Published online Mar 13, 2023.
https://doi.org/10.4048/jbc.2023.26.e10
A Preliminary Experience of Endoscopic Total Mastectomy With Immediate Free Abdominal-Based Perforator Flap Reconstruction Using Minimal Incisions, and Literature Review
Abstract
Purpose
Endoscopic total mastectomy (ETM) is predominantly performed with reconstruction using prostheses, lipofilling, omental flaps, latissimus dorsi flaps, or a combination of these techniques. Common approaches include minimal incisions, e.g., periareolar, inframammary, axillary, or mid-axillary line, which limit the technical ability to perform autologous flap insets and microvascular anastomoses, as such the ETM with free abdominal-based perforator flap reconstruction has not been robustly explored.
Methods
We studied female patients with breast cancer who underwent ETM and abdominal-based flap reconstruction. Clinical-radiological-pathological characteristics, surgery, complications, recurrence rates, and aesthetic outcomes were reviewed.
Results
Twelve patients underwent ETM with abdominal-based flap reconstruction. The mean age was 53.4 years (range 36–65). Of the patients, 33.3% were surgically treated for stage I, 58.4% for stage II, and 8.3% for stage III cancer. Mean tumor size was 35.4 mm (range 1–67). Mean specimen weight was 458.75 g (range 242–800). Of the patients, 92.3% successfully received endoscopic nipple-sparing mastectomy and 7.7% underwent intraoperative conversion to skin-sparing mastectomy after carcinoma was reported on frozen section of the nipple base. Mean operative time for ETM was 139 minutes (92–198), and the average ischemic time was 37.3 minutes (range 22–50). Fifty percent of patients underwent deep inferior epigastric perforator, 33.4% underwent MS-2 transverse rectus abdominis musculocutaneous (TRAM), 8.3% underwent MS-1 TRAM, and 8.3% underwent pedicled TRAM flap reconstruction. No cases required re-exploration, no flap failure occurred, margins were clear, and no skin or nipple-areolar complex ischemia/necrosis developed. In the aesthetic outcome evaluation, 16.7% were excellent, 75% good, 8.3% fair, and none were unsatisfactory. No recurrences were observed.
Conclusion
ETM through a minimal-access inferior mammary or mid-axillary line approach, followed by immediate pedicled TRAM or free abdominal-based perforator flap reconstruction, can be a safe means of achieving an “aesthetically scarless” mastectomy and reconstruction through minimal incisions.
INTRODUCTION
Complete oncological resection, safety, function, and aesthetics are integral components of comprehensive breast cancer surgical care. For any patient facing a mastectomy, requisite therapeutic components include skin and nipple-areolar complex (NAC) preservation (when possible) [1, 2], consideration of breast reconstruction [3], and it has been established that both aesthetic and functional outcomes contribute toward overall patient satisfaction and are considered major determinants of quality of life. The community now recognizes that autologous reconstruction establishes enduring natural aesthetics and tactile results, and the free abdominal-based perforator flap has become the preferred method that allows close-to-ideal breast defect restoration while minimizing abdominal donor site morbidity. Alternative flaps, prosthetics, and adjunctive procedures continue to provide options for patients presenting with challenging clinical scenarios, those with unavailable or inadequate abdominal donor sites, or as a component of patient choice [3].
Minimally invasive breast surgery (MIBS) evolved to push the conventional boundaries of aesthetic outcomes, as surgical breast oncologists sought the holy grail of resection-restoration in as discrete a manner as technically possible [4, 5, 6, 7, 8, 9]. The most obvious advantage of the endoscopic or robotic minimally invasive breast technique is that the surgeon is empowered to make smaller inconspicuous incisions that can even be sited off the main mound of the breast. At present, MIBS and its commonly associated reconstructive techniques are predominantly considered more suitable for women with small- to moderate-sized breasts (i.e., A and B cup size) [8] and are more commonly offered along with whole breast reconstruction utilizing mammary prostheses [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26], lipofilling [27], omental flap reconstruction [28], latissimus dorsi (LD) flap reconstruction [21, 23, 29, 30, 31, 32, 33], or a combination of techniques [28, 33]. Meanwhile, MIBS with immediate free abdominal-based perforator flap reconstruction has not been robustly explored because of two major limitations of the minimal-access approach: challenges with flap inset and shaping and the need for access to recipient vessels for microvascular anastomoses, namely the internal mammary artery (IMA), thoracodorsal (TD), or lateral thoracic artery. We report our preliminary experience with the endoscopic total mastectomy (ETM) through a minimal-access inframammary or mid-axillary line approach, followed by immediate pedicled transverse rectus abdominis musculocutaneous (ETM-TRAM) or free deep inferior epigastric perforator (DIEP) flap reconstruction, as a means of a safe and “aesthetically scarless” mastectomy and reconstruction with respect to the region surrounding the breast.
METHODS
We studied all consecutive female patients with newly diagnosed breast cancer who underwent ETM and pedicled TRAM or DIEP flap reconstruction performed by a single breast surgeon and a team of three plastic and reconstructive microsurgeons at the Sengkang General Hospital Breast Centre. We examined the patients’ clinical-radiological-pathological characteristics, type and details of breast and reconstructive surgery, and the recurrence rate. Complications were defined as surgical site infection (according to the CDC and ASEPSIS score [34]), perioperative hemorrhage (referred as the need for transfusion, compression, percutaneous or surgical evacuation, and hemostasis), skin and/or NAC ischemia or necrosis (epidermolysis, partial- or full-thickness ischemia and/or necrosis), and flap failure (complete necrosis of the flap). Aesthetic outcomes were evaluated using clinical and photography-based assessments, objectively by the breast and plastic and reconstructive microsurgeons, and subjectively by the patients at the 1-month and 6-month postoperative follow-ups. The aesthetic outcomes were deemed 1) “excellent” if the reconstructed and normal breasts were perfectly symmetrical and equal in size and morphology, the patients were extremely satisfied, and perceived that the results were nearly identical to their preoperative state; 2) “good” if there was some, but no significant difference in symmetry and the patients were satisfied; 3) “fair” if asymmetry was distinct and the patients were satisfied; and 4) “unsatisfactory” if there was significant asymmetry or distortion and the patients were dissatisfied. Ethics approval for this study was obtained from the Centralized Institutional Review Board (CRIB) SingHealth (CIRB Ref:2019/2419). Informed consent was obtained from all patients.
Patient selection
All patients considered for endoscopic nipple-sparing mastectomy (NSM) and abdominal-based flap reconstruction were assessed preoperatively for oncological safety and technical feasibility, and a consensus was reached on the approach and incision placement via the inframammary fold (IMF) or mid-axillary line by the breast surgeon and attending plastic and reconstructive microsurgeon. Patients were fully counseled on appropriate alternative surgical therapies, including other reconstructive options. The selection criteria included female patients with early breast cancer and those with clinical and/or radiological response to neoadjuvant therapy for locally advanced breast cancer. Preoperatively, the cancers should have been considered technically resectable based on clinical and radiological assessments; in particular, there should be a low likelihood of skin or NAC involvement and reasonable confidence of an R0 resection. Voluminous breasts with ptosis (i.e., cup-size C and larger and/or Regnault ptosis grade 2 and 3), smoking, high body mass index, prior cesarean sections and abdominoplasty, diabetes mellitus, and prior radiotherapy are considered relative contraindications for free abdominal-based flaps; however, they do not prevent our team from making an assessment nor our patients from making an informed choice for free-flap reconstruction at our collaborative breast center [35, 36, 37, 38, 39, 40].
Operative technique
ETM was performed through a 5 cm minimal-access inframammary incision or a mid-axillary line incision with the goal of skin and NAC preservation. An operative rigid endoscope was used to dissect the entire premuscular-subfascial pocket. Dissection of the subcutaneous skin flap followed the plane of the superficial fascia, performed under direct vision by cautery followed by sharp surgical scissor dissection after injection of tumescent fluid. The farthest aspect of the breast, that is, the superior-lateral aspect for the IMF incision and the medial aspect for the mid-axillary line incision, was transected using electrocautery or an energy device under direct endoscopic vision. Nipple-base sampling was performed in all patients, and frozen section results guided the preservation of the NAC versus its resection and intraoperative conversion to skin-sparing mastectomy (SSM). We routinely perform intraoperative skin flap viability assessments, whereby mastectomy skin flap perfusion is measured using indocyanine green angiography.
The choice of abdominal-based flap and its risks and benefits were discussed with patients prior to surgery, and consensus decisions were made with regard to the preference for either a pedicled or free flap. If a pedicled abdominal-based flap was preferred, an ipsilateral TRAM would be raised with the deep inferior epigastric artery (DIEA) pedicle kept long. A subcutaneous tunnel was made towards the chest pocket, ensuring that the abdominal skin remained thick for adequate perfusion. The flap was rotated into the breast pocket through this subcutaneous tunnel. We routinely perform vascular augmentation of the TRAM flap via the midaxillary line incision or a small axillary incision created for sentinel lymph node biopsy or axillary dissection. The typical recipient vessels are branches of the TD or lateral thoracic artery pedicle. If a free abdominal flap was preferred, an ipsilateral free DIEP flap or free muscle-sparing TRAM flap was raised, with the DIEA pedicle kept long. The recipient of choice is the IMA system owing to its good size match, pristine plane, and negative thoracic pressures generated during respiration that can assist with flap venous drainage. To access the IMA system, an inferior mammary incision may require a medial extension along the IMF. In cases where a mid-axillary line incision is planned, the TD pedicle is the system of choice. For free flaps, we routinely performed one arterial microanastomosis and two venous microanastomoses. The free flap was transferred to the pocket for microanastomosis, and anchoring sutures were applied superiorly and medially to recreate an aesthetically pleasing breast mound. We routinely leave a small skin paddle for free-flap monitoring. This skin paddle was excised on postoperative day 7 to produce an aesthetically pleasing curvilinear IMF or a longitudinal mid-axillary line scar in an “aesthetically scarless” position, “off the breast.”
RESULTS
A total of 12 female patients underwent ETM, sentinel lymph node biopsy, targeted and/or axillary lymph node dissection, followed by pedicled TRAM or free DIEP flap reconstruction between May 2020 and August 2022. The mean age of the patients was 53.4 years (range 36–65 years) and 41.7% had chronic comorbidities. The mean tumor size was 35.4 mm (range 1– 67 mm). Of the patients, 33.3% were surgically treated for stage I cancer, 58.4% for stage II cancer, and 8.3% for stage III cancer. 58.3% of patients underwent upfront surgery and 41.7% underwent neoadjuvant systemic therapy (Table 1).
Table 1
Patient and tumour characteristics (n = 12)
Endoscopic NSM was successfully performed in 91.7% of patients, and 8.3% underwent intraoperative conversion to SSM after carcinoma was reported in the frozen section results of the nipple base sample. The one patient who underwent SSM received neoadjuvant systemic therapy, had no prior clinical-radiological evidence of NAC cancer involvement, and the final histology confirmed carcinoma involving the areolar and nipple, in addition to the nipple base sample. The mean specimen weight was 458.75 g (range 242–800 g). The mean operative time for ETM was 139 minutes (range 92–198 minutes). Fifty percent of the patients underwent DIEP, 33.4% underwent MS-2 TRAM, 8.3% underwent MS-1 TRAM, and 8.3% underwent pedicled TRAM flap reconstruction. The mean total operative time was 595 minutes (range 430–828 minutes). For cases in which free flaps were performed, the average ischemic time was 37.3 minutes (range 22–50 minutes). Tumor-free margins were obtained in all cases. None of the patients required reexploration and there were no events of flap failure. No skin or NAC ischemia or necrosis was observed (Table 2). In the aesthetic outcome evaluation, ETM-TRAM or ETM-DIEP flap reconstruction was associated with 16.7% excellent (Figure 1), 75% good, 8.3% fair, and 0% unsatisfactory results. The average follow-up time was 15.4 months (range 1–31 months). No locoregional or distant recurrences were observed during the follow-up period (Table 2).
Table 2
Surgical outcomes (n = 12)
Figure 1
Endoscopic nipple-sparing mastectomy and abdominal-based pedicled and free-perforator flap reconstruction.
TRAM = transverse rectus abdominis musculocutaneous; DIEP = deep inferior epigastric perforator.
DISCUSSION
NSM and immediate reconstruction has been established in routine clinical practice, albeit with evolving applications and without a universally agreed consensus regarding indications and oncological safety [1]. The Oncoplastic Breast Consortium consensus conference in 2018 concluded that the oncological safety of the NSM is comparable to that of conventional mastectomy and SSM if cases are selected appropriately. There was consensus among the group that NSM can be performed for any tumor size that does not involve the skin or NAC, independent of axillary status. Clinical signs of nipple involvement, R1 resection at the nipple margin, and positive retroareolar margins are the main contraindications for nipple preservation [2]. Developed to advance this practice since 1995 [4, 5, 6], MIBS has been widely acknowledged as an oncologically safe approach in modern breast surgical oncology [16, 23, 41, 42, 43, 44]. Dissection and precise hemostasis can occur under clear camera-led direct visualization; the use of endoscopic-robotic instruments affords us smaller inconspicuous incisions that cause minimal scarring, postoperative pain, and greater patient satisfaction; and wound complications are reported to be rare [4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 41, 42, 43, 44, 45, 46, 47, 48]. One developing concept that encapsulates the value of the minimally invasive breast ideology is the principle of “aesthetically scarless” breast surgery, which describes an operation whereby incisions and “scars” remain but are secured out of sight and minimized within the range of oncologic safety [45]. Incisions for MIBS can be designed at the periareolar region, axillary region, mid-axillary line (where it may be hidden by the adducted arms), or IMF [4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 41, 42, 43, 44, 45, 46, 47, 48]. The major advantage of endoscopic and robotic breast surgery over “conventional” surgery is that the operative incision can be both small and planned “away from the breast,” allowing this principle to be successfully applied [45].
A strong collective of at least 24 cohort studies and selected case series involving oncological ETM [4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 22, 23, 24, 25, 26, 27, 28, 29, 30, 46] and 8 studies of robotic-assisted mastectomy [18, 19, 20, 21, 31, 32, 33, 48] have demonstrated comparable surgical and short- or medium-term oncologic outcomes to conventional breast surgery. However, publications scarcely depict the use of MIBS alongside autologous flap reconstruction, as opposed to 23 publications depicting implant-based reconstruction techniques [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26] and/or lipofilling [27]. The overarching reasons include the inherently small incision size in MIBS and the resulting challenges associated with autologous flap inset, breast shaping, and vascular anastomosis. A review of the literature suggests only four publications describing tissue reconstruction following ETM (i.e., two LD, one omental, and one pedicled TRAM flap reconstruction) and four studies involving the robot (i.e., three LD and one abdominal-based perforator flap reconstruction) [28, 29, 30, 31, 32, 33, 46, 48] (Tables 3 and 4). This collective excludes studies purely describing the endoscopic or robotic management of gynecomastia, breast-conserving surgery, or flap harvesting without the component of MIBS. In this context, the most commonly reported autologous flap is the LD, which has limitations in terms of desired volume, shaping, and functional preservation [29, 30, 33]. The inherent limitation of the omental flap is its restriction to small breast volumes, with one study suggesting that it would likely be insufficient on its own if the breast volume was > 150 mL, requiring the addition of a prosthetic [28]. To date, there have been no publications describing the combination of endoscopic mastectomy and free abdominal-based flap reconstruction; however, two groups have explored the use of the endoscope with the TRAM flap [46] and the robot with free perforator flap reconstruction [48] (Tables 3 and 4).
Table 3
Cohort studies, case series, and case reports on minimally invasive breast surgery (endoscopic or robotic assisted) mastectomy and immediate autologous flap reconstruction
Table 4
Detailed findings of cohort studies, case series, and case reports on minimally invasive breast surgery (endoscopic or robotic assisted) mastectomy and immediate autologous flap reconstruction
In 2015, Lai et al. [46] first reported encouraging results in 49 cases of ETM followed by immediate pedicled TRAM flap reconstruction in 48 patients. NSM was performed in 79.6% of the patients and the rest underwent SSM; 48 cases were unipedicled, and one was a bipedicled TRAM flap. The reported mean mastectomy weight was 487±120g, implying that the upper limit was 607 g. They encountered a low 10.3% partial NAC ischemia/necrosis rate and 98% good-to-fair aesthetic outcomes at 3-month follow-up (Tables 3 and 4). Kuo et al. [47] published a technical feasibility report in 2020, and the same team led by Huang et al. [48] conducted a 2021 retrospective cohort study demonstrating the innovative use of robotic approaches in 22 cases of robotic-assisted NSM (R-NSM) via an anterior axillary line incision, followed by immediate microsurgical free-flap reconstruction. In the R-NSM group, 20/22 cases received an incision length ≤ 5 cm, and the mean mastectomy weight was 350.4 ± 158.1 g, implying an upper limit of 508.5 g. Of the patients, 86.4% received a DIEP and 13.6% received a profunda artery perforator flap reconstruction. The authors utilized the TD (81.8%) or lateral thoracic (18.2%) vessels as recipient vessels in the majority of the 22 robotic mastectomies and free perforator flap reconstructions [48] and acknowledged the expected challenges in microvascular anastomosis and flap inset and shaping [47, 48, 49]. In a separate report, the group described a technique of transcutaneous medial fixation suturing that aids flap in-setting and creation and maintenance of the medial breast border [49]. The rate of positive resection margins was 4.55%; the rates of skin and NAC necrosis were 21.1% and 5.3%, respectively; 5.3% encountered partial flap failure; and no flap loss was reported. Ultimately, they concluded that R-NSM and perforator flap reconstruction presented equal surgical and oncological safety to conventional NSM and demonstrated superior aesthetic results in terms of scar length, location, and visibility, and symmetry of the breast IMF [48].
The learning curve involved in performing MIBS with an immediate pedicled TRAM flap is undoubtedly gentler than that of free perforator flap reconstruction. There are technically fewer “surgical access” demands for the inset of a pedicled TRAM flap, which is inherently a flap with a long pedicle and arc of rotation, brought from abdomen to chest via a subcutaneous tunnel. Owing to the relative ease of flap entry, MIBS-TRAM can be performed through almost any of the aforementioned incisions, including the periareolar, axillary, mid-axillary line, and IMF. However, creating a subcutaneous tunnel from the abdomen to the breast pocket can potentially affect the inferomedial blood supply to the mastectomy skin, explaining the increased association with skin or nipple necrosis. Meanwhile, the free abdominal-based flap does not require a subcutaneously dissected tunnel, involves minimal rectus abdominis muscle sacrifice, decreases donor-site morbidity such as abdominal bulge and herniae, and increases general satisfaction [3, 50], and studies have demonstrated increased cost-effectiveness [51].
Preservation of the NAC is undeniably valuable because the NAC represents a geometric and aesthetic focal point of the breast [52], retains significant psycho-emotional importance [53], and its presence has been linked to increased sexual functioning and patient satisfaction [54]. Complications involving the skin and NAC contribute to an increased need for prolonged wound management, additional interventions, potential delays in adjuvant therapy, cosmetic scarring and distortion, and financial and psychosocial morbidity [55]. Meanwhile, NAC reconstruction to perfection remains challenging [56]. The IMF or midaxillary line ETM approach ensures that the nipple is not at the watershed zone of ischemia, as opposed to periareolar-based incisions, improving its chances of survival [57, 58]. Low levels of skin or nipple necrosis should be expected with the MIBS-free perforator flap, and our early clinical results with IMF or midaxillary line ETM-DIEP flap reconstruction demonstrate the same.
Robotic systems offer many benefits including three-dimensional magnified visualization and improved dexterity and ergonomics; however, it may be acknowledged that access to a robotic system can be limited by country, healthcare funding model, center availability, resource allocation, and financial considerations. Robust uptake has been somewhat dampened by limited data on cost-effectiveness and clear evidence of superiority over endoscopic or even conventional approaches [59, 60]. In comparison, the endoscopic MIBS approach utilizes a relatively affordable rigid endoscope and laparoscopic equipment that are readily available as part of almost any general surgical practice, making the technique easily accessible to surgeons and extendable to the patients they serve. Both robotic and endoscopic systems continue to be valuable technologies and luxuries in modern practice and have proven their value in our patient population. That said, for these reasons, our team finds value in incorporating the practice of ETM-DIEP flap reconstruction into routine public institution practice to serve our patient population.
We also hypothesize that the expansion of the MIBS repertoire into abdominal-based flaps could potentially push arbitrary upper-limit boundaries for application in larger ptotic breasts. Traditional MIBS-associated reconstructive techniques, such as direct-to-mammary prostheses [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26], lipofilling [27], omental flap [28], and LD flap reconstruction [21, 23, 29, 30, 31, 32, 33], have generally been described to be more suited to women with small-to moderate-sized breasts (i.e., A and B cup size), as opposed to larger ptotic morphology [9]. The reasons are multifactorial, including the preference for case selection of small- to average-sized breasts for NSM in general (both conventional and MIBS), a ceiling of limitation when it comes to available implant sizes, and the expected volume limitation for potential autologous tissue harvested from these sites. Authors postulate that this may be why most reports on MIBS occur out of Asian countries, such as Japan, Korea, and China, because of the relatively petite sizing in the average Asian breast and body habitus and the general limitation of autologous tissue volume for replacement [42].
Morphological factors such as C cup size and beyond and/or Regnault ptosis grades 2 and 3 are still considered relative contraindications for performing NSM. It is generally believed that larger breasts require a larger surface area of perfusion, there is likely to be a longer distance between the nipple and the surrounding blood supply from the chest wall, increased manipulation of the breast skin envelope during flap dissection can endanger the blood supply to the delicate subdermal nipple plexus, and increased traction on skin flaps in larger ptotic breasts can contribute to additional stress that accentuates the imbalance between demand and supply of the NAC, which is now critically dependent on the dermal microvasculature [35, 36, 37, 38, 39]. The nipple necrosis rate in patients with a C-cup or larger breast has been reported upwards of 30% [39], which is significantly higher than the necrosis rate in patients with A- or B-cup breasts, which can fall under 10% [61]. Our early and limited experience suggests that ETM can be performed in moderate to large breasts up to a mastectomy specimen weight of 800 g, although we caution that there may instead be a role for skin-reducing mastectomy in women with considerably larger and more ptotic breasts [62].
When planning a combined MIBS-reconstruction approach, it is useful to note that patients with small and non-ptotic breasts may be aesthetically better suited for incisions in the axilla or mid-axillary line. While technically feasible, these women may still have a visible scar in the straight-on view as opposed to women with more voluminous ptotic breasts that may hide a well-placed IMF scar. If the priority of the patient and surgical team is primarily to achieve an “aesthetically scarless” result, a laterally placed mid-axillary line approach would be our preferred ETM incision. It is technically feasible to utilize the TD pedicle as the system of choice. Flap inset and medial fixation are more challenging but can be enhanced using an endoscope.
This small case series and audit is inherently limited in its ability to make statistically significant conclusions regarding this surgical approach, and we do not claim to make any conclusive statements regarding safety; however, we hope that our cases may strengthen the literature and reaffirm like-minded colleagues who are presently challenging similar preconceived restrictions related to endoscopic breast surgery. Successful ventures in ETM and immediate perforator flap reconstruction require a close working relationship and dynamic and cooperative efforts among breast and plastic and reconstructive microsurgeons and the multidisciplinary team. Overall, our experience with the minimal-access inferior mammary fold and mid-axillary line ETM approach demonstrates the technical feasibility of these incisions and strategies alongside free abdominal-based flap reconstruction, or pedicled when preferred or not technically feasible. The end results can attain ideal cosmetic outcomes, given that most of these scars are ultimately hidden nearly in the patient’s “bra line” by the patient’s ptotic reconstructed breasts or by the arm upon adduction.
In conclusion, ETM through a minimal inferior mammary or mid-axillary line incision, followed by immediate pedicled TRAM or free abdominal-based perforator flap reconstruction, can be a safe means of achieving an “aesthetically scarless” mastectomy and breast reconstruction.
Conflict of Interest:The authors declare that they have no competing interests.
Author Contributions:
Conceptualization: Ngaserin S, Wong AWJ, Feng JJ, Tan BKT.
Data curation: Ngaserin S, Wong AWJ, Leong FQH, Tan BKT.
Formal analysis: Ngaserin S.
Investigation: Ngaserin S, Wong AWJ.
Methodology: Ngaserin S, Wong AWJ, Feng JJ.
Project administration: Ngaserin S.
Resources: Ngaserin S, Tan BKT.
Supervision: Ngaserin S.
Validation: Ngaserin S.
Writing - original draft: Ngaserin S, Wong AWJ.
Writing - review & editing: Ngaserin S, Wong AWJ, Leong FQH, Feng JJ, Kok YO, Tan BKT.
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