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Abstract
Objective This study aimed to analyze the prognostic factors for overall and progression-free survival in patients with vulvar cancer.
Methods This international, multicenter, retrospective study included 2453 patients diagnosed with vulvar cancer at 100 different institutions. Inclusion criteria were institutional review board approval from each collaborating center, pathologic diagnosis of invasive carcinoma of the vulva, and primary treatment performed at the participating center. Patients with intraepithelial neoplasia or primary treatment at non-participating centers were excluded. Global survival analysis and squamous cell histology subanalysis was performed.
Results After excluding patients due to incomplete data entry, 1727 patients treated for vulvar cancer between January 2001 and December 2005 were registered for analysis (1535 squamous, 42 melanomas, 38 Paget’s disease and 112 other histologic types). Melanomas had the worse prognosis (p=0.02). In squamous vulvar tumors, independent factors for increase in local recurrence of vulvar cancer were: no prior radiotherapy (p<0.001) or chemotherapy (p=0.006), and for distant recurrence were the number of positive inguinal nodes (p=0.025), and not having undergone lymphadenectomy (p=0.03) or radiotherapy (p<0.001), with a HR of 1.1 (95% CI 1.2 to 1.21), 2.9 (95% CI 1.4 to 6.1), and 3.1 (95% CI 1.7 to 5.7), respectively. Number of positive nodes (p=0.008), FIGO stage (p<0.001), adjuvant chemotherapy (p=0.001), tumor resection margins (p=0.045), and stromal invasion >5 mm (p=0.001) were correlated with poor overall survival, and large case volume (≥9 vs <9 cases per year) correlated with more favorable overall survival (p=0.05).
Conclusions Advanced patient age, number of positive inguinal lymph nodes, and lack of adjuvant treatment are significantly associated with a higher risk of relapse in patients with squamous cell vulvar cancer. Case volume per treating institution, FIGO stage, and stromal invasion appear to impact overall survival significantly. Future prospective trials are warranted to establish these prognostic factors for vulvar cancer.
- vulvar and vaginal cancer
- vulvar neoplasms
- neoplasm recurrence, local
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HIGHLIGHTS
The 5-year overall survival for squamous cell carcinoma of the vulva was 61.4% and disease-free survival was 48.1%.
Centers with nine or more cases per year had higher survival rates.
Risk of mortality increased with increasing number of metastatic lymph nodes.
Introduction
Vulvar cancer accounts for 5.1% of all gynecologic cancers and usually affects women aged 65–70 years,1 2 although the incidence of vulvar cancer in women aged <50 years is increasing at an alarming rate.3 Squamous cell carcinomas account for 90% of all malignant vulvar tumors; among them, 50% are asymptomatic.4 Inguinal nodal status at the time of diagnosis is the most important prognostic factor. Nodal involvement decreases the 5-year overall survival rate from 90% to 50%.5 The most important risk factors for nodal metastasis are the clinical nodal status, age, differentiation grade, tumor stage and size, depth of stromal invasion, and the presence of lymphovascular space invasion. Conversely, data regarding the pattern of recurrence in vulvar cancer are very limited, with the majority of relapses confined to the vulvar region.6
The survival rate in patients with vulvar cancer has increased during the last decades, probably due to the performance of more radical surgeries; however, in the last 10 years, the surgical approach has shifted towards a more conservative management, with an overall trend towards a more tailored and individualized approach.7
Currently, one of the main controversies is the the extension of free margins from the tumor resection edge, since this is considered one of the most important prognostic factors for local recurrence.8 Different guidelines recommend obtaining free surgical margins of 10–20 mm for the local control of vulvar cancer, though the data are still very heterogeneous: Heaps et al9 showed a 50% local recurrence in patients whose surgical margins were <8 mm and De Hullu et al also demonstrated an increased risk of recurrence in patients with margins <8 mm (p=0∙002).10 Currently there is no agreement as to what it is considered to be free margins, setting a cut-off from a minimum free surgical margin of 8 mm to 20 mm for the local control of vulvar cancer.9–11
Large multicenter studies are need to confirm the factors associated with survival in vulvar cancer and so allow a tailored management of vulvar cancer. The aim of this study was to determine the prognostic factors associated with the recurrence rate and overall survival in patients with vulvar cancer, and specifically in squamous cell carcinoma,within an international, multicenter, retrospective study design — the VULCAN study.
Methods
After obtaining institutional review board (IRB) approval in every participating institution, an international, multicenter, retrospective study of patients diagnosed with vulvar cancer was performed. The study collated 2453 vulvar cancer patients from 100 different centers; of these, only 1772 vulvar cancer cases (72.2%) were registered in an encrypted online database where investigators included their cases (a review of the quality of data was performed), 31 cases did not fulfill the inclusion criteria, and 14 had data entry errors. Data for the remaining 1727 (70.4%) patients treated between January 2001 and December 2005, including all histologic subtypes, were collected (Figure 1). Of these patients, 1535 (62.6%) had squamous cell histology, were melanomas (n=42), Paget’s disease (n=38), and other histologic types (n=112).
Flow chart of VULCAN study data collection.
Participation in the study was offered to all oncologic European centers via email using the European Network of Young Gynae Oncologists (ENYGO) mailing list. Distribution according to country is shown in Online supplementary figure 1. Inclusion criteria were IRB approval from each collaborating center, pathologic diagnosis of invasive carcinoma of the vulva, and primary treatment performed at the participating center. Patients with intraepithelial neoplasia or primary treatment at non-participating centers were excluded. Disease staging was based on the Federation of Gynecology and Obstetrics (FIGO) 2009 classification system.12 Uniform criteria for surgical procedure nomenclature, pathologic variables, and sites of recurrence were established. Superficial lymphadenectomy was defined as inguinal dissection up to the level of cribiform fascia, while deep inguinal lymphadenectomy was categorized if lymph nodes under the cribiform fascia were removed. Sentinel node biopsy was performed in centers where the technique was available and validated . Radiotherapy in vulva and groins was considered pre-operatively in FIGO stage IV; while it was administered post-operatively in the same fields if tumor size ≥30 mm, free margin <8 mm, lymphovascular space invasion present, ≥2 inguinal lymph nodes involved, or when there was complete replacement or extra capsular spread in any node, and in FIGO stage III. Chemotherapy was indicated in FIGO stages III-IV.
Supplemental material
All surgical specimens were fixed in formalin and embedded in paraffin prior to examination. The distance of margins was determined from tissue sections stained with haemotoxylin and eosin (H&E). Surgical margin was defined as the distance between the lesion and the surgical incision, which corresponds to disease-free tissue. Patient follow-up was carried out every 3 months for the first year, every 6 months until the fifth year, and after that annually, discharging the patient after 10 years. Data collection was performed via a web-based encrypted database located in the local host of the Catalan Institute of Oncology (ICO) at URL: http://bioinfo.iconcologia.net/sego/. This manuscript was prepared in accordance with STROBE (Strengthening The Reporting of OBservational Studies in Epidemiology) guidelines.13
Normally distributed quantitative data are presented as mean±SD, whereas asymmetrically distributed data are presented as median (range). Qualitative variables are presented as absolute value and percentage. Quantitative data were compared between groups using Student’s t-test and analysis of variance (ANOVA). Categorical variables were compared using the chi-square test and multivariate analysis (logistic regression) was performed in order to identify potential risk factors. Survival analysis was performed using Kaplan–Meier curves and the Cox proportional hazards model. Hazard ratios (HRs) with a corresponding 95% CI were calculated to represent the magnitude of association between prognostic factors and the risk for recurrence of vulvar cancer. All comparisons were two-tailed and the alpha error was set at 5%. All data were analyzed using SPSS version 15.0 (SPSS, Inc., Chicago, IL, USA) and SAS 8.0 (SAS Institute, Inc., Madrid, Spain).
Results
Baseline characteristics of all 1727 eligible patients included in the VULCAN study (1535 squamous and 192 non-squamous cases) are shown in Table 1. Mean patient age was 70±5.1 years. Some 91.7% of patients were post-menopausal at the time of diagnosis. Excluding unreported cases, 13% of patients were smokers, and 57.4% of patients did not have a history of vulvar pathology prior to the diagnosis of vulvar cancer, 24.9% had lichen sclerosus, and 17.8% presented with vulvar intraepithelial neoplasia. One-fifth (21.5%) of the patients tested positive for human papillomavirus. Main symptoms at the time of diagnosis included a vulvar mass (50%) or itching (25%); the remaining patients (25%) were asymptomatic. Most (84.5%) of the patients had a single lesion. The tumor involved the midline in 43.3% of the patients and 30.7% of the patients had palpable inguinal nodes.
Clinicopathologic and surgical characteristics of patients with vulvar cancer (n=1727)
Surgical treatments were as follows: wide local excision (19.1%), radical vulvectomy (72.1%), pelvic exenteration (0.6%), and no surgical treatment (8.2%). Tumor margins <15 mm were observed in 20.7% of squamous cell tumors. Radiotherapy was administered in 39.1% of the patients; 25.2% received it pre-operatively, and the remaining 74.8% after surgery. In total, 10.5% of the patients received adjuvant chemotherapy. Sentinel node biopsy was performed in 14.1% of the patients and inguinal lymphadenectomy in 60.1%. Of all the lymph node dissections, 78.7% were bilateral and 21.3% were unilateral. Deep dissection was performed in 68.6% of the patients versus superficial dissection in 31.4%. In all, 82.3% of the patients underwent separate incisions. The mean number of resected lymph nodes was 13.3±8.1 nodes. In the case of macroscopic extracapsular node involvement, all the tumoral mass was removed and no inguinal residual tumor was observed after lymph node dissection in all patients. No pelvic lymphadenectomies were reported.
Overall, median follow-up in the VULCAN study was 73 (IQR 14–130) months. A total of 714 (41.3%) patients recurred, 483 (27.9%) were local relapses and 231 (13.4%) distant relapses. Median time to onset of local recurrence from completion of treatment for all histologic types was 30.3 (range 27–33) months versus 29.1 (range 26–32) months for squamous cell carcinoma, 41 (range 22–60) months for melanomas, 37 (range 24–50) months) for Paget's disease, and 39.5 (range 24–54) months) for other histologic types. There were no significant differences according to histologic type in disease-free interval (p=0.11) (Figure 2). Median overall survival for all histologic types was 104.8 (range 101–107) months. Significant differences (p=0.02) in overall survival according to histologic type were observed: 102.2 (range 99–105) months for squamous cell carcinoma, 85 (range 65–104) months for melanomas, 121 (range 113–128) m0nths for Paget's disease, and 117.7 (range 109–126) months for other histologic types (Figure 3).
Disease-free survival of all histologic types. Kaplan–Meier survival curve according to recurrence (p=0.11).
Overall survival related to histologic type. Kaplan–Meier curve (p=0.02).
As overall survival differed significantly according to histologic type, only 1535 squamous carcinoma cases were analyzed in the multivariate analysis, which accounted for 88.9% of the patients. Multivariate analysis showed that five characteristics were significantly associated with the risk of global recurrence of squamous cell vulvar carcinoma when adjusted to FIGO stage: patient age (p=0.031), number of positive inguinal nodes (p<0.001), tumoral resection margins (p=0.029), not undergoing chemotherapy (p=0.018), and not undergoing any radiotherapy (p<0.001). Regarding the impact of positive groin lymph nodes, disease-free survival was 28.1 months when only one node was affected versus 16.1 months (p<0.001) when more than one node was positive.
Factors significantly associated with an increase in local recurrence of squamous cell vulvar cancer were not undergoing radiotherapy (p<0.001) and not undergoing chemotherapy (p=0.006). Tumor resection margins had no prognostic significance (p=0.059) nor did the number of positive nodes (p=0.83) (Table 2).
Multivariate analysis of prognostic factors for local recurrence in 1535 cases of squamous vulvar cancer
Factors significantly associated with distant metastasis were presence of metastatic lymph nodes (p=0.025), not undergoing lymphadenectomy (p=0.003), and not undergoing radiotherapy (p<0.001). Adjuvant chemotherapy and tumor resection margins were not significantly associated with the presence of distant metastasis (p=0.137 and p=0.103, respectively) (Table 3).
Multivariate analysis of prognostic factors for distant recurrence in 1535 cases of squamous vulvar cancer
Finally, factors significantly associated with overall survival were: case volume of the participating center (p=0.05), number of positive nodes (p=0.008), FIGO stage (p<0.001), administration of chemotherapy (p=0.001), tumor resection margins (p=0.045), and stromal invasion >5 mm (p=0.001). Centers with ≥9 cases per year had higher survival rates with a HR of 0.9 (95% CI 0.9 to 1) (p=0.05). The risk of mortality increased 1.1-fold for each positive node, 1.6-fold for each increase in FIGO stage, and 2.2-fold in patients that did not undergo chemotherapy. During the follow-up period, a tumoral resection margin >15 mm correlated with a 10% mortality rate, a free margin >18 mm was associated with a 9.1% mortality rate, and a free margin >20 mm was associated with a 6.5% mortality rate. Stromal invasion >5 mm increased the risk of mortality 1.9-fold. The 5-year overall survival rate for squamous cell carcinoma of the vulva was 61.4% and 5-year disease-free survival was 48.1%. Median time from initial treatment to relapse was 73 months, but the median time for survival was not reached.
Discussion
Most studies reporting prognosis of patients with vulvar cancer focus on specific factors such as local recurrence, lymph node spread, treatment outcome, or adjuvant treatment.14 15 Such studies are often characterized by single institution reviews of small case series. The present study is one of the largest multicentric studies conducted in vulvar cancer, and it found that there are significant differences in overall survival among all histotypes of vulvar cancer, vulvar melanoma being the one with the worst results followed by squamous cell tumors. In vulvar squamous cancers, the main prognostic factors seem to be the FIGO stage, number of metastatic inguinal lymph nodes, and the lack of adjuvant treatment. In addition, case volume per treating institution impacts on survival, which has not previously been reported in the literature.
The most important factors predicting recurrent disease in vulvar cancer are: tumor stage, number of metastatic nodes, tumor size, depth of invasion, lymphovascular space invasion, tumor location, and tumor resection margins.16 An Italian multicenter study reported 187 (37.3%) recurrences in 502 patients with vulvar cancer. Multivariate analysis showed that there were only three factors statistically associated with the risk of recurrence: FIGO stage >II (p=0.029), positive lymph node metastasis (p=0.009), and lymphovascular space invasion (p=0.004).15 Burger et al17 analyzed the prognostic factors for survival in 190 patients with squamous vulvar carcinoma. Patients with lymph node metastasis had a higher relative mortality than those without nodal infiltration. In fact, a retrospective analysis of 389 patients with vulvar cancer by Raspagliesi et al18 reported that among all tumor-related variables, nodal status was the most significant prognostic factor. In contrast to these findings, a retrospective population-based study by Rhodes et al19 reported that overall survival was not negatively affected in patients with positive inguinal lymph nodes. Chan and colleagues11 reported that tumor stage, tumor size, pathologic margins, and >2 infiltrated lymph nodes were independent factors for recurrence, although they did not analyze the effect of adjuvant treatment. Finally, the AGO-CaRE 1, a retrospective study of 1249 patients diagnosed with squamous vulvar cancer, suggested an improved prognosis in node-positive patients with adjuvant therapy irrespective of the number of positive nodes, which could balance the unfavorable prognosis of this cohort of patients.20
In the present study, the factors associated with local recurrence were not undergoing radiotherapy (p<0.01) and not undergoing chemotherapy (p=0.006). Adjuvant radiotherapy should be administered when ≥2 lymph nodes are involved or when there is complete replacement or extracapsular spread in any node.21 22 An additional factor associated with poor prognosis in the present study was stromal invasion; stromal invasion >5 mm increased the risk of mortality 1.9-fold. The FIGO staging system12 sets the cut-off point to upgrade from stage IA to IB as 1 mm, which was also supported by Yoder et al;23 however, Maggino et al15 considered 3 mm to be a better cut-off, with a relative risk of 1.5 (p=0.03). Likewise, Chan11 observed that 5-year overall survival was 92.2% for stromal invasion <4 mm versus 73.2% for stromal invasion ≥4 mm.
A surgical margin of >8 mm is broadly considered to be oncologically safe. De Hullu10 observed that a surgical macroscopic margin of 10 mm corresponded with a microscopic margin of 8 mm in 50% of cases. Due to this discrepancy, most researchers recommend a surgical margin ≥20 mm.24–26 Palaia et al16 reported that the microscopic cut-off of 8 mm was obtained in 83% of cases if the macroscopic margin was 10 mm, in 91% if 15 mm, and in 98% if 20 mm. Moreover, in patients reported in that study with margins >8 mm (67%) no recurrence were observed. In contrast to these reports, Woelber et al14 did not observe an increase in the risk of relapse in patients with free margins <8 mm. In the present study, a free margin >15 mm correlated with a 10% mortality rate, >18 mm with a 9.1% mortality rate, and >20 mm with a 6.5% mortality rate. When considering extending the tumor resection margins one should highlight the associated higher surgical morbidity, especially in cases where the tumor is close to the clitoris, urethra, or perianal region. Therefore, a more radical surgical approach that aims to achieve higher oncologic safety should be carefully balanced with a higher surgical morbidity and potential impairment of the patient’s quality of life.
The worse prognosis was seen in patients with bilateral lymph node involvement, and this could be explained by the higher number of total positive nodes regardless of the bilaterality.26 Our low rate of contralateral lymph node metastasis (0.7%) could indicate that contralateral lymphadenectomy is not indicated in the absence of ipsilateral metastasis. Although prospective studies reported the feasibility and reliability of sentinel node biopsy in vulvar carcinoma,27 the findings in our study showed that the majority of surgeons did not routinely use sentinel node biopsy for the management of squamous cell vulvar cancers; however, one must consider that this trend may have changed during the last 5 years. The 5-year overall survival rate in our study was 61.4% and 5-year disease-free survival was 48.1% across all tumor stages, both of which are comparable with earlier findings.18 25
The current study is one of the largest studies evaluating patients with vulvar cancer and correlating prognostic indicators with oncologic outcomes. In addition, the study represents outcomes from numerous institutions in Europe. However, we do recognize a number of limitations that include the retrospective study design, which introduced variations in data collection, auditing, and follow-up. There was also no central pathology review to confirm the diagnosis or the risk factors. Lastly, there was no information provided on treatment complications.
In conclusion, the most important predictors of recurrence in patients with vulvar cancer were number of positive nodes and not undergoing radiotherapy or chemotherapy. Factors that impacted overall survival were the case volume at each center, number of positive nodes, FIGO stage, treatment with chemotherapy, surgical margins, and stromal invasion >5 mm.
References
Footnotes
Correction notice This article has been corrected since it was first published. There were typographical errors in Tables 2 and 3; the third and fourth columns in Tables 2 and 3 were unintentionally switched, so the hazard ratios and confidence intervals from Table 2 corresponded to Table 3; and those from Table 3 corresponded to Table 2. This has now been corrected.
Collaborators Arones M (Complejo Hospitalario Universitario de Albacete, Spain), Arque M (Hospital de Santa Creu i Sant Pau, Spain), Barbara Gardella B (Fondazione IRCCS Policlinico San Matteo, Pavia, Italy), Bartusevicius A (Lithuanian University of Health Sciences, Kaunas, Lithuania), Bernal MT (Hospital de Donostia, San Sebastian, Spain), Blecharz P (Maria Sklodowska-Curie Memorial Cancer Center, Krakow, Poland), Ceccaroni M (Sacred Heart Hospital Negrar, Verona, Italy), Covo-Pinto L (Hospital Universitario Marques de Valdecilla, Santander, Spain), Cristobal I (Hospital Sanitas La Zarzuela, Madrid, Spain), Cruz JL (Hospital de la Ribera, Spain), De la Torre J (Hospital Universitario de Canarias, Spain), De Iaco P (Policlinico S. Orsola-Malpighi, Bologna, Italy), Diaz M (Complejo Hospitalario Universitario Juan Canalejo, La Coruña, Spain), Diaz-De la Noval B (Hospital Universitario Central de Asturias, Spain), DiFiore H (Fundacion Jimenez Diaz, Madrid, Spain), Dogan A (Ege Gynaecology Training and Research Hospital, Turkey), Etxabe I (Instituto Oncologico de Gipuzkoa, Spain), Fargas F (Instituto Universitario Dexeus, Barcelona, Spain), Franco S (Hospital Vall D'Hebron, Barcelona, Spain), Feijoo L (Hospital Virgen de la Concha, Zamora, Spain), Fernandez A (Hospital Costa del Sol, Marbella, Spain), Fernandez L (Hospital de Cabueñes, Gijon, Spain), Fernandez M (Complejo Hospitalario de Pontevedra, Spain), Festi A (Policlinico G.B. Rossi, Verona, Italy), Garcia-Casals C, (Complejo Hospitalario Juan Canalejo, La Coruña, Spain), Garrido RA (Complejo Hospitalario de Ciudad Real, Spain), Gil-Ibañez B (Corporacion Sanitaria Parc Tauli, Spain), Gil-Moreno A (Hospital Vall D'Hebron, Barcelona, Spain), Gines A (Hospital Universitario Puerto Real, Spain), Gomez AI (Complejo Asistencial de Segovia, Spain), Gomez I (Hospital Virgen del Camino, Pamplona, Spain), Gonçalves E (Unidade Local Saude Alto Minho, Portugal), Gonzalez L (Hospital Universitario Marques de Valdecilla, Santander, Spain),Grane N (Hospital General de Vic, Spain), Grigoriadis C (University of Athens, Aretaieion Hospital, Greece), Gutierrez L (Hospital Universitario Nuestra Señora de Valme, Spain), Haidopoulos D (Alexandra Hospital, Athens, Greece), Herraiz N (Hospital Clinico Universitario Lozano Blesa, Zaragoza, Spain), Irslinger E (UFK Tuebingen, Germany), Iyibozkurt A (Istanbul Faculty of Medicine, Istanbul University, Turkey), Joigneau L (Hospital de Fuenlabrada, Madrid, Spain), Karlsson H (Hospital Virgen del Camino, Pamplona, Spain), Kondi-Pafiti A (Aretaieion University Hospital, Athens, Greece), Mardas M (Poznan University of Medical Sciences, Poland), Marino M (Complejo Hospitalario de Caceres, Spain), Martinez A (Hospital de Sant Pau i Santa Tecla, Tarragona, Spain), Martinez C (Hospital Son Dureta, Mallorca, Spain), Martos MA (Hospital Universitario Puerta de Hierro-Majadahonda, Madrid, Spain), Menjon S (Hospital Universitario Virgen de las Nieves, Spain), Mitsopoulos V (Metaxa Memorial Cancer Hospital, Athens, Greece), Mora P (Hospital Clinico Universitario, Valladolid, Spain), Morales S (Hospital de Poniente, Spain), Moreno A (Hospital de la Linea de la Concepción, Spain), Nieto A (Complejo Hospitalario de Caceres, Spain), Novo A (Complejo Hospitalario Santiago de Compostela, Spain), Oehler M (Royal Adelaide Hospital, Adelaide), Otero M (Complejo Hospitalario de Leon, Spain), Papatheodorou DC, (Metaxa Memorial Cancer Hospital, Athens, Greece), Perez I (Fundacion Jimenez Diaz, Madrid, Spain), Piek J (VU University Medical Center, Amsterdam, The Netherlands), Polterauer S (Medical University of Vienna, Austria), Prado A (Hospital Costa del Sol, Marbella, Spain), Reula MC (Hospital de Santa Creu i Sant Pau, Spain), Robles M (Complejo Hospitalario de Pontevedra, Spain), Romeo M (Hospital Universitario Germans Trias i Pujol, Spain), Rosado C (Hospital de Mataro, Barcelona, Spain), Rubio P (Hospital Universitario Miguel Servet, Zaragoza, Spain), Rzepka J (MSC Memorial Cancer Center Warsaw, Poland), Sanz R (Fundacion Jimenez Diaz, Madrid, Spain), Sehouli J (Charité, Campus Virchow Clinic, University Hospital, Berlin, Germany), Soler C (Hospital de Santa Creu i Sant Pau, Spain), Sukhin V (Grigoriev Institute of Medical Radiology, Ukraine), Tsolakidis D (General Hospital Papageorgiou, Greece), Vieira-Baptista P (Hospital de Sao Joao, Portugal), Yildirim Y (Ege Gynaecology Training and Research Hospital, Izmir, Turkey), Zuñiga MA (Complejo Hospitalario Torrecardenas, Almeria, Spain).
Contributors This publication does not include any figures or tables from any other publications.
Funding The Spanish Society of Obstetrics and Gynecology partially funded the online database used for data collection.
Disclaimer No other funding agency or sponsor played a role in the design or performance of the study, including the collection, management, analysis, and interpretation of the data, and preparation, review, and approval of the report.
Competing interests None declared.
Patient consent for publication Not required.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in the article or uploaded as supplementary information.