Diagnostic utility of cryobiopsy for invasive mucinous lung adenocarcinoma

Introduction

Background

Lung cancer remains the leading cause of cancer-related mortality, with adenocarcinoma accounting for approximately 50–60% of cases, making it the most common histopathological subtype (1). Lung adenocarcinoma is classified into five major predominant subtypes (lepidic, acinar, papillary, micropapillary, and solid) and several variant subtypes (invasive mucinous, colloid, fetal, and enteric) (2). This classification system is recognized for its correlation with clinical characteristics and is widely utilized in clinical practice and research.

Invasive mucinous lung adenocarcinoma (IMA), previously termed mucinous bronchoalveolar carcinoma (BAC), was reclassified as an independent histologic subtype of adenocarcinoma in the 2015 World Health Organization (WHO) classification (3). Under earlier classification systems, mucinous BAC had a poorer prognosis than non-mucinous BAC (4). Consequently, the 2015 WHO classification differentiated mucinous BAC from its non-mucinous counterpart, redefining it as either mucinous adenocarcinoma in situ (AIS)/minimally invasive adenocarcinoma (MIA) or IMA, while non-mucinous BAC was reclassified as non-mucinous AIS/MIA or lepidic-predominant adenocarcinoma. In cases where IMA coexists with non-mucinous components constituting ≥10% of the tumor, the diagnosis should be mixed invasive mucinous and non-mucinous adenocarcinoma.

IMA accounts for approximately 2–5% of lung adenocarcinoma cases and predominantly affects older adults, with a slightly higher prevalence in women than in men. There is reportedly no significant association between IMA and smoking (5). Notably, IMA exhibits distinct histopathological and radiological characteristics. IMA tumor cells display a goblet or columnar cell morphology with abundant intracytoplasmic and interalveolar mucins. The basally localized nuclei, a hallmark of IMA, are rarely observed in other lung adenocarcinomas. Multiple growth patterns, including acinar, papillary, micropapillary, and lepidic, can be observed, with the lepidic growth pattern being the most common. As mucin-producing tumor cells spread throughout the alveoli, computed tomography (CT) images typically reveal alveolar consolidation, which can resemble pneumonia or other benign lung diseases (6-9). Other radiographic features of IMA include a solitary nodule or mass, which may mimic benign tumors due to its slow growth rate and limited ¹⁸F-fluorodeoxyglucose uptake on positron emission tomography (10). The imaging diagnosis of IMA is often challenging at initial presentation, potentially leading to missed opportunities for surgical resection.

Rationale and knowledge gap

To distinguish IMA from other lung diseases, several studies have evaluated the utility of transthoracic needle biopsy (TTNB) and transbronchial biopsy (TBB). Although TTNB has a higher diagnostic yield than TBB, it carries an increased risk of complications, including pneumothorax and potentially fatal events such as systemic air embolism or tumor seeding (11,12). Conversely, TBB has a lower complication rate but also a lower diagnostic yield compared to TTNB (13). Despite the use of these conventional biopsy techniques, the diagnostic yield for IMA remains insufficient, as small biopsy or cytology specimens often contain low cellularity, non-diagnostic alveolar mucin pools, poorly atypical tumor cells, and crush artifacts, limiting their diagnostic accuracy (14-16).

Cryobiopsy is a relatively new biopsy technique that allows for a biopsy of the entire circumference of the contacted area, facilitating high-quality and quantity tissue specimens. Therefore, cryobiopsy reportedly has a higher diagnostic yield for lung cancer compared to conventional biopsy (17-25). The introduction of cryobiopsy has improved the diagnostic yield of TBB and shown promising results comparable to TTNB (26,27).

Objective

We hypothesized that the high qualitative and quantitative cryobiopsy specimens would contribute to the diagnostic success of IMA. Although our previous study showed that cryobiopsy specimens are also helpful in identifying the histopathological subtype in lung adenocarcinoma (28), the diagnostic yield for IMA has not been examined yet. Thus, this retrospective study aimed to elucidate whether cryobiopsy will be a valid technique for diagnosing IMA. We present this article in accordance with the STARD reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-865/rc).

Methods

Patients

We retrospectively reviewed consecutive patients who underwent surgical resection for lung cancer at our institution between March 2017 and July 2024. Among them, patients diagnosed with IMA, including those with mixed non-mucinous components, were selected. Patients who had not undergone preoperative diagnostic bronchoscopy or had undergone only endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) were excluded. Finally, patients who had undergone bronchoscopy for peripheral pulmonary lesions (PPLs) were included in the analyses. These patients were categorized into either the cryo group or the conventional group based on whether cryobiopsy was performed.

This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments and the Ethical Guidelines for Epidemiological Research in Japan. The study protocol was approved by the National Cancer Center Institutional Review Board (approval No. 2018-090). Written informed consent for the clinical procedure was obtained from all patients.

Bronchoscopic procedures

Prior to bronchoscopy, virtual bronchoscopic navigation (VBN) was reconstructed from high-resolution CT (HRCT) data using a workstation (Ziostation2^®; Ziosoft, Tokyo, Japan) (29). In addition to VBN, virtual fluoroscopy (VF) was also reconstructed on the workstation (30).

All bronchoscopic procedures were performed under local anesthesia with lidocaine and moderate-to-deep sedation using fentanyl or pethidine in combination with midazolam or propofol. Most bronchoscopic procedures were performed in an outpatient setting. One of the following bronchoscopes (P260F, P290, 1T260, or 1TQ290; Olympus, Tokyo, Japan) was inserted and navigated using VBN/VF, after which a radial endobronchial ultrasound (R-EBUS) probe (UM-S20-17S or UM-S20-20R; Olympus) was advanced under X-ray fluoroscopic guidance (VersiFlex VISTA^®; Hitachi Ltd., Tokyo, Japan). R-EBUS detection was then classified based on the locational relationship between the probe and the lesion as within, adjacent to, or invisible (31-33). Subsequently, forceps biopsy and/or needle aspiration were performed.

In the cryo group, all patients were intubated with an 8.0-mm inner diameter tracheal tube (Portex^® Uncuffed Ivory PVC, oral/nasal tracheal tube; Smiths Medical, Minneapolis, MN, USA). A 1.9-mm reusable or 1.7-mm single-use cryoprobe, in combination with the ERBECRYO^® 2 system (Erbe Elektromedizin GmbH, Tübingen, Germany), was inserted through the working channel and advanced to the location where R-EBUS detected the lesion under X-ray fluoroscopic guidance. After freezing for 3–6 s, the cryoprobe was removed together with the bronchoscope. Immediately thereafter, another therapeutic bronchoscope was inserted through the tracheal tube and wedged into the biopsy site to control bleeding (i.e., the two-scope technique) (34). The frozen specimens were thawed in saline and transferred to 10% neutral buffered formalin for histopathological examination. The remaining saline was used as a fluid specimen for cytological and microbiological analyses. In some cases, cryobiopsy was performed following forceps biopsy and/or needle aspiration.

Rapid on-site cytological evaluation was performed in most cases (35). Multiple aspects of the tissue sample were imprinted onto two glass slides. One slide was air-dried and stained using the Diff-Quik stain, while the other was fixed with ethanol and subjected to Papanicolaou staining.

Radiological evaluation

All patients underwent HRCT scans prior to bronchoscopy. The lesion size, lesion location, distance from the costal pleura, lesion morphology, radiographic feature, and bronchus sign were assessed using axial HRCT images. The maximum diameter of each lesion was measured to determine lesion size. The lesion location was categorized as (I) outer, defined as lesions in the outer third of the lung; and (II) inner, referring to lesions in the inner two-thirds of the lung. The distance from the costal pleura was measured as the shortest perpendicular length from the lateral border of the lesion to the pleural surface. The lesion morphology was classified as solid, part-solid, or pure ground-glass based on the presence and extent of ground-glass opacity. The radiographic feature was categorized into a solitary or pneumonic type. The solitary type was defined as a solitary nodule or mass with a well-defined shape, whereas the pneumonic type was characterized by a consolidation with an irregular margin (Figure 1). Two board-certified respiratory endoscopists (H.F. and T.T.) independently conducted the classification, and in cases of disagreement, a consensus decision was reached. The bronchus sign was considered positive if a bronchus led directly to the lesion. Additionally, the visibility of the lesion on chest radiographs was assessed.

Figure 1 Radiographic features of IMA (arrows). The radiographic feature was categorized into a solitary or pneumonic type. The solitary type was defined as a solitary nodule or mass with a well-defined shape (A-D), whereas the pneumonic type was defined as a consolidation with an irregular margin (E-H). Forceps biopsy (A,B,E,F) or cryobiopsy (C,D,G,H) were performed, with successful diagnoses in (A,E,C,G) and unsuccessful diagnoses in (B,D,F,H), respectively. IMA, invasive mucinous lung adenocarcinoma.

Outcomes

The primary outcome was to compare the diagnostic yield between the cryo and conventional groups. The diagnostic yield was calculated as the percentage of cases successfully diagnosed with carcinoma based on bronchoscopic biopsy specimens. Additionally, factors influencing the diagnostic yield were analyzed.

The secondary outcome was to compare the safety profiles between the two groups. Complications were defined as any adverse events occurring during or after bronchoscopy that could be attributed to the procedure. Bleeding severity was assessed using the Nashville Bleeding Scale (36).

Statistical analysis

Differences in the distribution of patient characteristics between the two groups were analyzed using Fisher’s exact test or Pearson’s Chi-squared test for categorical variables and the Mann-Whitney U test for continuous variables, as appropriate. A two-sided P value of less than 0.05 was considered statistically significant. A multivariable analysis using a logistic regression model was conducted to identify independent predictive factors associated with diagnostic yield. The criterion for variable exclusion in multivariable analysis was a P value greater than 0.20. All statistical analyses were performed using R statistical software version 3.6.1 (R Foundation for Statistical Computing, Vienna, Austria).

Results

During the study period, 5,053 lung cancer surgeries were performed, and 3,038 patients (60.1%) were diagnosed with adenocarcinoma, of whom 201 (6.7%) had IMA (Figure 2). Among these, 106 patients had undergone bronchoscopy for PPLs prior to surgery and were included in the analyses: 46 in the cryo group and 60 in the conventional group.

Figure 2 Patient selection flow diagram. EBUS-TBNA, endobronchial ultrasound-guided transbronchial needle aspiration.

The patient characteristics are summarized in Table 1. The median age was 72 years (range, 39–84 years), with 53.8% of patients older than 70 years. Fifty-eight patients (54.7%) were male, and 39 (36.8%) had a smoking history of >20 pack-years. The median lesion size was 26.7 mm (range, 8.6–100.5 mm), with 36.8% of lesions measuring ≤20 mm. Solid, part-solid, and pure ground-glass lesions were observed in 46.2%, 52.8%, and 0.9% of cases, respectively. Meanwhile, 28.3% of lesions were bronchus sign-negative, and 36.8% were invisible on chest radiographs. Regarding radiographic features, 65.1% of lesions were categorized as solitary type.

Next-generation sequencing (NGS) was performed in 27 patients, of whom 21 (77.8%) harbored KRAS mutations. The most common variant was G12D, detected in 11 patients (40.7%), followed by G12V in 4 (14.8%), G13C in 2 (7.4%), and G12A, G12C, G12F, and Q61H each in 1 patient (3.7%). Additionally, a BRAF mutation (D594N) was identified in 1 patient (3.7%). Other single-plex tests for EGFR mutations and ROS1 fusions were performed in five patients, and for ALK fusions in 12 patients (including duplicates); all results were negative.

Compared to the conventional group, the cryo group had a higher proportion of subsolid lesions (part-solid: 69.6% vs. 40.0%; pure ground-glass: 2.2% vs. 0%, P=0.002). All other factors, including age, sex, smoking status, lesion size, lesion location, distance from the costal pleura, lesion morphology, radiographic features, bronchus sign, and lesion visibility on chest radiographs, were comparable between the two groups.

The overall diagnostic yield was 82.1%. The diagnostic yield was significantly higher in the cryo group than in the conventional group (93.5% vs. 73.3%, P=0.01). The univariable analysis demonstrated that the diagnostic yield was significantly higher in patients with lesion size ≥20 mm (89.6% vs. 69.2%, P=0.02) and those with pneumonic-type radiographic features (94.6% vs. 75.4%, P=0.02), in addition to the biopsy group (Table 2). Furthermore, the multivariable analysis revealed that cryobiopsy was the only independent factor significantly associated with a higher diagnostic yield [adjusted odds ratio (OR), 5.07; 95% confidence interval (CI): 1.33–19.40; P=0.02]. A representative case successfully diagnosed using cryobiopsy is presented in Figure 3.

Figure 3 A representative case of a 73-year-old male in whom cryobiopsy diagnosed IMA. HRCT reveals a 23.0-mm part-solid nodule in the left lower lobe (A) (arrow: positive bronchus sign). The radiographic feature was categorized as a solitary type. Referring to the path on VF (B,C), at the location where an R-EBUS showed within the lesion (D), cryobiopsy was performed using a 1.7-mm single-use cryoprobe (E). Histopathological analysis reveals uncrushed tumor cells with goblet and columnar cell morphology, abundant intracytoplasmic and interalveolar mucins (arrowheads). Hematoxylin and eosin stain; (F) ×10 magnification; (G) ×100 magnification. The yellow box indicates the region shown at higher magnification in panel (G). A, anterior; HRCT, high-resolution computed tomography; IMA, invasive mucinous lung adenocarcinoma; L, left; R-EBUS, radial endobronchial ultrasound; VF, virtual fluoroscopy.

Complications are summarized in Table 3. Although there were no cases of grade 4 bleeding in either group, grade 2 and grade 3 bleeding were more frequent in the cryo group than in the conventional group (41.3% vs. 10.0% and 4.3% vs. 0%, P<0.001). Additionally, pneumonia was more commonly observed in the cryo group.

Discussion

Key findings

The present study investigated the utility of cryobiopsy in the diagnosis of IMA by comparing it with conventional biopsy. The results demonstrated that the cryo group had a significantly higher diagnostic yield than did the conventional group (93.5% vs. 73.3%, P=0.01). Multivariable analysis revealed that cryobiopsy was the only independent factor significantly associated with a higher diagnostic yield (adjusted OR, 5.07; 95% CI: 1.33–19.40; P=0.02).

Strengths and limitations

Although previous studies have evaluated the efficacy of cryobiopsy for PPLs (17-25), limited research has specifically examined its diagnostic yield for IMA. We identified only one prior case report describing an instance where cryobiopsy successfully established a pathological diagnosis of IMA after forceps biopsy had failed (15). To our knowledge, this is the first comparative study demonstrating that cryobiopsy is superior to conventional biopsy for the diagnosis of IMA.

Our study has some limitations. First, this was a retrospective study conducted at a single institution. The conventional group may have included cases where cryobiopsy was technically challenging, potentially introducing selection bias. Second, the study population included only patients who underwent surgical resection, meaning the true overall diagnostic yield was not comprehensively assessed. This is because not all lung adenocarcinoma subtypes were specified among cases diagnosed based on bronchoscopic biopsy specimens, and some cases of IMA may have been present among non-diagnostic cases. Finally, in some cases, cryobiopsy was performed following conventional biopsy, meaning that the diagnostic yield reported for the cryo group was not solely based on cryobiopsy.

Comparison with similar research and explanations of findings

IMA is histopathologically characterized by goblet or columnar cell morphology with basally located nuclei and abundant cytoplasm. The alveolar cavity surrounding the tumor is typically filled with mucins (5). Diagnosing IMA in bronchoscopic specimens has historically been challenging due to non-diagnostic alveolar mucin pools, poorly atypical tumor cells, or crush artifacts (14). In this study, the diagnostic yield in the conventional group was 73.3%. This yield is comparable to the cumulative diagnostic yield reported for R-EBUS-guided TBB (13), indicating that the accuracy of the conventional biopsy method was not inherently low. However, cryobiopsy collects larger tissue samples with fewer crush artifacts than conventional forceps biopsy (17-25). Nasu et al. reported that the mean sample size obtained by cryobiopsy was significantly larger than that obtained by forceps biopsy (14.1 vs. 2.62 mm², P<0.001) (19). Schuhmann et al. and Udagawa et al. also demonstrated that the sample size was significantly larger with cryobiopsy compared to forceps biopsy (20,24). Moreover, Franke et al. showed that cryobiopsy samples exhibited fewer artifacts and a significantly higher percentage of alveolar tissue (53.6% vs. 25.4%, P<0.001) (25). The larger volume of the cryobiopsy samples allows for the detection of minute tumor lesions that might be missed in smaller samples. This is particularly important for mucinous tumors, which are often difficult to diagnose using small samples due to the presence of abundant extracellular mucin. Therefore, cryobiopsy theoretically enhances the histopathological diagnosis of IMA. As a result, the cryo group demonstrated a significantly higher diagnostic yield of 93.5%.

While multivariable analysis identified the cryo group as the only independent factor significantly associated with a higher diagnostic yield, the univariable analysis demonstrated that larger lesion size and pneumonic-type radiographic features were also significantly associated with diagnostic success. Previous studies have reported that pneumonic-type lesions tend to be larger than solitary-type lesions (6-9). In these studies, the radiographic feature of IMA was classified into two categories: solitary type, which appears as a nodule or mass on CT images, and pneumonic type, which is characterized by consolidation opacities reflecting interalveolar mucins or tumor cells. The spread of mucins or tumor cells is believed to contribute to lesion size; however, reported lesion dimensions vary widely across different studies. The median lesion size in this study was 26.7 mm, which aligns with typical findings; however, the range (8.6–100.5 mm) indicates considerable variation. This variability suggests a potential confounding effect between lesion size and radiographic feature, possibly offsetting their significance in the multivariable analysis. Nevertheless, cryobiopsy remained the only significant factor, reinforcing its role as a robust diagnostic tool. Conversely, while the bronchus sign has been widely reported as a significant predictor of diagnostic yield for PPLs (19,21,32), it was not found to be significant in this study. In some cases, the abundant mucin surrounding IMA may have resulted in a pseudo-positive bronchus sign by disrupting bronchial continuity on HRCT.

Distinguishing IMA from other lung adenocarcinoma subtypes is critical for determining appropriate surgical treatment strategies. The clinical prognosis of IMA has been reported to be poorer than that of other subtypes due to its higher infiltrative and spreading potential (37,38). In recent years, there has been growing interest in limited resection (i.e., wedge resection and segmentectomy) for patients with early-stage lung cancer (39,40). Some studies explored the association between the extent of lung resection and the prognosis in patients with IMA (41-43). Although no definitive conclusion has been reached regarding whether lobectomy or sublobar resection is the optimal approach for IMA, the highly infiltrative and spreading nature of IMA suggests that limited resection should be approached with caution to minimize the risk of local recurrence. We believe that preoperative identification of the IMA is critical for guiding surgical strategies. Conversely, some studies have suggested that IMA is associated with a lower rate of nodal metastases, which could contribute to improved local control and better overall survival compared to other subtypes, potentially leading to more favorable surgical outcomes in selected patients (44,45). Only two cases in which EBUS-TBNA was performed were included in the target population (Figure 2), suggesting that EBUS-TBNA is rarely utilized for the preoperative diagnosis of IMA. Therefore, the preoperative diagnosis of IMA is expected to primarily rely on TBB. Given this dependency, the findings of this study, demonstrating the superior diagnostic performance of cryobiopsy compared to conventional biopsy, hold significant clinical relevance. In particular, the 93.5% diagnostic yield underscores transbronchial cryobiopsy as a highly promising diagnostic method for IMA.

In non-surgical cases, beyond obtaining a definitive diagnosis, molecular profiling is essential for determining treatment strategies, particularly as targeted therapies continue to advance. IMA exhibits distinct genotypic features compared to non-mucinous adenocarcinoma, characterized by a low prevalence of EGFR mutations and a high frequency of KRAS mutations (46,47). In this study, among the cases analyzed using NGS, KRAS mutations were detected in 77.8% of cases, while EGFR mutations and ALK fusions were entirely absent, including results from single-plex assays. Currently, targeted therapy is available only for the KRAS G12C variant (48), but investigational drugs are also being developed for other variants, such as G12D. Additionally, it is crucial to assess co-mutations, such as STK11 and KEAP1, which have been reported to negatively influence the efficacy of immune checkpoint inhibitors in KRAS-mutant lung cancer (49,50). Our molecular findings were obtained using NGS with a relatively small gene panel on resected specimens. However, to achieve comprehensive molecular profiling in clinical practice, it is necessary to perform NGS with a broader gene panel on biopsy specimens. Cryobiopsy has been demonstrated to be well-suited for NGS in diagnosing PPLs (51,52), and its utility is expected to be even greater in IMA, where obtaining an adequate number of tumor cells remains a significant challenge.

Implications and actions needed

A major concern with cryobiopsy is the technical challenge and risk of complications. The utility of earlier reusable cryoprobes was restricted due to the thickness and stiffness of their tip, particularly when targeting lesions in the right upper lobe (RUL) and left upper segment (LUS). However, with increased procedural experience, we successfully accessed RUL/LUS lesions even using reusable cryoprobes (21,23). Further improvements have been achieved with the introduction of thinner, single-use cryoprobes, which enhance navigability and lesion accessibility (22). As a result, despite a higher proportion of RUL/LUS lesions in the cryo group compared to the conventional group, the diagnostic yield was significantly higher. This finding supports the utility of cryobiopsy for diagnosing IMA, irrespective of lesion location.

Regarding complications, the incidence of bleeding was significantly higher in the cryo group than in the conventional group, consistent with prior findings (17-23). Grade 3 bleeding was observed in two cases, both involving solitary-type lesions in the right lower lobe, located considerably distant from the costal pleura (38.7 and 50.4 mm, respectively). To mitigate the risk of fatal bleeding, we employed the two-scope technique (34). However, for lesions in the middle lesion location, which have been associated with an increased risk of bleeding, the use of a balloon blocker may be advisable (18). Additionally, pneumonia occurred in 10.9% of the cryo group, whereas no cases were observed in the conventional group. Although cavitation, low-density areas, and bronchial stenosis have been identified as risk factors for post-TBB infection (53), none were observed in this study. Instead, the excessive mucus production characteristic of IMA may have compromised airway drainage, particularly when combined with post-biopsy bleeding, potentially contributing to the higher pneumonia incidence in the cryo group. Although no direct evidence exists, pneumonia carries the risk of delaying surgery, suggesting that prophylactic antibiotic administration should be considered when performing cryobiopsy for PPLs suspected to be IMA.

Conclusions

Cryobiopsy demonstrated a high diagnostic yield and was superior to conventional biopsy for diagnosing IMA. These findings support cryobiopsy as a valuable diagnostic technique for IMA, offering greater accuracy and tissue preservation compared to conventional biopsy methods.

Acknowledgments

We would like to thank Editage (https://www.editage.jp/) for English language editing.

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-865/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-865/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-865/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-865/coif). Y.M. received grants from Hitachi, Ltd., and personal fees from INTUITIVE, Olympus, AMCO, Thermo Fisher Scientific, Erbe Elektromedizin GmbH, and Fujifilm. H.F. received grants from Hitachi High-Tech Corporation, and personal fees from AstraZeneca, Erbe Elektromedizin GmbH, DNA Chip Research Inc. Yukihiro Yoshida received grants from Chugai Pharmaceutical Co., Ltd., Takahata Precision Co., Ltd., AstraZeneca plc. Yasushi Yatabe received grants from Merk Biopharma, Chugai-pharma, Konica-Minolta REALM, Optieum Biotechnologies, Eizai, and personal fees from AstraZeneca, MSD, AbbVie, Novartis, Amgen, Daiichi-Sankyo, Janssen Pharma, Konica-Minolta REALM, MSD, Chugai-pharma, AstraZeneca, Merk Biopharma, Novartis, Amgen, Daiichi-Sankyo, Thermo Fisher Science. T.T. received grants from JSPS KAKENHI Grant, and personal fees from Nippon Medical School Foundation, Hamamatsu University School of Medicine. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments and the Ethical Guidelines for Epidemiological Research in Japan. The study protocol was reviewed and approved by the National Cancer Center Institutional Review Board (approval No. 2018-090). Written informed consent for the clinical procedure was obtained from all patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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