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. 2021 Jan 5;16(1):e0245076. doi: 10.1371/journal.pone.0245076

Safety and efficacy study: Short-term application of radiofrequency ablation and stereotactic body radiotherapy for Barcelona Clinical Liver Cancer stage 0–B1 hepatocellular carcinoma

Feiqian Wang 1,2, Kazushi Numata 1,*, Atsuya Takeda 3, Katsuaki Ogushi 1, Hiroyuki Fukuda 1, Koji Hara 1, Makoto Chuma 1, Takahisa Eriguchi 3, Yuichirou Tsurugai 3, Shin Maeda 4
Editor: Stephen Chun5
PMCID: PMC7785216  PMID: 33400718

Abstract

Aim

To evaluate the safety and efficacy of the administration of radiofrequency ablation (RFA) and stereotactic body radiotherapy (SBRT) in the short term to the same patients in Barcelona Clinical Liver Cancer (BCLC) stages 0–B1.

Methods

From April 2014 to June 2019, we retrospectively reviewed BCLC stage 0–B1 patients with fresh hepatocellular carcinoma (HCC) lesions that were repeatedly treated by RFA (control group, n = 72), and by RFA and subsequent SBRT (case group, n = 26). Propensity score matching (PSM) was performed to reduce the selection bias between two groups. Recurrence, survival, Child–Pugh scores and short-term side effects (fever, bleeding, skin change, abdominal pain and fatigue) were recorded and analyzed.

Results

After PSM, 21 patients remained in each group. Seventeen and 20 patients in the case and control groups experienced recurrence. For these patients, the median times to progression and follow-up were 10.7 and 35.8 months, respectively. After PSM, the 1-year progression-free survival rate in case and control groups were 66.7% and 52.4%, respectively (P = 0.313). The inter-group overall survival (OS) was comparable (3 and 5-year OS rates in case groups were 87.3% and 74.8%, while rates in control groups were 73.7% and 46.3%, respectively; P = 0.090). The short-term side effects were mild, and the incidence showed no inter-group difference. The 1-year rates of the Child–Pugh score deterioration of ≥2 in case and control groups were 23.8% and 33.3% (P > 0.05), respectively.

Conclusion

The short-term administration of RFA and SBRT to the same BCLC stage 0–B1 patients may be feasible and effective because of their good prognosis and safety.

Introduction

Patients in Barcelona Clinical Liver Cancer (BCLC) stages 0–B1 have well-preserved liver function and are expected to receive curative treatment [1]. Radiofrequency ablation (RFA) is regarded as a favorable or even first-line treatment option for inoperable hepatocellular carcinoma (HCC) at these stages [1, 2]. However, there are many conditions for which RFA is unable to be performed or would predictably have an inferior outcome; for example, a risky location of lesions (lesions adjacent to vessels, diaphragm or heart or protruding from the hepatic surface), invisibility on ultrasound and lesions with a large size [3]. Stereotactic body radiotherapy (SBRT) is not recommended as the therapy for early-stage HCC. However, it has been frequently used in early-stage HCC patients with a reported good prognosis. The overall survival (OS), progression-free survival (PFS) and tumor response of SBRT are comparable with RFA [3, 4]. In addition, for tumors with a size of over 2 cm, SBRT outperformed RFA in local PFS. For lesions of all sizes, SBRT showed a relatively higher 1-year OS and lower incidence of acute grade 3+ complications [5]. In view of the above reported facts, on the premise of the priority of RFA as the primary treatment for BLCL stage 0–B1 patients with inoperable HCCs, we aimed to introduce SBRT as a novel alternative treatment strategy for lesions that were ineligible for RFA treatment. RFA and subsequent SBRT treatment included multifocal lesions treated with RFA and SBRT separately, and local/distant recurrent lesions after RFA treatment were treated with SBRT. For the latter case, a few studies exhibited the feasibility of using SBRT on residual RFA-treated lesions with encouraging outcomes [6, 7]. Regrettably, however, there are no data available on the conditions of the short-term application of RFA and SBRT in the same patient for multifocal lesions or intrahepatic distant recurrence, which is very common in the progress of HCC. The survival benefits obtained from the additional treatment of SBRT after initial RFA need to be weighed against the relative possible risks of the procedure-related cumulative toxicities of the two modalities. In other words, both the feasibility and safety of this novel treatment strategy should be evaluated in detail before it is put into clinical practice.

To address this issue, in the present study, we investigated the outcomes and safety of performing RFA and SBRT methods in the short term. We made a comparison with the already well-recognized therapy of repeated RFA [8] in order to determine the feasibility and effectiveness of our novel RFA and SBRT therapy for BLCL stage 0–B1 patients.

Materials and methods

Patient enrollment

From April 2014 to June 2019, we retrospectively reviewed patients with BCLC stages 0–B1 with fresh HCC lesions. According to the Japan Society of Hepatology Guideline [9], the diagnosis of HCC was either based on typical radiological criteria or proven by histopathology. All the RFA procedures were performed at Yokohama City University Medical Center (YCUMC), while SBRT treatment was undertaken at Ofuna Chuo Hospital. Notably, the first diagnoses of HCC of these patients were done in YCUMC. If the patients needed to be treated with SBRT, their receiving doctors at YCUMC would contact Ofuna Chuo Hospital and refer the patients to Ofuna Chuo Hospital for SBRT treatment. Clinical information (such as gender, age, etiology of HCC, Child–Pugh classification), imaging data, histology reports and treatment processes were retrospectively collected from a review of the electronic medical- recording system, the radiology database and pathology records from YCUMC, respectively. Data collection and the analysis of all enrolled patients and lesions in our retrospective study were approved by the institutional review board of YCUMC (the original approved number was “B180200054” and the updated number is “B200300052”) and were in compliance with the principles of the Declaration of Helsinki; the requirement for informed consent was waived. The information from all patients was fully anonymized. When downloading, copying and using the patient’s information, a CD-R or USB rather than a network was used, and a password was set for the database.

First, we included the patients in a general policy: (1) in BCLC stage 0–B1, patients were supposed to receive curative rather than palliative treatment; (2) the primary treatment was RFA; (3) the next treatment (multifocal HCC lesions in the same patients or local/intrahepatic recurrence after the initial RFA) would be either RFA or SBRT; and (4) the time interval between the primary treatment (RFA) and the next treatment (RFA or SBRT) would not be more than three months.

The case group and the control group were not randomly assigned. If RFA was predicted to be performed well, RFA was preferred as the first choice. Otherwise, SBRT therapy was performed instead of RFA. The case group had to satisfy the conditions that the subsequent treatment in a short period (within three months) would be SBRT, regardless of the recurrence of RFA-treated lesions or another fresh lesion. The control group enrolled patients with lesions, all of which were treated by RFA in a short period, including multifocal lesions, recurrent RFA-treated lesions and other fresh lesions. The selection of the study population is presented in a flow chart below (Fig 1).

Fig 1. Flowchart of the study population.

Fig 1

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In total, 48 lesions from 21 patients in the case group and 37 lesions from 21 patients in the control group, respectively, were finally used for data analysis after PSM. PSM: propensity score matching; RFA: radiofrequency ablation; SBRT: stereotactic body radiotherapy; HCC: hepatocellular carcinoma; BLCL: Barcelona Clinical Liver Cancer.

Treatment modalities

RFA procedure

As Wang F’s study described [10], RFA was performed using a 480 kHz generator (VIVA RF generator; STARmed, Gyeonggi, Korea) and a 17-gauge, internally cooled, adjustable RF electrode (Proteus; STARmed, Gyeonggi, Korea). The lengths of the active tips of the electrodes were selected mainly according to the location and size of the lesion. For example, for lesion with a diameter of no more than 2 cm, an electrode with a 2 cm tip was selected and ablated at 20 W. Otherwise (for sizes larger than 2 cm), an electrode with a 3 cm tip and ablation at 40 W was selected. The median duration and temperature of one ablation was 12 minutes and maintained above 60°C, respectively. Post-operative contrast-enhanced ultrasound (CEUS) examination was undertaken to evaluate the adequacy of ablation. A complete ablation was defined as no perfusion of contrast agent into the ablative area (which completely covered the lesion area as a whole), showing a completely black appearance with a distinct boundary. When the ablation was completed, the needle tip was kept hot when the needle was retracted to prevent bleeding from thermal coagulation, seeding along the puncture route.

SBRT procedure

The process was performed according to Takeda A’s study [11]. Multiarc, dynamic conformal radiation was planned using a radiation treatment planning system (FOCUS XiO, version 4.2.0–4.3.3: Computerized Medical Systems, St Louis, Mo) and was performed using x-rays from a 6 MV linear accelerator (Clinac 2100C; Varian Medical Systems Inc, Palo Alto, Calif). Generally, SBRT procedures with total doses of 35 Gy were delivered in five fractions over 5–7 days. Notably, for lesions which were close to the gastrointestinal tract, the dose for a 10 cc area of the gastrointestinal tract was limited to <25 Gy. In this context, the treatment strategy was hypofractionated radiotherapy: a total dose of 36–45 Gy in 12–15 fractions over 16–21 days. Treatment was planned to enclose the planning target volume with a maximal dose of 60–80% of isodose line.

Follow-up and evaluations of treatment outcomes

The endpoint of overall survival assessment was determined as the patient’s death, loss in follow-up or most recent imaging examination of any kind (US, CEUS, CT or MRI). Local recurrence was defined when a radiological enhancement/ hypervascularity was detected at the treated ablated sites of RFA and a 95% isodose line of SBRT. Intrahepatic recurrence was defined as a newly detected lesion outside of the ablation site (RFA) or planning target volume (SBRT). Follow-up times were calculated from the start date of the prior treatment within this study period, even in cases with previous treatment history for HCC. Short-term side effects of RFA and SBRT were evaluated from the day to one week after treatment. Events of side effects were named and graded according to the National Institutes of Health-defined Common Terminology Criteria for Adverse Events. To assess treatment-related effects on liver function, we analyzed Child–Pugh scores after the latter treatment (RFA for the control group and SBRT for the case group) in one, three and six month and one year follow-ups. The changes compared with baseline Child–Pugh scores were recorded.

Statistical analyses

Propensity score matching (PSM) was performed to reduce potential confounders and selection biases between the case group and the control group. Propensity scores were calculated using a multivariable logistic regression model including sex, baseline age (with a median value of 70 years-old as a cutoff value), viral etiology (hepatitis B virus (HBV) and/or hepatitis C virus (HCV) versus non-hepatitis B non-hepatitis C (NBNC)), Child–Pugh class (A versus B), pre-treatment alpha-fetoprotein and albumin (normal versus abnormal) and tumor size (with 3 cm as a cutoff value). Following the estimation of propensity scores, the tumors were matched using the 1:1 nearest-neighbor matching algorithm with an optimal caliper of 0.02 and without replacement. Non-matching results were discarded.

After PSM, to compare differences in the baseline characteristics and parameters of outcomes between the two groups, the Pearson’s Chi-square or Fisher’s exact test was used to analyze categorical variables and the Mann–Whitney U test (non-normally distributed data) or Student’s t-test (normally distributed data) was employed to compare continuous variables. All statistical analyses were two-sided, and values of P <0.05 were considered statistically significant. Analyses were performed using the statistical software SPSS 24.0 (Inc, Chicago, IL). The survival curves were depicted using the Kaplan–Meier method. Differences of PFS and OS were calculated using log-rank tests.

Results

Baseline characteristics

Before PSM, 98 consecutive patients with 196 lesions fulfilled the eligibility criteria and entered the study. Apart from the HCC history, other baseline characters revealed no differences between the two groups. After PSM, 21 patients in each group were retained for afterwards analysis. Eighty-five target fresh HCC lesions from 42 patients were diagnosed between June, 2013 and June, 2019. All the baseline characteristics showed no difference between groups. Baseline characteristics before and after PSM are summarized in Table 1.

Table 1. Baseline characteristics of patients and tumors before and after PSM1.

Before PSM After PSM
Parameters Control group Case group Total P Control group Case group Total P
No. of patients/lesions 72/135 26/61 98/196 / 21/37 21/48 42/85 /
BLCL stage (0/A/B1) 15/52/5 6/17/3 21/69/8 0.717 6/14/1 4/14/3 16/22/4 0.493
Child–Pugh (A/B) 64/8 25/1 89/2 0.272 20/1 20/1 40/2 1.000
HCC history (No/yes) 26/46 18/8 44/54 0.004 10/11 16/5 26/16 0.057
Age, mean ± S.D. (year) 71.4 ± 9.4 68.3 ± 11.7 10.6 ± 10.1 0.171 68.1 ± 10.4 69.5 ± 10.6 68.8 ± 10.4 0.672
Gender (Male/female) 58/14 19/7 77/21 0.426 17/4 16/5 33/9 0.707
Etiology (HCV/HBV/NBNC) 55/5/12 19/1/6 74/6/18 0.686 13/2/6 16/1/4 29/3/10 0.593
AFP, mean ± S.D. (ng/mL) 125.3 ± 400.4 72.1 ± 152.4 111.1 ± 352.0 0.512 18.8 ± 41.4 50.7 ± 121.8 34.8 ± 91.3 0.267
Albumin, mean ± S.D. (g/dL) 3.8 ± 0.6 4.0 ± 0.6 3.8 ± 0.6 0.065 3.9 ± 0.4 4.0 ± 0.6 4.0 ± 0.5 0.793
Lesion number (1/2/(3–5)) 11/47/14 5/14/7 16/61/21 0.582 7/12/2 5/9/7 12/21/9 0.170
Types of treatment (single lesion/multi-lesions)2 11/61 5/21 16/82 0.640 7/14 5/16 12/30 0.495
Lesion size, mean ± S.D. (mm) 16.3 ± 5.5 16.7 ± 9.0 16.4 ± 6.6 0.778 17.1 ± 5.7 17.5 ± 9.7 17.3 ± 7.9 0.847
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1 PSM: propensity score matching; BLCL: Barcelona Clinical Liver Cancer; HCV: hepatitis C virus; HBV: hepatitis B virus; NBNC: non-hepatitis B non-hepatitis C; AFP: alpha-fetoprotein.

2 A single lesion means both that primary and secondary therapy were performed on the same lesion. The latter treatment (SBRT or RFA) aimed to treat the LTP or RFA-incomplete treated lesions. Multi-lesions show that primary and secondary therapy were performed on the different lesions. The latter therapy (SBRT or RFA) aimed to treat the IDR lesion or multifocal lesions that were entirely treated with RFA or separately with RFA and SBRT.

Recurrence and survival after PSM

The endpoint of follow-up was August 20, 2020. The median follow-up time was 35.8 months (range, 2.6–80.5 months) for all patients, and almost equal between the case group (the value of the mean and standard deviation was “40.0 ± 19.7” months) and control group (40.0 ± 22.9 months) (P = 0.091). The median time to progression was 10.7 months (with a range of 1.1–62.2 months) for all patients. The case group (13.9 ± 12.3 months) have slightly longer time to progression than the control group (8.3 ± 9.2 months), however, there was no statistical difference between groups (P = 0.105). As a whole, 17 patients in the case group (six exhibited local tumor progression while 11 exhibited intrahepatic tumor recurrence) and 20 of 21 patients in the control group (five exhibited local tumor progression while 15 exhibited intrahepatic tumor recurrence) experienced recurrence. The 1- and 2-year PFS rates in the case groups (66.7% and 31.4%) were slightly higher than the control group (52.4% and 28.6%) but demonstrated no statistical significance (P = 0.313) (Fig 2c). The 1, 3 and 5 year OS rates in the case groups were 95.2%, 87.3% and 74.8%, and thus comparable with those in the control groups, at 90.5%, 73.7% and 46.3%, respectively (P = 0.090) (Fig 2d). The 1 year cumulative intrahepatic recurrence rates in case and control groups were 33.3% and 29.5%, respectively (P = 0.968) (Fig 3c). In a 1 year follow-up, there was no local recurrent lesion in the case group, while 25.7% patients in the control group experienced local recurrence (P = 0.064) (Fig 3d). The cumulative numbers of HCC related-deaths (e.g., liver failure, multiple organ metastasis and failure) during the entire follow-up were 1 and 4 for the case group and the control group, respectively.

Fig 2. Progression-free and overall survival rate curves for both groups.

Fig 2

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(a) Progression-free survival before PSM. (b) Overall survival before PSM. (c) Progression-free survival after PSM. (d) Overall survival after PSM.

Fig 3. Incidence curves of cumulative intrahepatic and local recurrence for both groups.

Fig 3

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(a) Cumulative incidence of intrahepatic recurrence before PSM. (b) Cumulative incidence of local recurrence before PSM. (c) Cumulative incidence of intrahepatic recurrence after PSM. (d) Cumulative incidence of local recurrence after PSM.

Toxicities after PSM

Within 1 year of follow-up after treatment, the cumulative rates of a Child–Pugh score deterioration ≥2 in case and control groups were 23.8% (five of 21 patients) and 33.3% (seven of 21 patients) (P > 0.05), respectively (Table 2). For the case group, there was no presence of radiation-induced gastrointestinal disorders such as bleeding, nausea and vomiting. One week after SBRT treatment, fatigue was reported in only one patient. The types of short-term side effects caused by RFA treatment were fever, bleeding of the operating area, skin erythema and edema, and abdominal pain. All of these side effects were mild and were relieved after observation or symptomatic treatment. There were no statistical differences between groups (P > 0.05) (Table 3). There were no grade 3+ adverse events in both groups.

Table 2. Change of Child–Pugh scores in follow-up after PSM1.

Groups Follow-up time Unchanged 1 2 3 4 -2 -1
Total patients One month 35 5 0 1 0 1 0
3 months 31 8 0 1 0 1 0
6 months 24 11 4 0 0 1 1
One year 23 9 2 1 1 0 2
Case group One month 19 2 0 0 0 0 0
3 months 16 5 0 0 0 0 0
6 months 12 7 1 0 0 0 1
One year 10 6 3 0 1 0 0
Control group One month 16 3 0 1 0 1 0
3 months 15 3 0 1 0 1 0
6 months 12 4 3 0 0 1 0
One year 13 3 1 1 0 0 2
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1 PSM: propensity score matching. A positive value indicates that, compared with the baseline Child–Pugh score, the Child–Pugh score increased after follow-up, while a negative value indicates a decline.

Table 3. Side effects related to RFA treatment after PSM1.

Side effect Control group (n = 21) Case group (n = 21) Total P
Fever 7 (33.3%) 6 (28.6%) 11 >0.05
Bleeding 5 (23.8%) 1 (4.8%) 5 >0.05
Skin erythema, edema 4 (19.0%) 0 (0.0%) 2 >0.05
Abdominal pain 6 (28.6%) 6 (28.6%) 12 >0.05
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1 RFA: radiofrequency ablation; PSM: propensity score matching.

Discussion

Previous studies have demonstrated that the significant risk factors for recurrence mainly include the size of HCC lesions (especially sizes >3 cm), etiology, serum albumin levels and serum alpha-fetoprotein, while factors affecting survival include the patient’s age and Child–Pugh stage [12–15]. Patient age is also considered to be related to post-treatment complications and disease morbidity [15, 16]. In that case, we performed a PSM analysis to reduce potential confounders and selection biases between the case group and the control group. After the PSM and baseline characteristics analysis of the enrolled patients and lesions, we managed to ensure that the inter-group difference of prognosis in afterwards statistics was caused by different treatments rather than the above risk factors.

As a locoregional treatment option, RFA is primarily recommended with curative intent for BLCL stage 0–A patients who have inoperable lesions and are not transplant candidates [2, 17]. Beneficial effects regarding OS and the safety of RFA treatment have been reported in BLCL stage B1 patients [18]. In a national 10 year survey in Japan of 20,659 HCC cases at BLCL stage A–B by RFA therapy, the 1, 3 and 5 year survival rates were 90.7–97%), (64.7–82.3%), and (43.6–63.8%), respectively [19]. The OS rate yielded from repeated RFA treatment in our study (1, 3 and 5 year OS rates were 90.5%, 73.7% and 46.3%, respectively) was consistent with the national survey data, consistently suggesting that the outcome of RFA treatment is considerable and stable. Nevertheless, in addition to the beneficial effects of OS and safety, the relative high recurrence rate and real demonstration of treatment failure (although with a low incidence rate) for RFA should also be taken seriously and dealt with appropriately. After the first RFA treatment for fresh HCCs, the rate of treatment failure was reported to be 1.2%. For the remaining 98.8% of lesions with complete responses, 6.2% of local recurrence and 35% of intrahepatic recurrence were detected [19]. There are many factors of patients and lesions that might contribute to an insufficient and/ or risky ablation, even for skilled operators. High-risk lesion locations (beneath the diaphragm, subphrenic or perivascular areas) are well-recognized factors that hamper the achievement of complete ablation with sufficient ablative margins [20]. Because of the minimally invasive nature of RFA, a bleeding-prone state of patients (with conditions such as hemodialysis requiring continuous use of anticoagulants) would be a contraindication for RFA. A large lesion size is also considered for a risky factor that affects the result of ablation. Large lesions are believed to more frequently have irregular borders, along with satellite lesions [21], and are thus not easy to completely ablate even after undertaking multiple RFA sessions. In addition, RFA performance-reduced complete necrosis was reportedly surprisingly high in 53.1% of HCC lesions smaller than 3 cm and 14.3% of lesions larger than 3 cm [22]. A recurrence might potentially be caused by incomplete necrosis and consequently lead to the presence of a residual tumor. For the above situation, which is not eligible for repeated RFA, no matter whether for recurrent lesions or lesions coexisting with RFA-treated lesions, an alternative modality other than RFA should be considered.

SBRT is usually indicated in patients with 1–3 lesions with a maximum diameter of 5–6 cm [23] (which is in agreement with BLCL stage 0–B1) which are not eligible for resection or other local therapies. RFA results in incomplete tumor control with increasing tumor size. In contrast, for tumors <5 cm, there is no size discrepancy in the recurrence for tumors treated with SBRT, and the quality of radiotherapy planning was satisfactory [24]. Unlike RFA, which is greatly restricted by the lesion location, there are very few geographic limitations for therapeutic approaches according to tumor location in image-guided radiotherapy-adopted SBRT [24] unless the tumor is adjacent to critical structures such as the bowel or heart, etc. A previous study showed significant improved local recurrence in patients treated with SBRT for larger lesions than possible with RFA [8]. Current practice, using SBRT as a sole treatment or in combination with other local therapies (transcatheter arterial chemoembolization, resection), irrespective of small retrospective cohorts or larger series and well-designed phase II trials, has consistently confirmed the value of SBRT in the treatment of HCCs by encouraging local control (1 year, 65–100%), OS rates (1 year, 32–94%) and low toxicity [14]. In line with the previous study, our novel therapy of administering short-term RFA and SBRT exhibited an acceptable PFS rate in one year (66.7%) and a stable and high OS rate in continuing follow-up years (the 1, 3 and 5 year OS rates in case groups were 95.2%, 87.3% and 74.8%). In particular, our new therapy showed no local recurrence in the first follow-up year while the repeated RFA treatment yielded a 1 year local recurrence rate of 25.7%. The fact that SBRT is superior to RFA in local recurrence has also been confirmed in much of the literature [4, 5, 25].

Although the prognosis for therapy with RFA and subsequent SBRT is comparable to or even better than that of repeated RFA, it does not come at the cost of reduced safety. SBRT is non-invasive and thus does not cause collateral thermal damage to adjacent structures. The most important dose-limiting factor of SBRT is liver damage. Fortunately, taking the advantages of SBRT’s characteristics of delivering high individual doses of radiation to target tumors and keeping rapid fall-off doses to surrounding normal tissues [26], there was no significant deterioration of liver function when compared with the control group (the patients’ Child–Pugh score deterioration rates of ≥2 in case and control groups were 5 and 7, respectively). In the case group, RFA was performed fewer times than in the control group. It is easy to understand the lower incidence of RFA-related complications than the cumulative incidence caused by repeated RFA performance in the control group. The incidence of radiation-related complications was low and almost negligible. Only one patient complained of fatigue, which was considered to be a typical syndrome of radiation hepatitis. Due to strict dose–volume constraints and proper patient selection, most previous SBRT studies have demonstrated a minimal risk of radiation hepatitis (a reported rate was 2.4%) [27]. No severe complications were encountered during the entire follow-up.

This study has several limitations. Firstly, our research has a retrospective nature, with a small number of patients and lesions. Therefore, selection bias was inevitable despite a PSM analysis. In other words, the patients and lesions in this study may not be representative of the HCC population in BLCL stage 0–B1. Secondly, multiple lesion types were treated with SBRT. For example, some lesions showed local recurrence or intrahepatic recurrence after RFA treatment, while others to which SBRT was applied co-existed with lesions treated by RFA. Due to the small number of cases, we were unable to conduct a subgroup analysis, so it is impossible to know whether the outcomes of RFA and SBRT were different in these different settings. Finally, our study was underpowered. Survival curves show that the study group had higher PFS and OS than the control group, yet there was no statistically significant difference. Additional retrospective chart reviews or—better yet—prospective, multicenter, large-scale trials are needed to determine whether the trends we found are in fact statistically significant.

Conclusion

In patients in BCLC stages 0–B1 with inoperable lesions and who are ineligible for RFA, SBRT may be a promising curative treatment option because of its comparable or even better prognosis and safety than the repeated use of RFA. Our novel therapy regarding initial RFA and subsequent SBRT treatment might provide new ideas for the curative treatment of HCC, but large-scale prospective studies are required before they can be definitively applied in clinical practice.

Supporting information

S1 File. The operation details of RFA and SBRT.

(DOCX)

Click here for additional data file. (14.2KB, docx)

Data Availability

Detailed data of patient information cannot be shared publicly because of the privacy of patients. Data are available from the Ethics Committee of Yokohama City University Medical Center (contact via https://yokohama-cu.bvits.com/rinri/login.aspx?ReturnUrl=%2frinri%2fCommon%2f) for researchers who meet the criteria for access to confidential data and electronic medical record system of Yokohama City University medical center.

Funding Statement

The author(s) received no specific funding for this work.

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Decision Letter 0

Stephen Chun

27 Nov 2020

PONE-D-20-33979

Safety and efficacy study: short-term application of radiofrequency ablation and stereotactic body radiotherapy for barcelona clinical liver cancer stage 0–B1 hepatocellular carcinoma

PLOS ONE

Dear Dr. Numata,

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"Our retrospective study design was approved by the institutional review board and in compliance with the principles of the Declaration of Helsinkii; the requirement for informed consent was waived."   

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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5. 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 manuscript requires extensive editing for grammatical errors as well as English language editing. For example, there are fragments in the Introduction and errors in tense in several sentence. Some of the terms used such as "fresh HCC" is not a term commonly used in the medical literature and would be more appropriately described as "previously untreated HCC."

There are inaccurate statements in the introduction misquoting other reports in the literature. For example, the sentence on line 53-54 in the introduction, which states SBRT outperformed RFA in overall survival (incorrect per that reference) and lower incidence of grade 3+ complications (the difference was not statistically significant), is misleading/false. This statement in the Introduction on lines 56-57, "we aimed to introduce SBRT as a novel alternative treatment strategy for lesions ineligible for RFA treatment" is not supported by the design of this retrospective study.

Because of the small sample size, none of the differences between the two groups are statistically significant; however, they are discussed as having differences between the therapies in the Abstract and Discussion.

Reviewer #2: This is a small retrospective study comparing patients with HCC who received SBRT and RFA in a short interval compared to RFA alone. The authors attempted to correct for possible differences between the groups by doing propensity score matching. The results showed that the case group had comparable outcomes and low toxicity.

1. Please try to edit the grammar of this manuscript, as there are several portions that are not written in clear and correct English. For example, the term "RFA and SBRT short-term performing" throughout the manuscript does not make sense grammatically and should be replaced. Other sentences that need to be re-written include: lines 61-64, 66-68, 248-249, 252 (intrahepatic spelling), 254-256, 264-266, 266-268, 294-298, 300-302, 311-312 ("respective"), 321-322.

2. Lines 172-173: You should mention the fact that there was an almost statistically significant difference in "HCC history" between the case and control groups, and perhaps discuss that in the discussion as a possible limitation.

3. Lines 274-277: there should be mention of the fact that SBRT tumor coverage can be limited (and possibly results in compromise in tumor control) if the tumor is adjacent to critical structures such as bowel or heart, etc.

Reviewer #3: I think this manuscript is excellent "as is," however I feel it could be made both more concise and slightly more clear.

Most importantly, to me "retrospectively enrolled" is a phrase I have not heard or read before - please do not use it. I would intend use "retrospectively reviewed" or "retrospective chart review." Did the authors do a retrospective chart review? Did you use an electronic medical record and/or artificial intelligence to select patients? I would like to see more in the methods section about this. I would also like to read more about PSM methods. I think Figure 1 is excellent and should be published as is.

I was glad to see in Methods section that you had IRB approval. Helsinki is spelled with only 1 i at the end in both Finnish and English, however - please correct this typo. I was concerned that you did not have any IRB approval or waiver when I read n/a in the Ethics Statement section - does that section need to be updated at all?

Please correct grammar. E.g. In Introduction page 3 line 46 "many conditions in which RFA is unable... inferior outcome; for example, large size, invisibility on ultrasound, and risky location, including close proximity to vessels, diaphragm or heart, and protrusion from the hepatic surface." There are a few other instances where "is" (singular) or "are" (plural) are used incorrectly, likely due to editing software error, for example page 5 line 89 should be "is not more than three months." I would use "First" instead of "Firstly" (page 5, line 84).

In Table 2, I would consider using - not / just because to me, - is a lot easier on my eyes. Also, please clarify, what is the difference between / (or preferably - ) and 0?

Table 3 could be shortened by eliminating the "no" rows and the "no / yes" column, instead of "Skin Change" label "Skin redness, swelling" or "Skin erythema, edema." All your numbers add up to 100% in each side effect category so it does not appear that a single patient was *not* asked about these side effects, hence the "no" rows being essentially useless. This is the only concern I have is that was every single "no" patient asked about every single side effect, every single time??? I appreciate this "no" data availability to me as a reviewer, but I think it need not be published.

Figures 2 and 3 are excellent. The y-axis labels especially are slightly blurry to me. Please ensure high enough resolution for publication because even on my regular monitor, it is blurry, and enlarging / zooming in only makes it appear more pixelated.

Discussion

Over all, this section could be made more concise. I am focusing my suggestions for improvement on the last paragraph of Discussion, as below:

Page 19 line 310 should be "inevitable despite PSM analysis." I would eliminate the world "well" in line 311. I believe the first word in line 312 should be "perspective" instead of "respective" (or perhaps you meant "retrospective"?) In lines 315-316, you could perhaps note that your study is underpowered to show statistically significant difference, despite the trends, hence the need for multi center, large-scale studies as you noted. In line 318 I would not the lesion types treated with SBRT were multiple, for example "local recurrence or intrahepatic recurrence...lesions treated by RFA."

I recommend re-ordering the last paragraph in the Discussion session as follows:

Keep lines 308 - end of line 311 the same. Then "Also, the lesion types treated with SBRT were multiple, for example... [as I wrote out in the paragraph immediately above]...lesions treated by RFA. Due to the small number of cases, we are unable to conduct a subgroup analysis, so it is impossible to know whether the outcomes of RFA and SBRT are different in these different settings. Finally, our study was underpowered. Survival curves show that the study group had higher PFS and OS than the control group, yet there is no statistically significant difference. Additional retrospective chart reviews or, better yet, prospective, multicenter, large-scale trials are needed to determine whether the trends we found are in fact statistically significant."

Conclusion

page 20 line 326 "tend" - what do you mean??

I would rewrite line 326 "SBRT is a promising and curative treatment option, because of its comparable or even improved prognosis and safety profile, compared to repeated RFA. Our study shows that initial RFA and subsequent SBRT is a safe and efficacious method for curative treatment of HCC, however larger-scale studies are required.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Liisa L. Bergmann, MD, MBA

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PLoS One. 2021 Jan 5;16(1):e0245076. doi: 10.1371/journal.pone.0245076.r002

Author response to Decision Letter 0

15 Dec 2020

Editor’s comments

Point: If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Reply I am quite sorry that I failed to upload our protocols to that identifier (DOI). Therefore, I wrote the steps of RFA and SBRT here as follows.

The operation details of RFA were as follows.

Instruments and equipment

RFA was performed under either grayscale US guidance (for HCC clearly detected by grayscale US) or contrast-enhanced US (CEUS) guidance (for US undetectable HCC). The LOGIQ E9 ultrasound system and a convex probe with a frequency of 1–6 MHz or a micro-convex probe with a frequency of 2–5 MHz were applied. To ablate all the tumors, RFA was performed using a 480 kHz generator (VIVA RF generator; STARmed, Gyeonggi, Korea), capable of producing a maximum power of 200 W and a specific size of 17-gauge internally cooled, adjustable RF electrode (Proteus; STARmed, Gyeonggi, Korea).

Important parameter setting

For CEUS guidance, in general, a low mechanical index (MI) mode (0.2–0.3) was used as the whole liver, which could be observed in real time, allows for repeated observations of the liver in a dynamic real-time manner because microbubbles stay static and undergo less destruction. Unfortunately, deep-located hepatic lesions might invisible with a low MI because of the attenuation of the US beam. If the lesion margin was indistinct (especially if the lesion was isoechogenic) on the grayscale US image and deep-located, high MI mode (0.8–1.2) and/or fusion imaging with enhanced CT/MRI were applied.

Operation tips

1)The lengths of the active tip of the electrodes were 5 mm, 1.0 cm, 1.5 cm, 2.0 cm or 3.0 cm. Each electrode was selected based on tumor size, tumor location, and operator preference. Briefly, for patients with a tumor diameter of 2 cm or smaller, an electrode with a 2 cm tip was inserted into the tumor and ablated at 20 W. For patients with a tumor diameter larger than 2 cm, an electrode with a 3-cm tip and ablation at 40 W was selected. For nodules with a relatively larger size (3–5 cm), the electrode was inserted at different sites.

2)In cases with hypervascular HCCs that exhibited hypervascular enhancement during the arterial phase and hypo-echoic enhancement during the post-vascular phase, we punctured the lesion during the post-vascular phase. In contrast, in cases with hypervascular HCCs that exhibited hypervascular enhancement during the arterial phase and iso-echoic enhancement during the postvascular phase, we punctured the lesion during the arterial phase of CEUS.

Operation steps

All patients received local anesthesia and analgesia before the procedure. In general, for lesion with big size, the first puncture of RFA targeted the center of the largest section of the lesion shown on the US images. Afterwards, the overlapping ablations were performed until the entire lesion was ablated. The ablation algorithm was based on elevations in tissue impedance. The mean duration of one ablation was 12 minutes, and the temperature of the ablated tissue was maintained above 60 ℃.

Immediately after ablation, the therapeutic response to RFA was evaluated by CEUS or fusion imaging to determine the adequacy of ablation, and whether additional ablation was needed. If sometimes the steam produced by radiofrequency ablation affects the visual field of ultrasound observation, wait for a moment for a clear field of vision before evaluating the adequacy of ablation. For RFA performed under the guidance of CEUS, post-operative CEUS examination was undertaken to see if the edge of the ablation area showed hypervascularity in AP. A complete ablation could be confirmed by no perfusion of contrast agent into the tumor as a whole, showing a “cavity” appearance with a distinct boundary. For RFA treatment guided by fusion imaging, the overlap function of fusion imaging was applied to observe whether the ablative area completely covered the lesion area shown on pre-operative enhanced MRI or CT images.

After ablation, the needle was retracted, maintaining its tip hot in order to prevent, by thermal coagulation, seeding or haemorrhage along the electrode track. Every procedure was aimed at obtaining a no less than 5 mm safety margin around the treated lesions.

The operation details of SBRT were as follows.

During free breathing of patients, Spiral, 4-phase, multidetector CT and/or dynamic contrast-enhanced MRI were conducted, and followed by fusion with a slow-scan CT scan (6–10 seconds per slice). The gross tumor volume (GTV), including enhanced tumor, was delineated with the slow-scan CT images. For the internal target volume, an internal margin (4–6 mm) was created around the clinical target volume (CTV) according to the respiratory movement of the diaphragm observed during fluoroscopy. For the planning target volume (PTV), individualized margins of 2 mm were applied around the internal target volume as a setup margin. Multiarc, dynamic conformal radiation was planned using a radiation treatment planning system (FOCUS XiO, version 4.2.0–4.3.3: Computerized Medical Systems, St Louis, Mo) and was performed using X-rays from a 6-MV linear accelerator (Clinac 2100C; Varian Medical Systems Inc, Palo Alto, Calif). Generally, SBRT with total doses of 35 Gy or 40 Gy were delivered in five fractions over 5–7 days.

For patients with Child-Pugh grade A–B grade, > 20% of the normal liver receiving > 20 Gy, and a total dose of 35 Gy was administered. For other patients, a total dose of 40 Gy were delivered. Notably, for the lesions which were close to the gastrointestinal tract, there was a policy that the dose to a 10-cc area of the gastrointestinal tract should be limited to <25 Gy. In this consideration, the treatment strategy was hypofractionated radiotherapy: a total dose of 36–45 Gy in 12–15 fractions over 16–21 days. Treatment was planned to enclose the planning target volume with a maximal dose of 60%–80% isodose line.

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Journal Requirements

Point 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Reply 1: Thank you very much for your kind reminding. We read the formatting sample carefully and made revision of our manuscript in respective of the font, font size and line spacing et. al to abstract, main text and title page accordingly.

Point 2. In the ethics statement in the manuscript and in the online submission form, please provide additional information about the patient records/samples used in your retrospective study, including:

a) whether all data were fully anonymized before you accessed them;

b) the date range (month and year) during which patients' medical records/samples were accessed.

Reply 2: Thank you for your valuable comments to perfect our manuscript.

a)We added the important information of ethics statement in the main text as follows(page 5, lines 85–99).

All the RFA performance were done at Yokohama City University Medical Center (YCUMC) while SBRT treatment at Ofuna Chuo Hospital. Notably, the first diagnoses of HCC of these patients were done in YCUMC. If the patients need to be treated with SBRT, their receiving doctors of YCUMC would contact Ofuna Chuo Hospital and refer the patients to Ofuna Chuo Hospital for SBRT treatment. Clinical information (such as gender, age, HCC etiology of HCC, Child–Pugh classification), imaging data, histology reports and treatment process were retrospectively collected from a review of the electronic- medical- recording system, the radiology database, and pathology records from our hospital, respectively. Data collection, analysis of all enrolled patients and lesions in our retrospective study were approved by the institutional review board of YCUMC (Original approved number was “180200054”, update number was “B200300052”) and in compliance with the principles of the Declaration of Helsinki; the requirement for informed consent was waived. All patients were fully anonymized of their information. When downloading, copying and using the patient's information, CD-R or USB rather than network were used, and the password was set for the database.

b)We evaluated the patients enrolled from April 2014 to June 2019 when the target lesions have just been determined as HCC and have not receive any treatment yet. The follow-up duration of these lesions were 2.6 to 98.9 months before PSM and 2.6 to 80.5 months after PSM, respectively. We carefully re-checked our data, the endpoint of follow-up was August 20, 2020. We added “endpoint of follow-up” in the result section of our main text (page 14, line 202).

Point 3. Please ensure reporting of 1-year progression-free survival in case group vs control group is reported correctly in the abstract.

Reply 3: Thank you very much for your valuable comments. We wrote in abstract that “After PSM, the 1-year progression-free survival rate in case and control groups were 66.7% and 52.4%, respectively (P = 0.313).” In result section of main text, we wrote “The 1- and 2-year PFS rate in case groups (66.7% and 31.4%) were slightly higher than control group (52.4% and 28.6%) but demonstrated no statistical significance (P = 0.313)”.

With careful checking of statistics and description, we believe our descriptions were correct and consistent among the whole main text.

Point 4. Thank you for including your ethics statement: “Our retrospective study design was approved by the institutional review board and in compliance with the principles of the Declaration of Helsinkii; the requirement for informed consent was waived.”

a. Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.

b. Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

Reply 4: The full name of the ethics institutional review board was Yokohama City University Medical Center. We have added this important information in the revised main text (page 5, line 93) as follows.

Data collection and the analysis of all enrolled patients and lesions in our retrospective study were approved by the institutional review board of YCUMC (the original approved number was “B180200054” and the updated number is “B200300052”) and were in compliance with the principles of the Declaration of Helsinki; the requirement for informed consent was waived.

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Review Comments

Response to reviewer 1

1-Point 1: The manuscript requires extensive editing for grammatical errors as well as English language editing. For example, there are fragments in the Introduction and errors in tense in several sentence. Some of the terms used such as "fresh HCC" is not a term commonly used in the medical literature and would be more appropriately described as “previously untreated HCC.”

1-Reply 1: We are quite sorry for grammatical errors. According to your suggestion, language presentation was improved with assistance from a native English speaker with appropriate research background.

1-Point 2. There are inaccurate statements in the introduction misquoting other reports in the literature. For example, the sentence on line 53-54 in the introduction, which states SBRT outperformed RFA in overall survival (incorrect per that reference) and lower incidence of grade 3+ complications (the difference was not statistically significant), is misleading/false. This statement in the Introduction on lines 56-57, "we aimed to introduce SBRT as a novel alternative treatment strategy for lesions ineligible for RFA treatment" is not supported by the design of this retrospective study.

1-Reply 2: Thank you very much for the reviewer’s insight comment. Our original sentence which the reviewer commented was “For tumors with size of over 2 cm, SBRT outperformed RFA in PFS, OS and yielded lower incidence of acute grade 3+ complications.” We referred it from the original research entitled Outcomes After Stereotactic Body Radiotherapy or Radiofrequency Ablation for Hepatocellular Carcinoma. This article reported that for lesions with size of over 2 cm, the HCC lesion group treated by SBRT have higher local PFS than RFA treated group (P=0.025). For lesions with all size, the lesions treated by SBRT showed higher one-year OS (no statistical difference but P value was not given) and yielded lower incidence of acute grade 3+ complications (P=0.31) than that by RFA.

Notably, we carefully stated “introduce SBRT as a novel alternative treatment strategy for lesions ineligible for RFA treatment” particularly “on the premise of the priority of RFA as the primary treatment for BLCL stage 0-B1 patients with inoperable HCCs”. We did not deny the usefulness and importance of RFA treatment. This statement was not only concluded from reference 5, but also from reference 3 (this article held the view that radiotherapy appears to be an acceptable alternative treatment option for patients who are not candidates for RFA) and 4 (this article held the view that SBRT therapy appears to be an effective alternative to RFA that should be considered when there is a higher risk of local recurrence or toxicity after RFA), which reported a comparable outcome (OS and PFS) of SBRT and RFA treated lesions.

We realized our expression of reference 5 was not rigorous and slightly incorrect, so we made revisions as follows (page 3, lines 58–59).

In addition, for tumors with size of over 2 cm, SBRT outperformed RFA in local PFS. For lesions with all size, SBRT showed a relatively higher 1-year OS and lower incidence of acute grade 3+ complications [5]. In view of the above reported facts, on the premise of the priority of RFA as the primary treatment for BLCL stage 0–B1 patients with inoperable HCCs, we aimed to introduce SBRT as a novel alternative treatment strategy for lesions ineligible for RFA treatment.

1-Point 3. Because of the small sample size, none of the differences between the two groups are statistically significant; however, they are discussed as having differences between the therapies in the Abstract and Discussion.

1-Reply 3: Thank you very much for the reviewer’s valuable comments. In describing this manuscript, we consistently take an objective view of the values and limitations of our research. In abstract, we described that “after PSM, the 1-year progression-free survival rate in case group (66.7%) were slightly higher than case group (52.4%) (P = 0.313)”. We just showed the difference between two values (66.7% and 52.4%) and particularly put the P value there. We did not write as “a statistical difference”. In results section, we demonstrated that there were higher values of “time to progression” and PFS rate (also some visible trend shown in survival curve) but no statistical inter-group difference in our results.

In revised limitation part of discussion section, we explained this indifference might due to small sample size and suggested the prospective multicenter, large-scale studies be organized to further verify our conclusion (page 22, lines 345–347).

Moreover, to make our expression in abstract more rigorous, we changed the original sentence of “After PSM, the 1-year progression-free survival rate in case group (66.7%) were slightly higher than case group (52.4%) (P = 0.313).” into “After PSM, the 1-year progression-free survival rate in case and control groups were 66.7% and 52.4%, respectively.” (page 2, lines 31–32)

Response to reviewer 2

This is a small retrospective study comparing patients with HCC who received SBRT and RFA in a short interval compared to RFA alone. The authors attempted to correct for possible differences between the groups by doing propensity score matching. The results showed that the case group had comparable outcomes and low toxicity.

2-Point 1: Please try to edit the grammar of this manuscript, as there are several portions that are not written in clear and correct English. For example, the term “RFA and SBRT short-term performing” throughout the manuscript does not make sense grammatically and should be replaced. Other sentences that need to be re-written include: lines 61-64, 66-68, 248-249, 252 (intrahepatic spelling), 254-256, 264-266, 266-268, 294-298, 300-302, 311-312 (“respective”), 321-322.

2-Reply 1: We apologize for the language problems in the original manuscript. We carefully checked the entire manuscript for typographic, grammatical and formatting errors by native speaker using the professional English editing service.

2-Point 2. Lines 172-173: You should mention the fact that there was an almost statistically significant difference in “HCC history” between the case and control groups, and perhaps discuss that in the discussion as a possible limitation.

2-Reply 2: Thank you very much for the reviewer’s insight comments. We have noticed the statistical difference of “HCC history” before PSM and described in lines 189–190 as “Apart from the HCC history, other baseline characters revealed no difference between two groups” and in lines 192–193 as “(after PSM) All the baseline characters have no difference between groups.” Most of our statistics analysis, results and discussion are based on the data after PSM, so we did not think the baseline indicator of “HCC history” is a limitation as it showed no inter-group difference.

2-Point 3. Lines 274-277: there should be mention of the fact that SBRT tumor coverage can be limited (and possibly results in compromise in tumor control) if the tumor is adjacent to critical structures such as bowel or heart, etc.

2-Reply 3: We quite agree with the comment and re-wrote the sentence in the revised manuscript as the following (page 20, lines 298–301):

Unlike RFA performance, which is much restricted by the lesion location, there is no very few geographic limitations for therapeutic approaches according to tumor location in image-guided radiotherapy-adopted SBRT [24] unless the tumor is adjacent to critical structures such as bowel or heart, etc.

Response to reviewer 3

I think this manuscript is excellent “as is,” however I feel it could be made both more concise and slightly more clear.

3-Point 1: a) Most importantly, to me “retrospectively enrolled” is a phrase I have not heard or read before - please do not use it. I would intend use “retrospectively reviewed” or “retrospective chart review.”

b) Did the authors do a retrospective chart review? Did you use an electronic medical record and/or artificial intelligence to select patients? I would like to see more in the methods section about this.

c) I would also like to read more about PSM methods.

d) I think Figure 1 is excellent and should be published as is.

3-Reply 1: a) We quite agreed with replacing “retrospectively enrolled” with “retrospectively reviewed”. We made revision in the main text accordingly (Page 2, line 21).

b) Clinical information (such as gender, age, HCC etiology of HCC, Child–Pugh classification), imaging data, histology reports and treatment process were retrospectively collected from a review of the electronic- medical- recording system, the radiology database, and pathology records from our hospital, respectively. For better understanding, we added the information in the method section of the main text. We used the classification and filtering function of excel and SPSS rather than artificial intelligence to select suitable patients.

c) We used SPSS to do the PSM analysis. We did 1:1 matching between the case group and control group (SPSS can only do the most commonly used 1:1 nearest neighbor matching algorithm). We set a cutoff caliper of 0.02 and without replacement, which was much accurate for matching compared with many published articles. We established multivariable logistic regression model including sex, baseline age (70 years-old as a cutoff value), viral etiology (HBV and/or HCV versus NBNC), Child-Pugh class (A versus B), pre-treatment alpha-fetoprotein and albumin (normal versus abnormal), and tumor size (3cm as a cutoff value). These baseline parameters were carefully referred from RFA, SBRT, PSM related published literature of high levels (For example, Koji Hara, et al, 2019, Hepatology; Nalee Kim, et al. 2020, Journal of Hepatology; Masatoshi Kudo, et al, 2019, Cancers). To make it easier for PSM to succeed (as an operating tip for PSM), all the data input the SPSS software for PSM performance should be enumeration data rather than measurement data, which was a pity that would possibly lose some information of the enrolled data. For the 26 patients in case group (RFA+SBRT), 21 were remained after PSM. Of these 21 patients, 13 were perfect matching (with all the enrolled parameters), while 8 were fuzzy matching. Other 5 patients were excluded after PSM because of failure in match.

d)We are much grateful for Prof. Liisa L. Bergmann’s positive evaluation of our research.

3-Point 2. I was glad to see in Methods section that you had IRB approval. Helsinki is spelled with only 1 i at the end in both Finnish and English, however - please correct this typo. I was concerned that you did not have any IRB approval or waiver when I read n/a in the Ethics Statement section - does that section need to be updated at all?

3-Reply 2: Thank you very much for Prof. Liisa L. Bergmann’s kind reminding of the spelling of helsinki. We have made revision accordingly.

In our research, all the RFA performance were done at Yokohama City University Medical Center (YCUMC) while SBRT treatment at Ofuna Chuo Hospital. Notably, the first diagnoses of HCC of these patients were done in YCUMC. If the patients needed to be treated with SBRT, their receiving doctors at YCUMC would contact Ofuna Chuo Hospital and refer the patients to Ofuna Chuo Hospital for SBRT treatment. Data collection and analysis of all enrolled patients and lesions in our retrospective study were approved by the institutional review board of YCUMC and the approval number was B180200054. (In 2018, a few content of the documents for ethical approval has been updated to make it more rigorous and consistent with the actual clinical situation. The updated number was B200300052. The main content was almost the same). We uploaded the documents and materials related to ethical review and approval in attached supplemental documents. Since all the materials are in Japanese, in order to make it easier to understand, we have translated some key sentences in the form of annotations.

3-Point 3. Please correct grammar. E.g. In Introduction page 3 line 46 "many conditions in which RFA is unable... inferior outcome; for example, large size, invisibility on ultrasound, and risky location, including close proximity to vessels, diaphragm or heart, and protrusion from the hepatic surface." There are a few other instances where "is" (singular) or "are" (plural) are used incorrectly, likely due to editing software error, for example page 5 line 89 should be "is not more than three months." I would use "First" instead of "Firstly" (page 5, line 84).

3-Reply 3: We are terribly sorry for the language problems in the original manuscript. We made revision to the mentioned places. In addition, we carefully checked the entire manuscript for typographic, grammatical and formatting errors by native speaker using the professional English editing service.

3-Point 4. In Table 2, I would consider using - not / just because to me, - is a lot easier on my eyes. Also, please clarify, what is the difference between / (or preferably - ) and 0?

3-Reply 4: Thank you very much for Prof. Liisa L. Bergmann’s valuable comments for perfect our manuscript. There is no difference between “/” and “0” as “/” in Table 2 means there is no such patient (have elevated or declined value of Child-Pugh score). To make it more clear in expression, we changed “/” into “0” in revised Table 2.

3-Point 5. Table 3 could be shortened by eliminating the "no" rows and the "no / yes" column, instead of "Skin Change" label "Skin redness, swelling" or "Skin erythema, edema." All your numbers add up to 100% in each side effect category so it does not appear that a single patient was *not* asked about these side effects, hence the "no" rows being essentially useless. This is the only concern I have is that was every single "no" patient asked about every single side effect, every single time??? I appreciate this "no" data availability to me as a reviewer, but I think it need not be published.

3-Reply 5: We much appreciate Prof. Liisa L. Bergmann’s insight teaching of Table 3. We made revision accordingly as follows. The nurse and/or the resident physician in charge of the patient would ask and check his/her temperature, symptoms and signs as a routine procedure after RFA.

Table 3. Side effects related to RFA treatment after PSM1

Side effect Control group (n=21) Case group (n=21) Total P

Fever 7(33.3%) 6(28.6%) 11 >0.05

Bleeding 5(23.8%) 1(4.8%) 5 >0.05

Skin erythema, edema 4(19.0%) 0(0.0%) 2 >0.05

Abdominal pain 6(28.6%) 6(28.6%) 12 >0.05

1 RFA: radiofrequency ablation; PSM: propensity score matching.

3-Point 6. Figures 2 and 3 are excellent. The y-axis labels especially are slightly blurry to me. Please ensure high enough resolution for publication because even on my regular monitor, it is blurry, and enlarging / zooming in only makes it appear more pixelated.

3-Reply 6: Thank you very much for your praise and kind reminding. We checked the submitted Figure 2 and 3. The pixels meet the magazine's requirements of 300 pixels and appeared clear in separated TIF forms. In this revision, we have uploaded a separate TIF table as a supplement.

3-Point 7. Discussion section: Over all, this section could be made more concise. I am focusing my suggestions for improvement on the last paragraph of Discussion, as below:

Page 19 line 310 should be "inevitable despite PSM analysis." I would eliminate the word "well" in line 311.

I believe the first word in line 312 should be "perspective" instead of "respective" (or perhaps you meant "retrospective"?)

In lines 315-316, you could perhaps note that your study is underpowered to show statistically significant difference, despite the trends, hence the need for multicenter, large-scale studies as you noted.

In line 318 I would not the lesion types treated with SBRT were multiple, for example “local recurrence or intrahepatic recurrence...lesions treated by RFA.”

3-Reply 7: We quite agree Prof. Liisa L. Bergmann’s constructive comments to perfect our manuscript. We made revision point-to-point of the above problems in our latest manuscript.

3-Point 8. I recommend re-ordering the last paragraph in the Discussion session as follows:

Keep lines 308 - end of line 311 the same. Then "Also, the lesion types treated with SBRT were multiple, for example... [as I wrote out in the paragraph immediately above]...lesions treated by RFA. Due to the small number of cases, we are unable to conduct a subgroup analysis, so it is impossible to know whether the outcomes of RFA and SBRT are different in these different settings. Finally, our study was underpowered. Survival curves show that the study group had higher PFS and OS than the control group, yet there is no statistically significant difference. Additional retrospective chart reviews or, better yet, prospective, multicenter, large-scale trials are needed to determine whether the trends we found are in fact statistically significant."

3-Reply 8: We much thank Prof. Liisa L. Bergmann’s teaching of the re-organization of our limitation part. The expression of revised limitation part is much clear, rigorous and easy to understand. We learned much and made revision accordingly.

3-Point 9. Conclusion section: page 20 line 326 "tend" - what do you mean??

I would rewrite line 326 “SBRT is a promising and curative treatment option, because of its comparable or even improved prognosis and safety profile, compared to repeated RFA. Our study shows that initial RFA and subsequent SBRT is a safe and efficacious method for curative treatment of HCC, however larger-scale studies are required”.

4-Reply 9: I am very sorry for my inappropriate expression of “purpose” or “aim”. I deleted “tend” and revised that sentence according to Prof. Liisa L. Bergmann’s excellent teaching as follows (page 23, line 350).

In patients in BCLC stages 0–B1 with inoperable lesions and who are ineligible for RFA, SBRT may be a promising curative treatment option...

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Revision by self-check of authors

Apart from reply to the comments of reviewer, we carefully re-checked our manuscript and data again. We made a few revisions to the latest version of submission as follows to perfect our manuscript.

Additional point 1. For figure 2, we are sorry that the data tables of "patient at risk" of a and c, b and d were mistakenly placed upside down. The revised figure 2 is as follows.

Additionally, to be more specific and easier for reading, we changed the insert “figure 2” , “figure 3” in the result section of main text into “figure 2c”, “figure 2d”, “figure 3c”, “figure 3d” as follows.

The 1- and 2-year PFS rate in case groups (66.7% and 31.4%) were slightly higher than control group (52.4% and 28.6%) but demonstrated no statistical significance (P = 0.313) (Fig 2c). The 1-, 3- and 5-year OS rate in case groups were 95.2%, 87.3% and 74.8%, comparable with those in the control groups with 90.5%, 73.7% and 46.3%, respectively (P = 0.090) (Fig 2d). The 1-year cumulative intrahepatic recurrence rate in case and control groups were 33.3% and 29.5%, respectively (P = 0.968) (Fig 3c). In a 1-year’s follow-up, there was no local recurrent lesion in the case group while 25.7% patients in the control group confronted local recurrence (P = 0.064) (Fig 3d).

Additional point 2. As the term of AFP, ALB and TACE have appeared only once in the main text, in principle, they are more suitable to use full name rather than abbreviations. Therefore, we made revision into alpha-fetoprotein and albumin in method section (page 10, lines 172–173); into “transcatheter arterial chemoembolization” in discussion section (page 21, lines 304–305). In addition, we added the full name of AFP in the footnote of Table 1 (page 12, line 189).

Additional point 3. We added “with a median value of” before “70 years-old as a cutoff” in statistical analyses of the method section. We wanted to demonstrate there that this cutoff value is based on median value of all the enrolled patients.

Additional point 4. As we known, without PSM, the results would be inaccurate without balancing the potential effects of baseline factors. Due to the limitation of manuscript space and in order not to confuse the reader with the “before” and “after” PSM results, the data we displayed in the results section are all after PSM performance. To make it clear, we added “after PSM” after the sub-title of “Recurrence and survival” (page 14, line 201) and “Toxicities” (page 16, line 233).

Additional point 5. We checked all the reference we cited. We corrected “BLCL b stage” to “BLCL B stage” in Ref. 1 and “japan” to “Japan” in both Ref. 9 and Ref.18.

Attachment

Submitted filename: 2020,12.15 1st round response to reviewers comments.docx

Click here for additional data file. (546.1KB, docx)

Decision Letter 1

Stephen Chun

22 Dec 2020

Safety and efficacy study: short-term application of radiofrequency ablation and stereotactic body radiotherapy for Barcelona Clinical Liver Cancer stage 0–B1 hepatocellular carcinoma

PONE-D-20-33979R1

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Acceptance letter

Stephen Chun

26 Dec 2020

PONE-D-20-33979R1

Safety and efficacy study: short-term application of radiofrequency ablation and stereotactic body radiotherapy for Barcelona Clinical Liver Cancer stage 0–B1 hepatocellular carcinoma

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File. The operation details of RFA and SBRT.

    (DOCX)

    Click here for additional data file. (14.2KB, docx)
    Attachment

    Submitted filename: 2020,12.15 1st round response to reviewers comments.docx

    Click here for additional data file. (546.1KB, docx)

    Data Availability Statement

    Detailed data of patient information cannot be shared publicly because of the privacy of patients. Data are available from the Ethics Committee of Yokohama City University Medical Center (contact via https://yokohama-cu.bvits.com/rinri/login.aspx?ReturnUrl=%2frinri%2fCommon%2f) for researchers who meet the criteria for access to confidential data and electronic medical record system of Yokohama City University medical center.

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