M
A
N
U
SC R
HR; Hazard ratio 95% CI; 95% confidence interval
IP T
Patient’s status
17