Abstract
To evaluate the clinical significance of lncRNAs in the resistance to cisplatin‐based chemoradiotherapy in esophageal squamous cell carcinoma (ESCC). We focused on lncRNAs which were frequently reported in ESCC or were involved in chemoradiotherapy resistance. LncRNA expressions were examined in paired cisplatin‐resistant and parental ESCC cell lines. Dysregulated lncRNAs were further measured in 162 pretreatment biopsy specimens of ESCC who received definitive chemoradiotherapy (dCRT). Then the correlations between lncRNA expression and response to dCRT and prognosis were analyzed. Three lncRNAs (AFAP1‐AS1, UCA1, HOTAIR) were found to be deregulated in cisplatin‐resistant cells compared with their parent cells. AFAP1‐AS1 was significantly up‐regulated in tumor tissues compared with adjacent normal tissues (P = 0.006). Furthermore, overexpression of AFAP1‐AS1 was closely associated with lymph node metastasis (P < 0.001), distant metastasis (P = 0.016), advanced clinical stage (P = 0.002), and response to dCRT (P < 0.001). Kaplan–Meier survival analysis revealed that high expression of AFAP1‐AS1 was significantly associated with shorter progression free survival (PFS) (median, 15 months vs. 27 months, P < 0.001) and overall survival (OS) (median, 29 months vs. 42 months, P < 0.001). In the multivariate analysis, high expression of AFAP1‐AS1 was found to be an independent risk factor to predict poor PFS (HR, 1.626; P = 0.027) and OS (HR, 1.888; P = 0.004). Thus, high expression of AFAP1‐AS1 could serve as a potential biomarker to predict tumor response and survival. Determination of this lncRNA expression might be useful for selection ESCC patients for dCRT. © 2016 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.
Keywords: esophageal neoplasms, long non‐coding RNA, AFAP1‐AS1, chemoradiotherapy
Abbreviations
- lncRNA
long non‐coding RNA
- ESCC
esophageal squamous cell carcinoma
- dCRT
definitive chemoradiotherapy
- OS
overall survival
- PFS
progression free survival
- qRT‐PCR
quantitative real‐time polymerase chain reaction
- TNM
tumor‐node‐metastasis
- 5‐FU
5‐fluorouracial
- PTX
paclitaxel
- CR
complete response
- PR
partial response
- PD
progressive disease
- NC
no changed
- IC50
50% inhibition concentration
- ROC
receiver operating characteristic curve
- AUC
area under the ROC curve
INTRODUCTION
Esophageal cancer is the eighth most common cancer and the sixth leading cause of cancer‐related death worldwide 1. Esophageal squamous cell carcinoma (ESCC) remains the predominant histological type of esophageal cancer in East Asia and China 2. Recently, despite remarkable improvements have been made in ESCC treatment and diagnosis, advanced stages of the disease are still difficult to manage 3. This malignancy is usually diagnosed at a locally advanced stage with obvious enlargement node, long lesion, and/or serious esophageal invasion 4. For these patients, the current standard treatment is surgical resection or concurrent definitive chemoradiotherapy (dCRT), or a combination of both 5, 6. Complete response (CR): defined by clinical disappearance of tumor, is used as indicator good response to dCRT. However, the combination of radiotherapy and concurrent chemotherapy has led to long‐term survival in only 25% of patients. 7. Thus, the emergence of chemoradiotherapy resistance is one of major obstacle in the management of ESCC patients. Therefore, the significance of detecting predictive biomarkers of therapeutic response should be emphasized.
A new insight into cancer pathogenesis emerged with the discovery of long non‐coding RNAs (lncRNAs), which are longer than 200 nt with no protein‐coding abilities but regulate expression of protein‐coding genes 8. To date, thousands of lncRNAs have been identified to have functional roles in a diverse range of cellular processes such as development, cell growth and apoptosis, and cancer metastasis 9. In addition, mounting evidence indicates that lncRNAs are frequently aberrant expression in numerous cancer types and some of them have been implicated in diagnosis and prognostication 10. Examples include lncRNA MALAT1 in prostate cancer, MVIH in hepatocellular carcinoma and FENDRR in gastric cancer, suggesting that lncRNAs could serve as a diagnostic and prognostic biomarkers for human malignancies 11, 12, 13. Currently, the mechanisms underlying resistance development to chemotherapeutic agents are still not fully understood. Recently, several studies have suggested that lncRNAs are likely to play crucial roles in the development of chemotherapy resistance in cancer 14. For example, W.P. Tsang demonstrated that lncRNA H19 could induce P‐glycoprotein expression and MDR‐1 associated drug resistance in liver cancer cells through regulation of MDR‐1 promoter methylation 15. Considering their critical roles in cancer, we hypothesized that the expression levels of lncRNAs in tumor could be associated with chemoradiotherapy resistance in patients with ESCC.
To test the hypothesis, 18 lncRNAs, which were frequently reported in esophageal cancer 16, 17, 18, 19, 20, 21, 22, 23, 24 or were involved in chemoradiotherapy resistance 8, 14, 15, 25, 26, 27, 28, 29, 30, were selected as candidates. They were examined in cisplatin resistant ESCC cell lines and patients treated with dCRT. Subsequently, the correlation between lncRNA (particularly lncRNA AFAP1‐AS1) expressions and patient clinical/prognostic factors were assessed to determine whether lncRNA expressions have predictive value of dCRT response and clinical outcome in patients with ESCC.
MATERIALS AND METHODS
Criteria for reporting recommendations for tumor markers in prognosis study (REMARK) were followed wherever possible.
Patient Information and Tissue Specimens
A total of 204 ESCC patients treated with dCRT between January 2008 and December 2009 in our hospital (Huai'an First Hospital, Nanjing Medical University, Jiangsu, China) were collected in the present study for survival analysis. Tumor tissue specimens and the matched normal esophageal mucosa tissues were obtained from patients by endoscopy before dCRT. And another 48 fresh ESCC tissues and paired adjacent normal tissues were obtained from patients undergoing surgery at Department of Thoracic Surgery between January 1 and May 30, 2014. These tissues were selected for qRT‐PCR analysis. All cases selected were based on the following criteria: histologically confirmed primary ESCC by available biopsy specimens; no previous local or systemic treatment; age less than 75 years; karnofsky≥70; adequate bone marrow, renal, pulmonary, and hepatic function; no significant medical disease. All biopsy tissue samples were snap frozen in liquid nitrogen and then stored at −80°C until RNA extraction. Tumor staging were determined according to the sixth edition of tumor‐node‐metastasis (TNM) classification for esophageal carcinoma (UICC, 2002).
The study was approved by Research Ethics Committee, Nanjing Medical University Huai'an First Hospital, and written informed consents were obtained from all patients.
Definitive Chemoradiotherapy
All the 204 patients treated with the same dCRT which included 5‐fluorouracial and cisplatin (FP) based regimens. Specifically, cisplatin was administered at 80 mg/m2 by intravenous infusion on day 1; and 5‐fluorouracial (5‐FU) 1000 mg/m2 was administered by continuous infusion for 24 h on days 1–4. Two courses of chemotherapy were used during radiotherapy at 4‐week intervals. Radiotherapy was initiated on day 1 of chemotherapy. All patients received external beam radiotherapy using 6 or 15Me LINAC (Siemens ONCOR). A total radiation dose of 60–70 Gy (1.8–2.0 Gy per day, 5 days per week) was delivered with 3‐ or 4‐field technique.
Clinical Response Evaluation and Follow‐Up
Four weeks after completion of dCRT, tumor responses to chemoradiotherapy were evaluated through endoscopy and CT scan. Briefly, the clinical responses were categorized as follows: complete response (CR) was defined as total regression of all assessable lesions; partial response (PR) was defined as more than 50% reduction in primary tumor size or more of the sum of the lesions and no progression of assessable lesions; progressive disease (PD) was defined as more than a 25% increase in primary tumor volume or appearance of new lesions; the remaining patients which did not meet the criteria of PD or PR were categorized as no changed (NC). Patients who were evaluated as CR and PR were included in the effective group; and the remaining patients were designated as the resistant group (NC + PD).
Post‐treatment follow‐up was performed 1 month after dCRT, followed by every 3 month during the first year, and then every 6 month for the second year, and finally annually until 5 yr after treatment.
Cell Lines and Cell Culture
The normal esophageal epithelial cell (Het‐1A) and human ESCC cells (KYSE30, KYSE70, KYSE150, KYSE450, KYSE510, and TE10) were maintained in RPMI‐1640 medium (Invitrogen, Carlsbad CA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY) at 37°C with 5% CO2. All cells were kind gifts from Prof. Zhi‐hua Liu (the State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China).
Establishment of Cisplatin‐Resistant Cell Lines
A cisplatin‐resistant ESCC cell line, KYSE30‐R, was established from KYSE30 cell line by exposure to gradually increasing concentrations of cisplatin (from 0.2 to 10 µmol/L) over a period of 5 months. Briefly, KYSE30 was exposed to an initial cisplatin concentration of 0.2 µmol/L in RPMI‐1640 plus 10% FBS. After 48 h, the treated cells were then washed three times with phosphate buffered saline and cultured in cisplatin‐free medium. Upon reaching of 70–80% confluence, the cells were grown in a higher drug concentration (10–20% increase per passage). The above treatment was then repeated until it reached a concentration of 10 µmol/L. We established cisplatin–resistant ESCC cell line in KYSE30, because the expressions of the majority of the lncRNAs were lower in KYSE30 when compared with other ESCC cell lines.
MTT Assay
The MTT assay was used to calculate the 50% inhibition concentration (IC50) for cisplatin and other anticancer agents. Briefly, cells (5 × 103/well) in 100 µl RPMI‐1640 with 10% FBS were plated into 96‐well plates in quadruplicate. After incubation overnight, they were treated with cisplatin at a concentration range of 0.3125–50 µmol/L (0.3125, 0.625, 1.25, 2.5, 5, 10, 25, and 50 µmol/L) for 6 h. Then the medium was removed, and 100 µl cisplatin‐free medium was added. After an additional 48 h, MTT solution (10 µl/well) was added, and the plate was incubated for 4 h. The blue dye taken up by cells was dissolved with dimethyl sulfoxide (100 µl /well), and the absorbance at 490 nm was measured using a microplate reader (Bio‐Rad Laboratories, California, USA). The IC50 of each anticancer drug was estimated by the dose–response curve.
Total RNA Extraction, Reverse Transcription and Quantitative Real‐Time PCR
Total RNA extraction was performed using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. RNA concentration and purity was assessed by measuring absorption (A260/A280) on spectrophotometry. Only samples with an A260/A280 ratio between 1.8 and 2.1 were considered for further experiments.
The reverse transcription reaction was performed with PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Dalian, China) in a 20 µl reaction volume.
SYBR® Premix Ex Tag™ II (Takara, Dalian, China) was used to examine the expression level of lncRNAs in tissue samples and cultured cells. All reactions were examined in triplicate and the specificity of each PCR reaction was confirmed by melt curve analyses. The expression levels of lncRNAs were calculated using △Ct method, where △Ct = Cttarget–Ctreference, smaller △Ct value indicates higher expression. Relative expression of lncRNAs was analyzed using 2−ΔΔCT method with GAPDH as the endogenous control to normalize the data. The primers used in this study are listed in Supplementary Table S1.
Statistical Analysis
The statistical significance of tissue and cell lncRNA levels between cancer and normal group was analyzed by Mann–Whitney or Student's t‐test. The Shapiro–Wilk test was used to verify if AFAP1‐AS1 expression follows a normal distribution. Correlations between AFAP1‐AS1 expression and various clinicopathological factors were evaluated by the χ2 test. Overall survival (OS) and progression free survival (PFS) were analyzed with the Kaplan–Meier method. OS and PFS were defined as the time from start of treatment to death and tumor progression. The cox proportional hazards regression model was used to test the prognostic values of clinical and biological variables. Statistical analyses were performed using SPSS software (version 20.0). All tests were two‐sided and P‐value less than 0.05 was considered significant.
RESULTS
Patient Characteristics
Of the 204 patients collected for survival analysis, 42 patients were excluded from the present study for the following reasons: lost to follow‐up (28 patients); fail to complete the dCRT (9 patients), and previous history of cancer (5 patients). Consequently, a total of 162 patients were selected for further investigation.
Of the 162 eligible patients, clinical stage included: stage I in 26 cases, stage II in 53 cases, stage III in 52 cases, and stage IV in 31 cases. The detailed information was listed in Table 1.
Table 1.
lncRNA AFAP1‐AS1 Expression and Clinicopathologic Characteristics
| a AFAP1‐AS1 expression (%) | Chi‐squared test | ||
|---|---|---|---|
| Characteristics | High | Low | P‐value |
| Age (year) | 0.641 | ||
| ≤55 | 9 (11.1) | 12 (14.8) | |
| >55 | 72 (88.9) | 69 (85.2) | |
| Gender | 0.463 | ||
| Male | 64 (79.0) | 59 (72.8) | |
| Female | 17 (21.0) | 22 (27.2) | |
| Tumor location | 1.000 | ||
| Proximal third | 7 (8.6) | 8 (9.9) | |
| Middle/distal third | 74 (91.4) | 73 (90.1) | |
| Tobacco use | 0.107 | ||
| Never | 44 (54.3) | 55 (67.9) | |
| Ever | 37 (45.7) | 26 (32.1) | |
| Alcohol use | 1.000 | ||
| Never | 60 (74.1) | 61 (75.3) | |
| Ever | 21 (25.9) | 20 (24.7) | |
| Primary tumor length (cm) | 0.738 | ||
| ≤5 | 53 (65.4) | 56 (69.1) | |
| >5 | 28 (24.6) | 25 (30.9) | |
| Histological differentiation | 0.890 | ||
| Well | 9 (11.1) | 11 (13.6) | |
| Moderate | 49 (60.5) | 48 (59.3) | |
| Poor | 23 (28.4) | 22 (27.1) | |
| Tumor depth | 0.185 | ||
| T1/T2 | 14 (17.2) | 22 (27.1) | |
| T3/T4 | 67 (82.8) | 59 (72.9) | |
| Lymph node metastasis | <0.001 | ||
| Node negative | 26 (32.1) | 49 (60.5) | |
| Node positive | 55 (67.9) | 32 (39.5) | |
| Distant metastasis | 0.016 | ||
| M0 | 59 (72.8) | 72 (88.9) | |
| b M1 | 22 (27.2) | 9 (11.1) | |
| TNM stage | 0.002 | ||
| I | 8 (9.9) | 18 (22.2) | |
| II | 20 (24.7) | 33 (40.8) | |
| III | 31 (38.3) | 21 (25.9) | |
| IV | 22 (27.1) | 9 (11.1) | |
| dCRT response | <0.001 | ||
| Effective (CR + PR) | 30 (37.0) | 68 (84.0) | |
| Resistant (CD + PD) | 51 (63.0) | 13 (16.0) | |
CR, complete response; PR, partial response; PD, progressive disease; NC, no changed.
Median expression level was used as a cut‐off to divide the 162 patients into AFAP1‐AS1‐high (n = 81) and AFAP1‐AS1‐low group (n = 81).
M1, there were 15 patients with cervical node metastasis, 9 patients with abdominal nodes, and 7 with metastasis in both nodes.
At the evaluation time, CR was achieved in 32 cases (19.8%), PR in 66 cases (40.7%), NC in 61 cases (37.7%), and PD in 3 cases (1.8%), respectively. After dCRT, 7 patients underwent esophagectomy, and 41 patients received adjuvant chemotherapy.
With a median follow‐up of 31 months (range 6–72 months), we identified 118 (73%) tumor progression and 122 (75%) deaths. Among the 122 dead patients, 110 (90%) died from tumor recurrence or distant metastasis, and 12 (10%) from other causes (three from heart disease, two from cirrhosis, two from kidney failure, two from bacterial pneumonia, two from second primary tumor, and one from cerebral hemorrhage). The 3‐ and 5‐yr OS rate was 48.1% and 24.7%, respectively.
lncRNA Expression Is Altered in Cisplatin‐Resistant ECSS Cell Lines
On the basis of previous studies, 18 lncRNAs (Table 2), which were frequently reported in esophageal cancer or were involved in chemoradiotherapy resistance, were selected in this study. To determine whether these lncRNAs are involved in the development of resistance to cisplatin in ESCC cells, we used qRT‐PCR to examine the expressions of the 18 lncRNAs in cisplatin–resistant ESCC cell line KYSE30‐R and its parental cell line KYSE30. The former was able to tolerate much higher concentrations (14‐fold) of cisplatin than its parental cell line (Table 3 and Figure 1). The IC50 values for KYSE30 and KYSE30‐R were 0.934 and 13.063 µmol/L, respectively. Simultaneously, KYSE30‐R also exhibited cross‐resistance to 5‐FU (∼threefold) and paclitaxel (∼fivefold) (Table 3 and Figure 1), two anticancer drugs that are widely used in combination with cisplatin for the treatment of ESCC. It was shown that the expressions of the majority of the lncRNAs remained unchanged (Table 2), three lncRNAs (AFAP1‐AS1, UCAT‐1, HOTAIR) were deregulated more than twofold in the paired cell lines (Figure 2A). To further identify changes in lncRNAs expression associated with cisplatin resistance, another pair of parental and cisplatin‐resistant ESCC cell line models (TE10 and T E10‐R) was also evaluated. As shown in Figure 2B, a similar result was also observed between TE10‐R and TE10. Of the three lncRNAs, AFAP1‐AS1 was chosen for further investigation because we found that its expression was up‐regulated in patients with local recurrence after dCRT (P = 0.009, Figure 2C).
Table 2.
List of lncRNAs Expression in KYSE30‐R Versus KYSE30 Cell
| lncRNA | Average fold change |
|---|---|
| AFAP1‐AS1 | 2.75 |
| UCA‐1 | 3.03 |
| HOTAIR | 2.23 |
| POU3F3 | 1.17 |
| HNF1A‐AS1 | 1.14 |
| SPRY4‐IT1 | 0.96 |
| PlncRNA1 | 1.21 |
| ENST00000435885.1 | 1.05 |
| ENST00000547963.1 | 1.12 |
| XLOC_013104 | 1.24 |
| 91H | 0.97 |
| LOC285194 | 0.92 |
| ARA | 1.31 |
| CCAT2 | 1.26 |
| AC006050.3‐003 | 1.19 |
| GAS5 | 1.22 |
| H19 | 1.09 |
| AK294004 | 1.21 |
Average fold was calculated from the delta‐delta Ct value (KYSE30‐R cell vs. KYSE30 cell) of 3 expts.
Table 3.
Multidrug Resistant Phenotype of KYSE30‐R Cells Compared to Its Parental KYSE30 Cells
| IC50 values (mean ± SD, µM) a | |||
|---|---|---|---|
| Drugs | KYSE30 | KYSE30‐R | * P‐value |
| Cisplatin | 0.934 ± 0.0277 | 13.063 ± 0.395 | <0.001 |
| 5‐FU | 10.717 ± 0.476 | 27.307 ± 0.272 | <0.001 |
| PTX | 0.217 ± 0.029 | 1.099 ± 0.089 | =0.001 |
IC50, 50% inhibition concentration; 5‐FU, 5‐fluorouracil; PTX, paclitaxel.
IC50 values were calculated by MTT assays as described in the materials and methods.
P‐value was determined by Student's t‐test.
Figure 1.
Dose response curves of KYSE30 and KYSE30‐R cells to cisplatin, 5‐fuorouracil (5‐fu), and paclitaxel (PTX). Cell viability was evaluated by MTT assay. The IC50 values to each drugs were calculated by SPSS 20.0. KYSE30‐R cells were more resistant to cisplatin (mean IC50, 13.063 vs. 0.934 µM, A), 5‐fu (mean IC50, 27.307 versus 10.717 µM, B), and PTX (mean IC50, 1.099 vs. 0.217 µM, C) than KYSE30 cells. Data are expressed as the mean ± SD.
Figure 2.
Cisplatin treatment increases lncRNA AFAP1‐AS1, UCA1, and HOTAIR expression. The expression levels of the three lncRNAs were significantly increased in cisplatin‐resistant sublines KYSE30‐R (A) and TE10‐R (B) compared with their parent cells. Data are presented mean ± SD. The Student's t‐test was used to compared the gene expression between the parental and cisplatin‐resistant cell lines (*P < 0.05). (C) AFAP1‐AS1 expression was up‐regulated in patients with local recurrence after definitive chemoradiotherapy (n = 20, P = 0.009). Statistical differences were analyzed using paired t test.
AFAP1‐AS1 Is Overexpressed in ESCC
Previous study has shown that AFAP1‐AS1 is overexpressed in barrett' esophagus and esophageal adenocarcinoma. In this study, We measured the expression levels of AFAP1‐AS1 by qRT‐PCR in 48 pairs ESCC samples and adjacent normal tissues (The detailed information was listed in Suppl. Table S2), and detected significantly higher expression of AFAP1‐AS1 in tumor specimens (39/48, 81%) compared to normal specimens (P = 0.006, Figure 3A). Moreover, we also examined the levels of AFAP1‐AS1 in ESCC cell lines, including KYSE30, KYSE70, KYSE150, KYSE450, KYSE510, TE10 cells and normal esophageal mucosa cell Het‐1A. AFAP1‐AS1 was up‐regulated in all of the six analyzed ESCC cell lines by 2.2‐ to 15‐fold (Figure 3B).
Figure 3.
qRT‐PCR analysis of lncRNA AFAP1‐AS1 expression in ESCC. (A) lncRNA AFAP1‐AS1 was significantly up‐regulated in 48 ESCC tumor samples compared with corresponding normal esophageal mucosa tissues (P = 0.006). △Ct method was used to measure the AFAP1‐AS1 expression, which was normalized to GAPDH. Smaller △Ct value indicates higher expression. Horizontal bars indicate median and interquartile range. The Student's t‐test was used to determine the significance of differences between groups. (B) AFAP1‐AS1 was up‐regulated in all of the six analyzed ESCC cell lines compared with normal esophageal mucosa cell Het‐1A. All data analyzed using Student's t‐test. *Significantly different from control (P < 0.05).
Correlation Between AFAP1‐AS1 Expression and Clinicopathological Variables
In light of these findings, we then determined whether there was a correlation between AFAP1‐AS1 expression and clinicopathological characteristics. We examined AFAP1‐AS1 expression in cancer tissues from another 162 ESCC patients independent of the 48 ESCC patients from cohort 1. According to the median value of relative AFAP1‐AS1 expression level (2.6‐fold, tumor/noncancerous tissues), the 162 patients were divided into two groups: AFAP1‐AS1‐high group (≥median, n = 81) and AFAP1‐AS1‐low group (<median, n = 81). As shown in Table 1, AFAP1‐AS1 up‐regulation was closely associated lymph node metastasis (P < 0.001), distant metastasis (P = 0.016), advanced clinical stage (P = 0.002), and lack of response to dCRT (P < 0.001, Table 1). However, there was no significant correlation between AFAP1‐AS1 expression and other clinicopathological variables (P > 0.05, Table 1).
Diagnostic Utility of AFAP1‐AS1
Analysis of the levels of AFAP1‐AS1 shows that it could be utilized to distinguish tumor samples from normal esophageal mucosa (Figure 3A). We therefore examined the diagnostic performance of AFAP1‐AS1. As shown in Figure 4A, AFAP1‐AS1 yield an area under curve (AUC) of 0.802 (95%CI: 0.765–849; P < 0.001) with 79.4% specificity and 73.3% sensitivity for distinguishing ESCC samples from normal esophageal mucosa.
Figure 4.
Tissue AFAP1‐AS1 may be a potential biomarker of ESCC. (A) AFAP1‐AS1 can clearly distinguish tumor samples from normal esophageal mucosa. The value of area under curve (AUC) to detect ESCC was 0.802 (P < 0.001). (B) AFAP1‐AS1 expression in patients with early ESCC (stage I + II, n = 79) was significantly higher than those of paired normal tissues (P < 0.001). AFAP1‐AS1 expression levels were calculated by △Ct method, and smaller ΔCt value indicates higher expression. Horizontal lines inside the box plots represent the median, boxes represent the interquartile range, and error bars represent 97.5th and 2.5th percentiles. Statistical differences were analyzed using Student's t‐test. (C) The value of AUC to detect early ESCC was 0.803.
Next, to explore the potential role of this lncRNA as a marker for early detection of ESCC, the AFAP1‐AS1 expression levels in tumor samples from early ESCC (stage I + II, n = 79) and paired normal tissues were then analyzed. As shown in Figure 4B, AFAP1‐AS1 expression in patients with early ESCC was significantly higher than those of paired normal tissues (P < 0.001). Figure 4C shows the diagnostic power of AFAP1‐AS1, the value of AUC used to detect early ESCC was 0.803 (95%CI, 0.735–870; P < 0.001). The diagnostic sensitivity and specificity were 44.6% and 92.3%. Our data provided evidence that AFAP1‐AS1 has great potential as a biomarker for early detection of ESCC.
Correlation Between Clinicopathological Parameters, AFAP1‐AS1 Expression and dCRT Response
Patients who were AFAP1‐AS1‐high on pretreatment cancer biopsies (n = 81), CR was achieved in 6 cases (7.4%), PR in 24 cases (29.6%), NC in 48 cases (59.3%), and PD in 3 cases (3.7%), respectively. In contrast, of the 81 patients with AFAP1‐AS1‐low expression, CR, PR, NC, and PD was achieved in 26 cases (32.1%), 42 cases (51.9%), 13 cases (16.0%), 0 cases (0%), respectively. AFAP1‐AS1 expression was the only factor that showed a significant association with CRT response (P < 0.001, Table 1). Unexpectedly, no clinicopathological parameter was detected to be correlated with dCRT response (P > 0.05, Suppl. Table S3).
AFAP1‐AS1 Expression and Clinical Outcomes
From Kaplan–Meier survival analysis, we found that high expression of AFAP1‐AS1 was significantly correlated with shorter PFS (P < 0.001, Figure 5A). The median PFS for patients with AFAP1‐AS1‐high tumor was 15 months compared with 27 months for patients with AFAP1‐AS1‐low expression. On univariate analysis, factor associated with PFS were: tumor depth (P = 0.005), lymphatic metastasis (P = 0.014), TNM stage (P = 0.008), dCRT response (P < 0.001), and AFAP1‐AS1 expression (P < 0.001), (Table 4). After adjustment for tumor depth, lymphatic metastasis, distant metastasis, TNM stage, and dCRT response, AFAP1‐AS1 expression remained an independent prognostic factor of PFS (HR, 1.626; 95%CI, 1.057–2.501; P = 0.027, Table 5). Furthermore, our multivariate analysis showed that tumor depth (HR, 1.942; 95%CI, 1.066–3.535; P = 0.030) and dCRT response (HR, 1.744; 95%CI, 1.160–2.624; P = 0.008) were also significantly independent prognostic factors for PFS (Table 5).
Figure 5.
Kaplan–Meier survival analysis of progression free survival (A) and overall survival (B) based on AFAP1‐AS1 expression in all 162 ESCC patients treated with definitive chemoradiotherapy (P < 0.001).
Table 4.
Univariate Cox Regression Analysis of PFS and OS
| PFS | OS | ||||||
|---|---|---|---|---|---|---|---|
| Prognostic factors | Case | HR | 95%CI | *P‐value | HR | 95%CI | * P‐value |
| Age (year) | 1.062 | 0.618–1.827 | 0.827 | 1.288 | 0.703–2.145 | 0.470 | |
| ≤55 | 21 | ||||||
| >55 | 141 | ||||||
| Gender | 0.925 | 0.602–1.421 | 0.721 | 0.936 | 0.616–1.422 | 0.756 | |
| Male | 123 | ||||||
| Female | 39 | ||||||
| Tumor location | 1.033 | 0.555–1.922 | 0.918 | 0.901 | 0.484–1.675 | 0.741 | |
| Proximal third | 15 | ||||||
| Middle/distal third | 147 | ||||||
| Tobacco use | 1.144 | 0.791–1.653 | 0.476 | 1.155 | 0.801–1.664 | 0.441 | |
| Never | 99 | ||||||
| Ever | 63 | ||||||
| Alcohol use | 0.886 | 0.583–1.348 | 0.572 | 0.988 | 0.654–1.494 | 0.954 | |
| Never | 121 | ||||||
| Ever | 41 | ||||||
| Primary tumor length (cm) | 1.051 | 0.716–1.542 | 0.801 | 1.234 | 0.846–1.800 | 0.276 | |
| ≤5 | 109 | ||||||
| >5 | 53 | ||||||
| Histological differentiation | 1.188 | 0.882–1.600 | 0.257 | 1.137 | 0.848–1.523 | 0.391 | |
| Well | 20 | ||||||
| Moderate | 97 | ||||||
| Poor | 45 | ||||||
| Tumor depth | 2.052 | 1.240–3.395 | 0.005 | 1.881 | 1.150–3.075 | 0.012 | |
| T1/T2 | 36 | ||||||
| T3/T4 | 126 | ||||||
| Lymph node metastasis | 1.582 | 1.096–2.285 | 0.014 | 1.779 | 1.231–2.571 | 0.002 | |
| Node negative | 75 | ||||||
| Node positive | 87 | ||||||
| Distant metastasis | 1.325 | 0.839–2.092 | 0.227 | 1.567 | 1.007–2.437 | 0.046 | |
| M0 | 131 | ||||||
| M1 | 31 | ||||||
| TNM stage | 1.645 | 1.141–2.371 | 0.008 | 1.796 | 1.247–2.586 | 0.002 | |
| I + II | 79 | ||||||
| III + IV | 83 | ||||||
| dCRT response | 2.117 | 1.470–3.051 | <0.001 | 2.170 | 1.511–3.117 | <0.001 | |
| Effective (CR + PR) | 98 | ||||||
| Resistant (CD + PD) | 64 | ||||||
| Adjuvant chemotherapy | 0.704 | 0.472–1.050 | 0.085 | 0.834 | 0.560–1.242 | 0.371 | |
| Yes | 41 | ||||||
| No | 121 | ||||||
| Total radiotherapy dose (Gy) | 1.031 | 0.692–1.535 | 0.882 | 0.877 | 0.587–1.309 | 0.520 | |
| 60 | 116 | ||||||
| >60 | 46 | ||||||
| AFAP1‐AS1 expression | 2.242 | 1.545–3.255 | <0.001 | 2.665 | 1.838–3.865 | <0.001 | |
| Low | 81 | ||||||
| High | 81 | ||||||
PFS, progression free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval; CR, complete response; PR, partial response; PD, progressive disease; NC, no changed.
P log‐rank test.
Table 5.
Multivariate Cox Regression Analysis of PFS and OS
| PFS | OS | |||||
|---|---|---|---|---|---|---|
| Prognostic factors | HR | 95%CI | P‐value | HR | 95%CI | P‐value |
| Tumor depth (T1/T2 vs. T3/T4) | 1.942 | 1.066–3.535 | 0.030 | 1.661 | 0.919–3.003 | 0.093 |
| Lymphatic metastasis (No vs. Yes) | 1.474 | 0.632–3.438 | 0.370 | 1.413 | 0.587–3.401 | 0.441 |
| Distant metastasis (M0 vs. M1) | 1.039 | 0.622–1.736 | 0.883 | 1.257 | 0.758–2.084 | 0.375 |
| TNM stage (I + II vs. III + IV) | 0.728 | 0.291–1.824 | 0.499 | 0.843 | 0.326–2.174 | 0.723 |
| dCRT response (Effective vs. Resistant) | 1.744 | 1.160–2.624 | 0.008 | 1.673 | 1.103–2.538 | 0.015 |
| Expression of AFAP1‐AS1(Low vs. High) | 1.626 | 1.057–2.501 | 0.027 | 1.888 | 1.223–2.915 | 0.004 |
PFS, progression free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval.
Similarly, patients with higher AFAP1‐AS1 expression had a poorer OS (P < 0.001, Figure 5B). The median OS for patients with AFAP1‐AS1‐high tumor was 29 months, compared with 42 months for those patients who had AFAP1‐AS1‐low expression. In univariate analysis, factors associated with OS were: tumor depth (P = 0.012), lymphatic metastasis (P = 0.002), distant metastasis (P = 0.046), TNM stage (P = 0.002), dCRT response (P < 0.001)), and AFAP1‐AS1 expression (P < 0.001), (Table 4). All the six clinicopathological characteristics were further applied for the multiple analyses. The multivariate cox proportional hazard regression analysis indicated that high expression of AFAP1‐AS1 was the most significantly unfavorable prognostic factor of OS (HR, 1.888; 95%CI, 1.223–2.915; P = 0.004) followed by lack of dCRT response (HR, 1.672; 95%CI, 1.103–2.538; P = 0.015, Table 5).
DISCUSSION
Recent studies have shown that certain lncRNAs (e.g., PVT1, MEG3, and HOTAIR) are involved in the resistance to cytotoxic drugs and ionizing radiation 31, 32, 33. In the present study, we identified alterations in lncRNAs expression during the development of cisplatin resistance on KYSE30‐R and its parental cell line. Among the 18 lncRNAs investigated, AFAP1‐AS1, previously reported as having oncogenic roles in Barrett' esophagus and esophageal adenocarcinoma, was significantly increased in cisplatin‐treated KYSE30‐R cell line compared its parent cell line. Furthermore, we also observed that its expression was up‐regulated in patients with local recurrence after dCRT. AFAP1‐AS1 is an lncRNA which was extremely hypomethylated and overexpressed in esophageal adenocarcinoma. Its silencing by small interfering RNA inhibited proliferation, induced apoptosis, and reduced tumor cells migration and invasion 16. In view of these findings, AFAP1‐AS1 was chosen for further investigation.
Recently, many biomarkers such as p53, Bax, hMLH1, EZH2 have been evaluated as possible predictive factors of tumor response to dCRT 34, 35, 36, 37. However, several of these data obtained by different studies is conflicting, such reliable tumor markers are still currently lacking. In this study, we observed that high expression of AFAP1‐AS1 was significantly correlated with poor response to dCRT in patients with ESCC. Several lncRNAs are known to be correlated with chemoradiotherapy sensitivity phenotypes in cancers. For example, over‐expression of HOTAIR could decrease the sensitivity of lung adenocarcinoma cells to cisplatin through regulation of p21 expression 27. Fan Y and his colleagues demonstrated that lncRNA UCA1 was up‐regulated in patients with metastatic bladder cancer after cisplatin‐based chemotherapy, and over‐expression of UCA1 significantly increased cell viability in cisplatin treatment by regulating wnt signaling 28. To the best of our knowledge, the correlation between AFAP1‐AS1 expression and resistance to chemoradiotherapy for ESCC has not been analyzed previously. The present results revealed a close relationship between AFAP1‐AS1 expression and ESCC chemoradiotherapy response. In other words, patients with high expression of AFAP1‐AS1 suggested resistance to dCRT. These results suggested that down‐regulation of AFAP1‐AS1 expression may have a new therapeutic application for ESCC patients in the future.
With a cohort of 162 randomly selected ESCC patients, we discovered that the AFAP1‐AS1 expression level was significantly up‐regulated in tumor samples compared with adjacent normal tissues. ROC curve analysis further indicated that AFAP1‐AS1 might be a good tumor marker for ESCC diagnosis. In recent years, growing evidence has indicated that noncoding RNAs, predominantly lncRNAs, could distinguish tumor patients from normal controls. Many lncRNAs are expressed in tissue specific manner compared with protein‐coding RNAs and have shown the feasibility of using them as molecular markers for diagnosis of cancer 38. For example, the lncRNA AC096655.1‐002 was significantly down‐regulated in gastric cancer tissues compared with paired adjacent normal tissues, and use of this lncRNA alone provided a remarkable improvement in the diagnosis of gastric cancer compared with classic tumor marker serum carcinoembryonic antigen 39. In prostate cancer, PCA3/DD3 was found to be upregulated more than sixty times in tumor tissue compared with normal prostate tissue. Such a large difference expressions in tumor compared to normal sample make PCA3/DD3 a promising biomarker for prostate cancer diagnosis 40.
The most important finding of the current study was the prognostic value of AFAP1‐AS1 expression in ESCC. To date, clinical complete response to treatment and TNM stage have been reported as the most important predictors of outcomes for patients with ESCC treated with definitive chemoradiotherapy 41. However, our multivariate analysis demonstrated that high expression of AFAP1‐AS1 was the most significantly unfavorable prognostic factor of OS surpassing the advanced TNM stage and the lack of primary CR. Consistently, similar results were also observed in pancreatic ductal adenocarcinoma 42. In addition, we found that patients with higher AFAP1‐AS1 level had significantly poorer PFS after dCRT. Therefore, AFAP1‐AS1 expression could be used as an attractive biomarker, in addition to other clinical parameters, in identifying patients with ESCC who are at a higher risk of tumor progression.
Lastly, it is worth mentioning that there were several potential limitations in our study, First of all, the present study was limited to retrospective assessments with a relatively small sample size. Second, we confirmed that AFAP1‐AS1 expression could predict resistance to chemoradiotherapy in patients with ESCC. However, the exact mechanisms were still unclear. Further functional experiments are thus required to elucidate which signaling pathway is involved between the high expression of AFAP1‐AS1 and chemoradiotherapy sensitivity in ESCC upon cellular level.
CONCLUSION
Our results demonstrated that AFAP1‐AS1 could represent a novel predictive biomarker of clinical outcomes (poorer PFS and OS) for ESSC patients treated with dCRT, for which overexpression of this lncRNA will indicate that these patients will not benefit from FP‐based dCRT.
Supporting information
Additional supporting information may be found in the online version of this article at the publisher's web‐site.
Supporting Information.
ACKNOWLEDGMENTS
We are grateful to Juan Tan for the assistance of preparing and submitting the manuscript. This work was supported by National Nature Science Foundation of China (Grant No.H1617/81372656).
REFERENCES
- 1. Pennathur A, Gibson MK, Jobe BA, Luketich JD. Oesophageal carcinoma. Lancet 2013; 381:400–412. [DOI] [PubMed] [Google Scholar]
- 2. Rustgi AK, El‐Serag HB. Esophageal carcinoma. N Engl J Med 2014; 371:2499–2509. [DOI] [PubMed] [Google Scholar]
- 3. Kausar T, Ahsan A, Hasan MR, Lin L, Beer DG, Ralhan R. Sperm protein 17 is a novel marker for predicting cisplatin response in esophageal squamous cancer cell lines. Int J Cancer 2010; 126:1494–1503. [DOI] [PubMed] [Google Scholar]
- 4. Zhang X, Li M, Meng X, et al. Involved‐field irradiation in definitive chemoradiotherapy for locally advanced esophageal squamous cell carcinoma. Radiat Oncol 2014; 9:64. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Kim MK, Cho KJ, Kwon GY, et al. ERCC1 predicting chemoradiation resistance and poor outcome in oesophageal cancer. Eur J Cancer 2008; 44:54–60. [DOI] [PubMed] [Google Scholar]
- 6. Matsuda S, Tsubosa Y, Niihara M, et al. Comparison of transthoracic esophagectomy with definitive chemoradiotherapy as initial treatment for patients with esophageal squamous cell carcinoma who could tolerate transthoracic esophagectomy. Ann Surg Oncol 2015; 22:1866–1873. [DOI] [PubMed] [Google Scholar]
- 7. Enzinger PC, Mayer RJ. Esophageal cancer. N Engl J Med 2003; 349:2241–2252. [DOI] [PubMed] [Google Scholar]
- 8. Redis RS, Sieuwerts AM, Look MP, et al. CCAT2, a novel long non‐coding RNA in breast cancer expression study and clinical correlations. Oncotarget 2013; 4:1748–1762. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Liu Q, Huang J, Zhou N, et al. LncRNA loc285194 is a p53‐regulated tumor suppressor. Nucleic Acids Res 2013; 41:4976–4987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Qi P, Du X. The long non‐coding RNAs, a new cancer diagnostic and therapeutic gold mine. Mod Pathol 2012; 26:155–165. [DOI] [PubMed] [Google Scholar]
- 11. Ren S, Wang F, Shen J, et al. Long non‐coding RNA metastasis associated in lung adenocarcinoma transcript 1 derived miniRNA as a novel plasma‐based biomarker for diagnosing prostate cancer. Eur J Cancer 2013; 49:2949–2959. [DOI] [PubMed] [Google Scholar]
- 12. Yuan S‐X, Yang F, Yang Y, et al. Long noncoding RNA associated with microvascular invasion in hepatocellular carcinoma promotes angiogenesis and serves as a predictor for hepatocellular carcinoma patients' poor recurrence‐free survival after hepatectomy. Hepatology 2012; 56:2231–2241. [DOI] [PubMed] [Google Scholar]
- 13. Xu TP, Huang MD, Xia R, et al. Decreased expression of the long non‐coding RNA FENDRR is associated with poor prognosis in gastric cancer and FENDRR regulates gastric cancer cell metastasis by affecting fibronectin1 expression. J Hematol Oncol 2014; 7:63. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Hou Z, Xu C, Xie H, et al. Long noncoding RNAs expression patterns associated with chemo response to cisplatin based chemotherapy in lung squamous cell carcinoma patients. PLoS ONE 2014; 9:e108133. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Tsang WP, Kwok TT. Riboregulator H19 induction of MDR1‐associated drug resistance in human hepatocellular carcinoma cells. Oncogene 2007; 26:4877–4881. [DOI] [PubMed] [Google Scholar]
- 16. Wu W, Bhagat TD, Yang X, et al. Hypomethylation of noncoding DNA regions and overexpression of the long noncoding RNA, AFAP1‐AS1, in Barrett's esophagus and esophageal adenocarcinoma. Gastroenterology 2013; 144:956–966 e954. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Gao T, He B, Pan Y, et al. Long non‐coding RNA 91H contributes to the occurrence and progression of esophageal squamous cell carcinoma by inhibiting IGF2 expression. Mol Carcinog 2014; 54:359–367. [DOI] [PubMed] [Google Scholar]
- 18. Yang X, Song JH, Cheng Y, et al. Long non‐coding RNA HNF1A‐AS1 regulates proliferation and migration in oesophageal adenocarcinoma cells. Gut 2014; 63:881–890. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Xie HW, Wu QQ, Zhu B, et al. Long noncoding RNA SPRY4‐IT1 is upregulated in esophageal squamous cell carcinoma and associated with poor prognosis. Tumor Biol 2014; 35:7743–7754. [DOI] [PubMed] [Google Scholar]
- 20. Chen F‐J, Sun M, Li S‐Q, et al. Upregulation of the long non‐coding rna hotair promotes esophageal squamous cell carcinoma metastasis and poor prognosis. Mol Carcinog 2013; 52:908–915. [DOI] [PubMed] [Google Scholar]
- 21. Wang CM, Wu QQ, Li SQ, et al. Upregulation of the long non‐coding RNA PlncRNA‐1 promotes esophageal squamous carcinoma cell proliferation and correlates with advanced clinical stage. Dig Dis Sci 2014; 59:591–597. [DOI] [PubMed] [Google Scholar]
- 22. Li J, Chen Z, Tian L, et al. LncRNA profile study reveals a three‐lncRNA signature associated with the survival of patients with oesophageal squamous cell carcinoma. Gut 2014; 63:1700–1710. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. Tong YS, Wang XW, Zhou XL, et al. Identification of the long non‐coding RNA POU3F3 in plasma as a novel biomarker for diagnosis fo ESCC. Mol Cancer 2015; 14:1–13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24. Li W, Zheng J, Deng J, et al. Increased levels of the long intergenic non‐protein coding RNA POU3F3 promote DNA methylation in esophageal squamous cell carcinoma cells. Gastroenterology 2014; 146:1714–1726 e1715. [DOI] [PubMed] [Google Scholar]
- 25. Tong YS, Zhou XL, Wang XW, et al. Association of decreased of lncRNA LOC285194 with chemoradioationtherapy resistance and poor prognosis in ESCC. J Transl Med 2014; 12:1–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Wang Q, Fan H, Liu Y, et al. Curcumin enhances the radiosensitivity in nasopharyngeal carcinoma cells involving the reversal of differentially expressed long non‐coding RNAs. Int J Oncol 2014; 44:858–864. [DOI] [PubMed] [Google Scholar]
- 27. Liu Z, Sun M, Lu K, et al. The long noncoding RNA HOTAIR contributes to cisplatin resistance of human lung adenocarcinoma cells via downregualtion of p21(WAF1/CIP1) expression. PLoS ONE 2013; 8:e77293. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Fan Y, Shen B, Tan M, et al. Long non‐coding RNA UCA1 increases chemoresistance of bladder cancer cells by regulating Wnt signaling. FEBS J 2014; 281:1750–1758. [DOI] [PubMed] [Google Scholar]
- 29. Jiang M, Huang O, Xie Z, et al. A novel long non‐coding RNA‐ARA: Adriamycin resistance‐associated. Biochem Pharmacol 2014; 87:254–283. [DOI] [PubMed] [Google Scholar]
- 30. Pickard MR, Williams GT. Regulation of apoptosis by long non‐coding RNA GAS5 in breast cancer cells: Implications for chemotherapy. Breast Cancer Res Treat 2014; 145:359–370. [DOI] [PubMed] [Google Scholar]
- 31. Zhang X‐w, Bu P, Liu L, Zhang X‐z, Li J. Overexpression of long non‐coding RNA PVT1 in gastric cancer cells promotes the development of multidrug resistance. Biochem Biophys Res Commun 2015; 462:227–232. [DOI] [PubMed] [Google Scholar]
- 32. Liu J, Wan L, Lu K, et al. The long noncoding RNA MEG3 contributes to cisplatin resistance of human lung adenocarcinoma. PLoS ONE 2015; 10:e0114586. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33. Malek E, Jagannathan S, Driscoll JJ. Correlation of long non‐coding RNA expression with metastasis, drug resistance and clinical outcomes in cancer patients. Oncotarget 2014; 5:8027–8038. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34. Ito T, Kaneko K, Makino R, et al. Prognostic value of p53 mutations in patients with locally advanced esophageal carcinoma treated with definitive chemoradiotherapy. J Gastroenterol 2001; 36:303–311. [DOI] [PubMed] [Google Scholar]
- 35. Kang SY, Han JH, Lee KJ, et al. Low expression of Bax predicts poor prognosis in patients with locally advanced esophageal cancer treated with definitive chemoradiotherapy. Clin Cancer Res 2007; 13:4146–4153. [DOI] [PubMed] [Google Scholar]
- 36. Nam TK, Lee JH, Cho SH, et al. Low hMLH1 expression prior to definitive chemoradiotherapy predicts poor prognosis in esophageal squamous cell carcinoma. Cancer Lett 2008; 260:109–117. [DOI] [PubMed] [Google Scholar]
- 37. He L‐R, Liu M‐Z, Li B‐K, et al. High expression of EZH2 is associated with tumor aggressiveness and poor prognosis in patients with esophageal squamous cell carcinoma treated with definitive chemoradiotherapy. Int J Cancer 2010; 127:138–147. [DOI] [PubMed] [Google Scholar]
- 38. Reis EM, Verjovski‐Almeida S. Perspectives of long non‐coding RNAs in cancer diagnostics. Front Genet 2012; 3:1–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39. Sun W, Wu Y, Yu X, et al. Decreased expression of long noncoding RNA AC096655. 1–002 in gastric cancer and its clinical significance. Tumor Biol 2013; 34:2697–2701. [DOI] [PubMed] [Google Scholar]
- 40. Marks LS, Bostwick DG. Prostate cancer specificity of PCA3 gene testing examples from clinical practice. Rev Urol 2008; 10:175–181. [PMC free article] [PubMed] [Google Scholar]
- 41. He LR, Liu MZ, Li BK, et al. Prognostic impact of H3K27me3 expression on locoregional progression after chemoradiotherapy in esophageal squamous cell carcinoma. BMC Cancer 2009; 9:461. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42. Ye Y, Chen J, Zhou Y, et al. High expression of AFAP1‐AS1 is associated with poor survival and short‐term recurrence in pancreatic ductal adenocarcinoma. J Transl Med 2015; 13:137. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Additional supporting information may be found in the online version of this article at the publisher's web‐site.
Supporting Information.