Hyperbaric oxygen therapy for radiation-induced tissue injury following sarcoma treatment: A retrospective analysis of a Dutch cohort.

BACKGROUND AND OBJECTIVES: Sarcomas are commonly managed by surgical resection combined with radiotherapy. Sarcoma treatment is frequently complicated by chronic wounds and late radiation tissue injury (LRTI). This study aims to gain insight in the use and results of hyperbaric oxygen therapy (HBOT) for radiation-induced complications following sarcoma treatment.
METHODS: All sarcoma patients treated between 2006 and 2017 in one of the five centers of the Institute for Hyperbaric Oxygen in the Netherlands were included for retrospective analysis.
RESULTS: Thirty patients were included, 18 (60.0%) patients were treated for chronic wounds and 12 (40.0%) for LRTI. Two patients with chronic wounds were excluded from analysis as HBOT was discontinued within five sessions. In 11 of 16 (68.8%) patients treated for chronic wounds, improved wound healing was seen. Nine of 12 (75.0%) patients treated for LRTI reported a decline in pain. Reduction of fibrosis was seen in five of eight patients (62.5%) treated for LRTI.
CONCLUSIONS: HBOT is safe and beneficial for treating chronic wounds and LRTI in the sarcoma population. Awaiting further prospective results, we recommend referring to HBOT centers more actively in patients experiencing impaired wound healing or symptoms of delayed radiation-induced tissue injury following multimodality sarcoma treatment.

Introduction

Sarcomas are malignant neoplasms originating from mesenchymal tissues, broadly divided by histology in soft-tissue and bone sarcomas, responsible for approximately 1% and 0.2% of all malignancies in adults, respectively [1]. The incidence of bone and soft-tissue sarcomas together is 900–1000 cases in the Netherlands per year [2].

Sarcoma management is complex and usually involves multiple modalities, such as radiotherapy (RT) and in some cases chemotherapy (CT). Surgical excision is the cornerstone of treatment and is essential to achieve local tumor control [3,4]. RT can be required to minimize local recurrence [5], but is frequently complicated by radiation-induced tissue injury [6-8]. Acute injury presents during or immediately following RT and is generally self-limiting. Late radiation tissue injury (LRTI) is characterized by a symptom-free latent period of three to six months or more, after which complications such as subcutaneous tissue fibrosis, edema, joint stiffness, osteoradionecrosis and fracture may occur [9,10].

The RT of choice in sarcoma management is external-beam radiotherapy (EBRT) and is administered in either the preoperative or postoperative setting. Both settings cause radiation-induced injury [8,11], but the timing influences the pattern and type of injury. The postoperative major wound complication rate is approximately 35% for preoperative EBRT, while postoperative EBRT leads to wound complications in 17% of patients [6-8]. Delayed wound healing, among others, is classified as a major wound complication [7]. Furthermore, high rates of LRTI have been reported in both preoperative and postoperative EBRT. Postoperative EBRT, which is characterized by larger radiation fields and higher radiation doses, more often results in LRTI [11-13]. Fibrosis occurs in around 48% of postoperatively irradiated patients and 32% of preoperatively irradiated patients, joint stiffness in 23% and 18% and edema in 23% and 15%, respectively [10,11,14]. Complications following sarcoma treatment result in detrimental functional outcome and general health status of patients [12,13].

Hyperbaric oxygen therapy (HBOT) is a therapy that comprises the administration of 100% oxygen at an elevated environmental pressure, which results in a direct increase in tissue oxygenation [15]. Also, HBOT mobilizes stem cells that enhance vasculogenesis [16,17]. These mechanisms result in improved tissue quality by neovascularization [9,18,19] and have shown to be beneficial in radiation proctitis and LRTI involving bone and soft tissues of the head and neck [9,20,21]. Furthermore, the application of HBOT after combined surgery and RT in head and neck tumors has shown to lead to improved healing of chronic wounds [22].

This retrospective cohort study aims to provide an overview of the use and results of HBOT for radiation-induced tissue injuries following multimodality sarcoma treatment. It is hypothesized that HBOT improves wound healing and LRTI for radiation-induced tissue injury in sarcoma patients.

Materials and methods

Patients

The Institutional Review Board of the University Medical Center Groningen (UMCG) approved this retrospective study (case number 2017.521). Informed consent was obtained from all patients included in the study. All sarcoma patients who were treated between January 2006 and October 2017 in one of the five centers of the Institute for Hyperbaric Medicine in the Netherlands were included. Patients were referred to the HBOT centers by medical specialists from various medical centers in the Netherlands for presence of chronic wounds or LRTI. Wounds were classified as chronic when not healed within three months. LRTI was defined as symptomatic tissue injury due to radiation toxicity after three months or more following completion of RT. The effects of HBOT were determined by either the patient or the doctor during follow up. Patients’ medical records were analyzed by a medical doctor to gain insight in patient and tumor characteristics, sarcoma treatment, indications for HBOT and HBOT results.

HBOT procedure

Patients underwent daily HBOT sessions (excluding weekends) for six to eight weeks in a hyperbaric chamber in one of the five facilities of the Institute of Hyperbaric Medicine in the Netherlands. In the chamber, the pressure was elevated to 2.5 atmospheres absolute (ATA) in ten minutes. When pressure reached 2.5 ATA, 100% oxygen was administrated in three sessions of 20 minutes by face mask, alternated with five minutes of room air, followed by a final 15 minutes of oxygen therapy. After this, pressure was lowered in ten minutes, with decompression on oxygen up to 1.3 ATA. This results in a total treatment duration of 110 minutes, with patients being exposed to hyperbaric oxygen at 2.5 ATA for a total of 90 minutes [21].

Statistical analysis

Quantitative analyses were performed using SPSS® Version 23.0 (IBM SPSS Statistics for Windows, Version 23.0 Armonk, NY: IBM Corp). Categorical variables were described using frequencies and percentages, numerical variables by median and interquartile ranges (IQR) or by mean and standard deviation (SD) as appropriate.

Results

Population

In total, 30 patients were included in this study, 18 men (60.0%) and 12 women (40.0%). The age of patients at initiation of HBOT ranged from 19 to 86 years and median age was 67.0 (55.3–74.5) years. The distribution of patients among different HBOT centers was as follows: 13 (43.3%) patients were treated in center A, seven (23.3%) in center B, five (16.7%) in center C, three (10.0%) in center D and two (6.7%) in center E. Time between surgery and referral was median 9 (2.8–26.8) months.

More than half of the patients, 16 (53.3%), had no history of smoking. Six (20.0%) patients had stopped smoking more than six months prior to therapy and two (6.7%) patients stopped smoking within six months prior to the start of HBOT. There were five (16.7%) patients still smoking at HBOT initiation. Diabetes mellitus (DM) was present in four (13.3%) patients and absent in 25 (83.3%) patients. Distinction between sarcoma groups was based on pathology results from the medical center from which the patient was referred. Type of sarcoma was bone in four (13.3%) and soft-tissue in 26 (86.7%) patients. Sarcomas were located in the trunk/thorax in seven (23.3%) patients, upper extremity in six (20.0%) patients, lower extremity in 16 (53.3%) and head/neck in one (3.3%) patient.

Sarcoma treatment consisted of only RT in one (3.3%) patient, RT and CT in one (3.3%) patient, RT, CT and resection in two (6.7%) patients and RT and resection in 26 (86.7%) patients. Wound closure after sarcoma resection was primary in 22 (73.3%), split skin graft (SSG) in one (3.3%) patient and by application of a skin flap in four (13.3%) patients. All patients were treated with EBRT. EBRT was administered in the preoperative setting in seven (23.3%) patients and postoperative in 22 (73.3%) patients. Preoperative patients were irradiated with a total dose of 50 Gy in 2 Gy fractions. The postoperative patients received a median total dose of 66.3 (60.0–70.0) Gy and all but two patients were treated in fractions of 2 Gy. These two patients were irradiated in fractions of 1.9 and 2.5 Gy. The patient that was treated by RT and CT received a total dose of 45.0 Gy in fractions of 1.8 Gy. Table 1 presents patient, tumor and treatment characteristics.

Table 1

Patient, tumor and sarcoma treatment characteristics.

Characteristic	N = 30
Gender
Male	18 (60.0%)
Female	12 (40.0%)
Age (years)	67.0 (55.3–74.5)
HBOT center
Center A	13 (43.3%)
Center B	7 (23.3%)
Center C	5 (16.7%)
Center D	3 (10.0%)
Center E	2 (6.7%)
Time between surgery and referral (months)	12.5 (2.8–26.8)
Smoking
Never	16 (53.3%)
Stopped since > 6 months	6 (20.0%)
Stopped since < 6 months	2 (6.7%)
Yes	5 (16.7%)
Missing	1 (3.3%)
DM
Yes	4 (13.3%)
No	25 (83.3%)
Missing	1 (3.3%)
Sarcoma type
Bone	4 (13.3%)
Soft-tissue	26 (86.7%)
Sarcoma location
Trunk/thorax	7 (23.3%)
Upper extremity	6 (20.0%)
Lower extremity	16 (53.3%)
Head/neck	1 (3.3%)
Sarcoma treatment
RT	1 (3.3%)
RT + CT	1 (3.3%)
RT + CT + resection	2 (6.7%)
RT + resection	26 (86.7%)
Wound closure
Primary	22 (73.3%)
Skin flap	4 (13.3%)
SSG	1 (3.3%)
Missing	3 (10.0%)
Timing of EBRT
Preoperative	7 (23.3%)
Postoperative	22 (73.3%)
Missing	1 (3.3%)
Total dosage of RT administered (Gy)
Preoperative	50 (50–50)
Postoperative	66.3 (60–70)

Data is presented as n (%), mean (SD) or median (IQR). Abbreviations. HBOT: hyperbaric oxygen therapy. DM: Diabetes Mellitus. EBRT: external-beam radiotherapy. SSG: split skin graft. RT: radiotherapy. CT: chemotherapy.

HBOT characteristics

Indications for HBOT were chronic wounds in 18 (60.0%) patients and LRTI in 12 (40.0%) patients. Of the 30 patients, 23 (76.7%) finished all sessions according to the treatment plan. Two patients ended HBOT because of diagnosis with lung metastases. Furthermore, presence of a lung embolus, treatment takeover by an academic center, claustrophobia, suspicion of a leg tumor and viral infection were reasons to stop for the other patients. The patients that did not continue HBOT because of claustrophobia and takeover by an academic center only underwent one and four sessions, respectively. These patients were therefore excluded from analysis of HBOT effects. The median of HBOT sessions was 40 (30–40) for the 28 remaining patients.

Adverse events

Twenty-two (73.3%) patients reported no adverse events, the other eight (26.7%) patients did. Barotrauma to the middle ear due to differences in ambient pressure was observed via otoscopy in five (55.5%) patients. Pressure equalization (PE) tubes were inserted in three of five patients. In one of the five patients, tympanocentesis for otitis media with effusion and vertigo was performed, which relieved the complaints. Myopia was reported two (22.2%) times. Fatigue was recorded one (11.1%) time. The patient that ended HBOT after one session reported claustrophobia (11.1%) as adverse event. One of the eight patients that experienced adverse events, had both myopia and ear problems for which a PE tube was placed.

Effects on chronic wounds and LRTI

Fifteen of the 16 chronic wounds were located at the site of the surgical wound. One wound spontaneously occurred in irradiated area near to the operation site. Of the 16 patients that received HBOT for chronic wounds, ten had data available on wound measurements before and after treatment. Improved wound healing was seen in all of these ten patients. In total, in 11 of 16 (68.8%) patients a trend towards healing of the wound was seen. Of these 11 patients that showed improved wound healing, complete wound healing, either (delayed) primary or secondary, was observed in seven patients. This corresponds with 43.8% of the total number of patients treated for chronic wounds. Table 2 presents effects of HBOT on chronic wounds. Fig 1 shows pictures of a patient referred from our medical center for a chronic wound in the upper leg before and after HBOT.

Table 2

Effects of HBOT on chronic wounds.

Patient	Sex	Age (years)	Smoking	DM	Timing of RT	Total dose of RT (Gy)	Number of HBOT sessions	Wound size prior to HBOT	Wound size after HBOT	Results
1	Female	21	No, never	No	Postoperative	Not recorded	20 + 10	Not recorded	3x4 cm	Wound closure by local mesoplastics.
2	Male	69	No, never	No	Postoperative	70	30	15.8x10.4 cm	Not recorded	Improved wound healing at 20 HBOT sessions.
3	Male	61	Yes	No	Postoperative	60	30 + 10	Not recorded	Not recorded	Cleaner, granulating wound, closure of the wound by free flap.
4	Male	84	No, never	No	Postoperative	50	40	3 cm deep	2.9 cm deep	Granulating wound.
5	Male	76	No, since > 6 months	No	Postoperative	64	45 + 20	14x14 cm	10x7 cm	Improved wound healing, wound closure by SSG.
6	Male	73	Yes	No	Postoperative	70	40	2x1 cm		Complete healing.
7	Female	68	No	No	Preoperative	50	30	2x3x10 cm	1x1 cm (4–5 cm beneath the skin)	Improved wound healing.
8	Male	70	No	No	Preoperative	50	30	6x2x1 cm		Complete healing.
9	Female	69	No	No	Postoperative	60	40	8 cm deep	Not recorded	Closure by tensor fascia lata free flap after 40 sessions.
10	Male	82	No	No	Preoperative	50	35	12x8x2 cm	5 cm in length	Superficial defect, improved wound healing.
11	Male	74	No	No	Preoperative	50	30	8x1x0.4 cm		Complete healing.
12	Male	86	No, since < 6 months	No	Postoperative	70	40	4x5 cm	3x4 cm	Improved wound healing, more granulation tissue.
13*	Female	69	No	Yes	Postoperative	Not recorded	26	5x2.5x3 cm	Not recorded	Cleaner wound, need for surgical closure (but impossible due to cardiac problems).
14	Female	63	No	Yes	Preoperative	50	27	8 cm deep	5 cm deep	Improved wound healing, clean wound.
15	Male	78	Yes	No	Preoperative	50	33	1 cm	Not recorded	Improved wound healing (wound almost closed), reduction of drain fluid.
16	Female	73	No, since < 6 months	Yes	Postoperative	60	36	2.5x0.2 cm	2x0.2 cm	Improved wound healing.

Abbreviations. DM: Diabetes Mellitus. RT: radiotherapy. HBOT: hyperbaric oxygen therapy. SSG: split skin graft. *This patient had a chronic wound near to the operation site in irradiated area. All other patients had non-healing surgical wounds.

Fig 1

Pictures of a patient referred from our medical center for a chronic wound of the medial upper leg after limb-salvage therapy, consisting of surgery and preoperative radiotherapy.

A, At HBOT initiation, 11 weeks after completion of limb-salvage therapy. B, Improved wound healing is seen after 40 sessions of HBOT.

Twelve patients were referred to HBOT centers for LRTI, including pain, fibrosis, osteoradionecrosis and radiation cystitis. Six of these patients were referred because of pain complaints. All of these patients reported a decrease in pain. In total, nine out of 12 (75.0%) patients experienced a decline in pain. Also, eight patients underwent HBOT for fibrosis and in five of eight (62.5%) patients an improvement of fibrosis was reported. Increase of joint function was reported twice. HBOT was indicated for osteoradionecrosis in one patient, but no objective results were reported. One patient received HBOT for hematuria resulting from radiation cystitis and hematuria resolved completely. Results of HBOT on indication for referral and other symptoms of LRTI are presented in Table 3.

Table 3

Effects of HBOT on LRTI.

Patient	Sex	Age (years)	Smoking	DM	Timing of RT	Total dose of RT (Gy)	Number of HBOT sessions	LRTI indication	Results on LRTI indication	Other results
17	Male	77	No, since > 6 months	No	Postoperative	70	30	Radiation-induced injury of soft tissues	Decrease of pain, edema and fibrosis.
18	Female	47	No, since > 6 months	No	Postoperative	70	40	Pain, fibrosis	Less pain.	Decrease in edema, erythema and increase in function of arm.
19	Female	62	Yes	No	Postoperative	66.5	40	Pain, fibrosis	Less pain, but pain medication was also altered during HBOT.	No change in joint function.
20	Female	66	No	No	Postoperative	70	40	Pain, fibrosis	Decrease in pain and fibrosis.	Increase in knee joint function.
21	Female	47	No, since > 6 months	No	Postoperative	66	40	Pain, fibrosis	Decrease in pain and fibrosis.
22	Male	64	No, since > 6 months	No	Postoperative	70	40	Pain, fibrosis	Decrease in pain and fibrosis.
23	Female	85	No	No	Postoperative	60	29	Pain	Slight improvement of pain.	HBOT was seen as intensive.
24	Female	65	Yes	No	Postoperative	70	40	Osteoradionecrosis	No objective results regarding to the osteoradionecrosis.	Less pain, decreased use of pain medication, improvement of defecation pattern.
25	Male	44	Not recorded	Not recorded	Postoperative	66	28 + 10	Fibrosis	Decrease in fibrosis.
26	Male	19	No	No	No operation	45	40	Radiation cystitis	Resolution of hematuria.
27	Male	61	No	No	Postoperative	70	40	Fibrosis	Decrease in fibrosis.	Decrease in pain.
28	Male	58	No, since > 6 months	Yes	Postoperative	60	40	Fibrosis		Decrease in pain. Less problems with defecation and micturition.

Abbreviations. DM: Diabetes Mellitus. RT: radiotherapy. HBOT: hyperbaric oxygen therapy. LRTI: late radiation tissue injury.

Discussion

No recent literature is available on HBOT outcome in chronic wounds and delayed radiation injury in the sarcoma population. In a case series of 17 patients receiving HBOT for chronic necrotic wounds in extremities after RT for a variety of malignancies, including eight soft-tissue sarcomas, Feldmeier et al. (2000) reported complete wound healing in 85% of patients when also taking into account delayed primary closed wounds [23]. In our cohort, complete wound healing was seen in seven of 16 (43.8%) patients. The difference in rates may result from dissimilar population characteristics. HBOT is more accepted as an adjunct to treatment in a variety of other malignancies treated with RT, both originating from soft-tissue and bone, such as head/neck tumors and tumors in the pelvic region [9,18,20,24,25]. In these studies, HBOT results in improvement of chronic wounds and LRTI.

In our medical center, HBOT is offered to a selection of patients suffering from radiation-induced tissue injuries in a late stadium and it is considered as a ‘last resort’ treatment option. Since median time between surgery and referral for HBOT was 9 months in the cohort, this view on HBOT is thought to be shared by specialists in the Netherlands. Of note, the onset of chronic wounds is frequently in the first few weeks after primary closure. In general, these wounds show inferior healing tendency rapidly after surgery and open shortly after sutures are removed. The restrained attitude towards HBOT is due to lack of evidence for efficacy of the therapy in sarcoma patients in our medical center. As a consequence, the patients referred to receive HBOT are usually the ones with the most severe complications. This results in a study population not representative of the actual patient population.

This retrospective cohort analysis has limitations resulting from the nature of the study. Data from patient records were gathered retrospectively, which leads to missing data, subjective outcomes and a limited population size. Outcomes are partly subjective, because in 10/16 patients treated for chronic wounds, wound measurements by a medical doctor before and after hyperbaric oxygen therapy are reported. Also, all sarcoma patients treated in over ten years in five centers in the Netherlands have been included for analysis. Another five centers facilitate HBOT in the Netherlands, however the Institute of Hyperbaric Medicine is the largest and its centers are widely dispersed over the Netherlands.

Radiation-induced tissue injury originates from either direct or indirect molecular effects of ionization, of which indirect effects result from emergence of radicals. Since completely intact DNA is needed for cell proliferation, damage to DNA and insufficient repair of DNA are frequent causes of cell death in radiated tissues. Therefore, cell survival in radiated tissue is based on cell proliferation frequency. Acute radiation injury is seen in tissues with high mitotic activity, such as tumors, but also in intestinal mucosa, the skin and hematopoietic stem cells [26]. Damage to blood vessels is a delayed type of radiation injury, occurs after several months and plays an important role in pathophysiology of chronic wounds and LRTI because of the creation of an hypoxic, hypocellular and hypovascular environment: “the 3H’s” [27,28]. Furthermore, release of cytokines, subsequent fibroblast proliferation and fibro-atrophic processes are hypothesized to form the basis of LRTI [29].

Marx et al. demonstrated that by administering oxygen at high pressure, vascularity and cellularity in tissues is increased [19]. Besides, HBOT has multiple other effects on tissue, such as improvement of white cell and fibroblast function and mobilization of stem cells [9,17,18]. These physiologic changes are hypothesized to result in the beneficial consequences of HBOT administration. There have been concerns about HBOT as being carcinogenic by promoting cell proliferation, but published literature does not support this [30]. Also, some adverse events are seen in HBOT, such as damage to the ears and temporary worsening of myopia [9]. In our population, HBOT was generally well tolerated (73.3% reported no adverse events) and complications were either treatable or not severe.

As survival rates for sarcomas ameliorate [31] and timing of RT shifts to being preoperatively administered [5], interest increases in finding new treatment options for radiation-induced tissue injuries. Preoperative RT leads to higher rates of postoperative wound complications (35% versus 17% for postoperative RT) [6-8] due to performing surgical intervention in already ischemic tissue, as a consequence of RT [29,32]. However, Davis et al. report similar results in functional outcome when comparing preoperative to postoperative RT at two years after treatment. Little is known about consequences of the shift of RT timing past those two years. Since wound complications, such as chronic wounds, are seen more frequently in the preoperatively irradiated population and LRTI is seen even after several years following RT, knowledge on the utilization of therapeutic options to reduce complications after sarcoma management continues to be relevant in the future. HBOT outcome on wound healing following preoperative RT and surgical intervention in sarcoma patients is currently being evaluated in a prospective, randomized study (NCT03144206) [33].

Conclusions

The aim of this retrospective analysis of a Dutch cohort is to gain insight in the use and results of HBOT for radiation-induced tissue injuries following multimodality sarcoma management. The patients referred to HBOT centers were the ones suffering from severe complications in a late stage after sarcoma treatment. HBOT is regarded as safe and beneficial for treating both chronic wounds and LRTI in these cases. Awaiting further prospective results, we recommend referring to HBOT centers more actively in an earlier stage in sarcoma patients experiencing impaired wound healing or symptoms of delayed radiation-induced tissue injury following multimodality sarcoma treatment.

27 Nov 2019

PONE-D-19-28754

Hyperbaric oxygen therapy for radiation-induced tissue injury following sarcoma treatment: a retrospective analysis of a Dutch cohort.

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This is a retrospective review of 30 patients with chronic radiation injury or chronic wounds after radiation for sarcoma. This report suffers the usual issues with retrospective reviews. In addition, the authors need to address the following points:

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Reviewer #1: Congratulations to the authors on a well-written, informative, and important manuscript. HBOT is a modality which we often discuss and occasionally employ at my institution. Although I would ultimately like to know the role for HBOT in the setting of NA RT *prior to* the onset of complications, this work provides an important first step.

I have a few small requests of the authors:

1) Lines 71-74, I would like to see separate references for the two claims - direct increase in tissue oxygenation (which is very well described) and release of bone marrow stem cells (which is a slightly more controversial topic). I do not agree that these two mechanisms should be lumped together in a single sentence with a single reference to support.

2) Lines 165-166. I was confused by the sentence "No barotrauma to the tympanic membrane was observed." It seems that if 55% of the patients had "ear problems" and 3 of those 5 patients required PE tube insertion then it is unlikely that there was any barotrauma to the TM. Can the authors please use more specific language to replace "ear problems"? And can they please clarify how they defined barotrauma? Perhaps they meant that 5 patients had symptoms related to equalization of pressure within the ear but there was no TM rupture. In my experience, most patients with symptoms referable to the ear have changes in the exam of the TM consistent with barotrauma, even if a rupture has not occurred. Clarification would be helfpul to the reader.

Aside from these small comments, the overall clarity of the authors' work - especially the Conclusions - adds to the value of this paper.

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Reviewer #1: Yes: William Eward

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4 Feb 2020

Thank you for the review of our manuscript “Hyperbaric oxygen therapy for radiation-induced tissue injury following sarcoma treatment: a retrospective analysis of a Dutch cohort.”. We have changed the manuscript according to the feedback and will discuss our considerations in detail in the following paragraphs.

The first request from the reviewer to the authors is to provide separate references for two claims that are made in lines 71-74 in the original manuscript, in which the mechanisms of hyperbaric oxygen therapy are explained. The first claim includes the direct increase of tissue oxygenation after treatment with hyperbaric oxygen therapy. The second claim involves the induction and mobilization of stem cells. Both mechanisms are thought to be at the base of the angiogenesis by hyperbaric oxygen therapy. For the first claim, the work of Hills (1999) is used, in which the physiologic mechanism behind the direct increase of tissue oxygenation is explained. For the second claim, the papers of Milovanova et al. (2009) and Thom et al. (2006) are used as a reference, in which the stem cell mobilization and pathway of vasculogenesis are researched by using murine models. Furthermore, to state the effects of hyperbaric oxygen therapy on angiogenesis, we refer to the paper of Marx et. al (1990) and two reviews; Feldmeier (2012) and the Cochrane review by Bennett et al (2016). Marx et al. (1990) demonstrates the increased vascular density after hyperbaric administration of oxygen, instead of normobaric oxygen.

The second request is to clarify the complications of hyperbaric oxygen therapy related to the ears. By requesting additional information from the researchers in the Institutes of Hyperbaric Medicine, we have improved this section. Fifty-five percent of patients that experienced adverse events had barotrauma to the middle ear due to changes in pressure. This was observed by the doctor via otoscopy.

Additional comments on the manuscript were made by the editor. These include the absence of a clear hypothesis, the absence of a control group, missing data, subjective outcomes and no justification of sample size. In the introduction of the revised manuscript, a more specific hypothesis has been added. Other comments are addressed in a new paragraph in the discussion. It is explained that this retrospective cohort study has limitations resulting from the nature of the study; the results should of course be interpreted bearing this in mind. We noticed the gap of knowledge on effects of hyperbaric oxygen therapy and the clinical relevance of finding treatment options for radiation-induced tissue injury in the sarcoma population. The use of hyperbaric oxygen therapy remains subject of debate in our medical center, which is also stated by the reviewer. We retrospectively analyzed patient records of patients that were referred to different centers of the Institute of Hyperbaric Oxygen as a ‘last resort’ option. Outcomes are partly subjective and there is no control group as data were gathered retrospectively. Of note, in 10/16 patients referred for chronic wounds, wound measurements by a medical doctor were reported before and after hyperbaric oxygen therapy (Table 2). In all of these patients, the effect of hyperbaric oxygen therapy can be objectivized. There was no sample size calculation, because our population size is completely dependent on the amount of referrals to the Institute of Hyperbaric Oxygen in more than a ten year period. We analyzed data of all sarcoma patients treated in these centers. We strongly believe that this manuscript is a first step in making hyperbaric oxygen therapy more accepted as an addition to the multidisciplinary treatment of sarcoma patients. It provides a clear overview of the effects and adverse events of hyperbaric oxygen therapy and it seems to be a safe and beneficial treatment option for this patient population that struggles with treatment-induced morbidity. The paper offers a new option to decrease the treatment-induced morbidity and incites new, prospective, research.

Further journal requirements involve style requirements, the ethical statement and data management. The style of the references has been adjusted to the templates provided. The ethical statement in the manuscript is elaborated by adding information on informed consent. Data were pseudo-anonimized by using an identification code, but place of treatment and several dates related to the patient could make it identifiable. So, the data cannot be made publicly available due to ethical restrictions. However, the result section includes two tables with anonimized patient data, to enable readers to objectively judge the rationale behind our conclusions. Requests on data can be sent to the corresponding author.

With this letter and the adjustments made in the manuscript, we hope to have adequately answered the questions and comments. We would be grateful if you would take this manuscript into consideration for publication.

2 Apr 2020

PONE-D-19-28754R1

Hyperbaric oxygen therapy for radiation-induced tissue injury following sarcoma treatment: a retrospective analysis of a Dutch cohort.

PLOS ONE

Dear Dr. Been,

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Brian E. Brigman

Academic Editor

PLOS ONE

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Please include the data requested by reviewer 2.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors should be congratulated on a well-revised manuscript which illustrates the possible utility in using HBOT to treat LRTI. This is a significant problem that causes great morbidity in patients treated with multimodal therapy for soft tissue sarcoma. This work lays the foundation for subsequent prospective studies.

Reviewer #2: This is a useful case series describing a group of patients treated with hyperbaric oxygen for wounds related to radiation injury. The only data needed is related to the onset of the wounds. It is unclear whether these wounds were present since surgery, or occurred some time afterward. A column should be added to Table 2 to provide the time of occurrence of the wound. In addition, the authors should state whether the wounds were at the site of surgery or somewhere else.

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Reviewer #1: Yes: William Eward

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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29 Apr 2020

Dear dr. B.E. Brigman and reviewers,

Thank you for the review of our manuscript “Hyperbaric oxygen therapy for radiation-induced tissue injury following sarcoma treatment: a retrospective analysis of a Dutch cohort.”. We have made adjustments to the manuscript according to the feedback provided.

Reviewer 2 commented the following: “This is a useful case series describing a group of patients treated with hyperbaric oxygen for wounds related to radiation injury. The only data needed is related to the onset of the wounds. It is unclear whether these wounds were present since surgery, or occurred some time afterward. A column should be added to Table 2 to provide the time of occurrence of the wound. In addition, the authors should state whether the wounds were at the site of surgery or somewhere else.” In 15 of 16 patients referred for chronic wounds, hyperbaric oxygen therapy was indicated for a chronic wound at the site of surgery. The remaining patient suffered from a wound that spontaneously occurred near to the operation site (in irradiated area). Adjustments have been made to the results-section and table 2 to clarify the site of chronic wounds.

Furthermore, time between surgery and referral to an Institute for Hyperbaric Medicine was less than 12 months for 14 of 16 patients suffering from chronic wounds. All of these 14 patients had a chronic wound at the site of surgery. In clinical practice, inferior healing tendency is seen rapidly after surgery, frequently soon after sutures are removed. Time to referral was more than 12 months for the other two patients. One of these patients was referred for a persistent wound more than two years after surgery and radiotherapy. The remaining patient is the one that was referred for a wound in irradiated area, in which the onset is unknown. The patient was referred more than four years after surgery.

During the review of patient files to provide more detailed data on location and onset of the chronic wounds, it was found out that date of surgery was incorrect for one patient in the dataset. The median time to referral was changed accordingly in the manuscript.

With this letter and the adjustments made in the manuscript, we hope to have adequately answered the questions and comments.

Yours sincerely on behalf of all authors,

L.B. Been, MD PhD

University of Groningen, University Medical Center Groningen

Department of Surgery, division of Surgical Oncology BA31

PO Box 30.001

9700 RB Groningen, The Netherlands

Phone: +31-503612317 ; Fax: +31-503611745

Email: l.b.been@umcg.nl

27 May 2020

Hyperbaric oxygen therapy for radiation-induced tissue injury following sarcoma treatment: a retrospective analysis of a Dutch cohort.

PONE-D-19-28754R2

Dear Dr. Been,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Brian E. Brigman

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I appreciate the efforts the authors have made to address my concerns and those of the other reviewers. The manuscript will be valuable to multiple different segments of the medical community.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes: Richard Moon, MD

29 May 2020

PONE-D-19-28754R2

Hyperbaric oxygen therapy for radiation-induced tissue injury following sarcoma treatment: a retrospective analysis of a Dutch cohort.

Dear Dr. Been:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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PLOS ONE