INTRODUCTION {#s1}
============

Esophageal cancer (EC) is the eighth most common and the sixth most deadly cancer worldwide \[[@R1]\]. Esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC) are two major histologic subtypes of EC. EAC is more common in western Europe and north America, whereas ESCC is more prevalent in south-eastern and central Asia, particularly China and Japan \[[@R2]\]. In China alone, 477,900 newly diagnosed EC cases and 375,000 deaths from EC were projected to occur in 2015 \[[@R3]\]. Despite recent advances in combination therapy strategies, including surgery, chemotherapy, and radiotherapy, the prognosis for EC patients remains unsatisfactory, especially for advanced-stage patients, and the 5-year overall survival rate remains at less than 20% \[[@R4]\]. The rapidly increasing incidence, demanding treatment, and poor outcomes of EC highlight the need for effective potential biomarkers for early diagnosis, prognosis prediction, and novel therapeutic targets.

The etiology and pathogenesis of EC involve complicated interactions between epigenetic, genetic, and environmental factors \[[@R5]--[@R7]\]. DNA methylation is a common form of epigenetic modification, which has a crucial role in human malignancies, such as breast \[[@R8]\], lung \[[@R9]\], and gastric \[[@R10]\] cancers. Abnormal methylation in the promoter region of tumor suppressor genes is one of the most common mechanisms of modification, and results in target gene transcriptional silencing. Moreover, with the introduction of precise and convenient methods of detection, DNA methylation has become a credible potential biomarker for early detection and diagnosis of cancer \[[@R11]\].

The cyclin-dependent kinase inhibitor 2A (*CDKN2A*) gene on chromosome 9p21 is a classical tumor suppressor gene \[[@R12]\]. It is responsible for inhibiting various cyclin-dependent kinases and plays an important role in cell cycle regulation by decelerating cell cycle progression at the G1/S phase \[[@R13], [@R14]\]. Hypermethylation of the *CDKN2A* gene promoter region, resulting in its inactivation, has been reported in several types of malignancy, including lung \[[@R15]\], head and neck \[[@R16]\], hepatocellular \[[@R17]\], breast \[[@R18]\], and esophageal \[[@R19]\] cancers. Although they have been frequently investigated, the association between *CDKN2A* promoter methylation and EC and the role of this modification in EC carcinogenesis, remain controversial. For example, one study reported that the methylation rate at the *CDKN2A* promoter was similar in EC patients and healthy controls \[[@R20]\]; however, another investigation identified a differential frequency of *CDKN2A* promoter methylation between these two groups \[[@R21]\]. In addition, the value for EC diagnosis of testing for *CDKN2A* methylation, particularly using blood samples, has been less intensely investigated.

Here, we performed a meta-analysis to generate a quantitative estimate of the association of *CDKN2A* methylation with EC risk and its role in EC carcinogenesis. Furthermore, we assessed whether there were associations between *CDKN2A* promoter methylation and the clinical characteristics of EC patients. In addition, we comprehensively evaluated the diagnostic utility of *CDKN2A* methylation for EC, to assess the future potential applicability of *CDKN2A* methylation testing for the prevention, diagnosis, and therapy of EC.

RESULTS {#s2}
=======

Study characteristics {#s2_1}
---------------------

The flow chart of the selection process for inclusion of studies in our analysis is presented in Figure [1](#F1){ref-type="fig"}. A total of 423 articles were initially identified, with 421 identified by database, and two by manual searching. After a careful initial review of the titles and abstracts, 237 duplicate and 83 irrelevant articles were excluded. Next, we reviewed the full text of the remaining articles. Among these, 61 articles were excluded, since 22 were focused on cell lines or animals trials, three were reviews, and 36 lacked methylation data. Finally, 42 articles including 44 studies (41 case-control and 3 cohort studies) were included in the meta-analysis. The basic characteristics of all eligible studies are presented in Table [1](#T1){ref-type="table"}.

![Flow diagram of the study search strategy for this meta-analysis](oncotarget-08-50071-g001){#F1}

###### The main characteristics of studies included in the analysis of the association of *CDKN2A* promoter methylation with esophageal cancer

  First author   Year   Country       Ethnicity   Method    Histology   Type     Carcinoma   Normal   Pre-carcinoma                   
  -------------- ------ ------------- ----------- --------- ----------- -------- ----------- -------- --------------- ----- ---- ---- ----
  Wang           2011   China         Asian       MSP       ESCC        Tissue   6           13       0               12    H    14   64
  Song           2009   China         Asian       MSP       ESCC        Tissue   41          130      18              260   A    Na   Na
  Song           2007   China         Asian       MSP       ESCC        Tissue   65          140      31              280   A    Na   Na
  Yan            2003   China         Asian       MSP       ESCC        Tissue   5           34       0               6     H    Na   Na
  Guo            2008   China         Asian       MSP       ESCC        Tissue   35          51       1               10    H    18   44
  Yu             2006   China         Asian       MSP       ESCC        Tissue   33          45       26              45    A    Na   Na
  Guo            2006   China         Asian       MSP       ESCC        Blood    14          51       0               10    H    0    44
  Zhao           2011   China         Asian       MSP       ESCC        Tissue   31          71       10              71    A    Na   Na
                                                                                                      2               71    H    Na   Na
  Wang           2012   China         Asian       qMSP      ESCC        Tissue   66          76       3               76    A    Na   Na
  Wang           2012   China         Asian       qMSP      ESCC        Blood    54          76       2               60    H    Na   Na
  Yao            2005   China         Asian       nMSP      ESCC        Blood    34          56       0               22    H    Na   Na
  Wang           1997   USA           Caucasian   MSP       EAC         Tissue   5           11       0               21    A    3    10
  Kempste        2000   Australia     Caucasian   MSP       ESCC        Tissue   3           7        0               7     A    Na   Na
                                                            EAC         Tissue   6           9        0               9     A    Na   Na
  Bian           2002   Switzerland   Caucasian   MS-SSCA   EAC         Tissue   18          22       0               10    H    10   33
  Nie            2002   China         Asian       MSP       ESCC        Tissue   7           21       0               25    A    5    13
  Sarbia         2004   Germany       Caucasian   qMSP      EAC         Tissue   27          50       0               50    A    Na   Na
  Zhang          2004   China         Asian       MSP       ESCC        Tissue   5           34       0               6     H    Na   Na
  Schulmann      2005   USA           Caucasian   qMSP      EAC         Tissue   34          76       2               64    A    14   93
  Abbaszadegan   2005   Iran          Caucasian   MSP       ESCC        Tissue   22          30       0               30    H    Na   Na
  Abbaszadegan   2005   Iran          Caucasian   MSP       ESCC        Blood    13          30       0               30    H    Na   Na
  Roth           2006   China         Asian       MSP       ESCC        Tissue   5           13       0               11    H    3    15
  Clement        2006   Switzerland   Caucasian   MS-SSCA   EAC         Tissue   13          27       0               16    H    Na   Na
  Guo            2006   China         Asian       MSP       ESCC        Tissue   36          69       0               17    H    20   60
  Ishii          2007   Japan         Asian       COBRA     ESCC        Tissue   22          56       18              56    A    8    21
                                                                        Tissue                        0               42    H    Na   Na
  Wang           2008   China         Asian       MSP       ESCC        Tissue   110         125      46              125   A    Na   Na
                                                                        Tissue                        0               10    H    Na   Na
  Salam          2009   India         African     MSP       ESCC        Tissue   36          69       2               69    A    Na   Na
  Wang           2009   USA           Caucasian   MSP       ESCC        Tissue   22          41       0               17    H    33   92
  Ganji          2010   Italy         Caucasian   MSP       ESCC        Tissue   12          44       0               19    H    Na   Na
  Taghavi        2010   Iran          Caucasian   MSP       ESCC        Tissue   31          50       2               50    A    Na   Na
  Lu             2011   China         Asian       MSP       ESCC        Tissue   106         120      46              120   A    Na   Na
  Ikoma          2007   Japan         Asian       MSP       ESCC        Blood    6           44       0               12    H    Na   Na
  Xing           1999   China         Asian       MSRE      ESCC        Tissue   17          34       3               34    A    Na   Na
  Brock          2003   Japan         Asian       MSP       EAC         Tissue   16          41       10              41    A    Na   Na
  Vieth          2004   Germany       Caucasian   MSP       EAC         Tissue   8           15       0               15    A    17   55
  Hardie         2005   UK            Caucasian   MSP       EAC         Tissue   18          21       9               21    H    14   18
  Li             2011   China         Asian       MSP       ESCC        Tissue   21          47       10              47    A    Na   Na
  Ling           2010   China         Asian       MSP       ESCC        Tissue   25          50       1               50    H    10   50
  Chen           2012   China         Asian       MSP       ESCC        Tissue   210         257      75              257   A    Na   Na
  Talukdar       2013   India         African     MSP       ESCC        Tissue   42          112      2               30    A    Na   Na
  Liao           2009   China         Asian       MSP       ESCC        Tissue   53          105      8               105   A    Na   Na
  Hoshimoto      2015   USA           Caucasian   qMSP      ESCC        Tissue   27          114      0               28    A    Na   Na
  Das            2014   India         African     MSP       ESCC        Blood    81          100      Na              Na    Na   Na   Na
  Ito            2007   Japan         Asian       MSP       ESCC        Tissue   29          38       Na              Na    Na   Na   Na
  Hibi           2001   Japan         Asian       MSP       ESCC        Blood    7           31       Na              Na    Na   Na   Na

M+, positive for *CDKN2A* methylation; MSP, methylation specific polymerase chain reaction; qMSP, quantitative methylation specific polymerase chain reaction; MSRE, methylation sensitive restriction endonuclease; nMSP, nested methylation specific polymerase chain reaction; MS-SSCA, methylation-sensitive-single-strand conformation analysis; COBRA, combined bisulfite restriction analysis; A, autologous (control sample from the same patients); H, heterogeneous (control samples from other individuals); ESCC, esophageal squamous cell carcinoma; EAC, esophageal adenocarcinoma; Na, not available.

Comparison of the frequency of methylation of the *CDKN2A* promoter in EC cases and healthy controls {#s2_2}
----------------------------------------------------------------------------------------------------

A total of 41 case-control studies, including 2487 EC cases and 2367 healthy controls, were included in the current meta-analysis. Our results indicated that the frequency of methylation of the *CDKN2A* promoter was significantly higher in EC cases than in healthy controls (odds ratio (OR) = 12.60, 95 % CI = 8.90--17.85; Figure [2A](#F2){ref-type="fig"}). Meta-regression and subgroup analysis were applied to investigate potential sources of the substantial heterogeneity that was detected among the studies. Although, meta-regression analysis did not identify any obvious source of heterogeneity (Table [2](#T2){ref-type="table"}), decreased heterogeneity was observed in several of the subgroup analyses (conducted according to ethnicity, sample source, control type, and detection method; Table [3](#T3){ref-type="table"}), suggesting that the heterogeneity may derive from multiple sources. Additionally, all subgroups showed that *CDKN2A* hypermethylation was significantly associated with EC. Moreover, the subgroup analysis showed that the OR of the EAC subgroup (OR = 19.03) was greater than that of the ESCC subgroup (OR = 12.00), and the OR of the Caucasian subgroup (OR = 32.67) was greater than that of the Asian (OR = 9.37) and African subgroups (OR = 17.59), as well as the OR of the blood sample subgroup (OR = 34.98) was greater than that of the tissue sample subgroup (OR = 11.60). To test the robustness of our results, a sensitivity analysis to determine the influence of single studies on overall pooled ORs was performed, and the results supported the stability and credibility of our data (Figure [3A](#F3){ref-type="fig"}). Application of Begg\'s funnel plot analysis indicated no significant publication bias among the studies (P = 0.47; Figure [4A](#F4){ref-type="fig"}).

![Pooled forest plot of *CDKN2A* methylation status during the carcinogenesis of esophageal cancer\
**(A)** Cancer vs. controls: OR = 12.60; 95 % CI, 8.90--17.85. **(B)** Cancer vs. precancerous lesions: OR = 2.89; 95% CI, 2.20--3.79. **(C)** Precancerous lesions vs. control: OR = 7.38; 95% CI, 4.31--12.66.](oncotarget-08-50071-g002){#F2}

###### Meta regression analyses of *CDKN2A* promoter methylation in esophageal cancer

  Heterogeneity source   Coefficient   95% CI   *P*     
  ---------------------- ------------- -------- ------- -------
  Publication year       0.031         −0.097   0.159   0.625
  Sample size            0.003         −0.005   0.011   0.449
  Ethnicity                                             
  Asian                  −0.831        −2.42    0.759   0.295
  Caucasian              0.672         −1.197   2.54    0.469
  Detection method                                      
  MSP                    −0.883        −1.79    0.024   0.056
  Sample source                                         
  Tissue                 −0.248        −1.901   1.405   0.762
  Histology                                             
  ESCC                   0.721         −0.581   2.022   0.268
  Control type                                          
  Heterogeneous          0.64          −0.426   1.706   0.23

###### Subgroup analyses of *CDKN2A* promoter methylation in esophageal cancer

  Subgroup           Case   Control   Pooled OR (95% CI)   Heterogeneity                                   
  ------------------ ------ --------- -------------------- --------------- ------------------------ ------ ---------
  Race                                                                                                     
  African            259    288       13                   374             17.59 (6.20--49.94)      47     0.17
  Caucasian          1023   736       310                  1571            32.67 (17.61--60.62)     0      0.93
  Asian              78     103       4                    95              9.37 (6.28--13.99)       68.7   \< 0.01
  Sample source                                                                                            
  Tissue             1239   991       325                  1908            11.60 (8.14--16.54)      61.8   \< 0.01
  Blood              121    136       2                    132             34.98 (12.01--101.85)    2      0.4
  Histology                                                                                                
  ESCC               1215   1000      306                  1814            12.00 (8.35--17.26)      60.9   \< 0.01
  EAC                145    127       21                   226             19.03 (6.03--60.11)      62.4   \< 0.01
  Control type                                                                                             
  Autologous         1017   764       312                  1573            9.75 (6.33--15.04)       75.5   \< 0.01
  Heterogeneous      506    452       15                   467             28.57 (16.96--48.12)     0      0.69
  Methods                                                                                                  
  MSP                1048   852       299                  1610            9.67 (5.46--27.95)       47     \< 0.01
  Not MSP            312    275       28                   430             31.50 (9.66--102.73)     78.7   \< 0.01
  Sample size                                                                                              
  \< 60              449    498       80                   644             5.64 (3.078--10.333)     57.7   \< 0.01
  ≥ 60               911    629       247                  1396            10.948 (1.247--96.116)   62.2   \< 0.01
  Publication year                                                                                         
  \< 2010            729    728       174                  1302            10.74 (7.00--16.49)      54.9   \< 0.01
  ≥ 2010             631    399       153                  738             16.83 (9.17--30.89)      68.9   \< 0.01

M, methylated; U, unmethylated.

![Sensitivity analysis of pooled odds ratios for *CDKN2A* methylation during the carcinogenesis of esophageal cancer\
**(A)** Cancer vs. controls. **(B)** Cancer vs. precancerous lesions. **(C)** Precancerous lesions vs. control.](oncotarget-08-50071-g003){#F3}

![Begg\'s funnel plots of publication bias for *CDKN2A* methylation during the carcinogenesis of esophageal cancer\
**(A)** Cancer vs. controls. **(B)** Cancer vs. precancerous lesions. **(C)** Precancerous lesions vs. control.](oncotarget-08-50071-g004){#F4}

Comparison of the frequency of methylation of the *CDKN2A* promoter in EC and precancerous lesions {#s2_3}
--------------------------------------------------------------------------------------------------

There were 14 studies involving 510 ECs and 612 precancerous lesions included in this analysis. In the absence of heterogeneity (I^2^ = 25%, P = 0.183), a fixed-effects model was applied to evaluate the association of between methylation of *CDKN2A* in EC and precancerous lesions. We observed that the frequency of *CDKN2A* methylation was significantly higher in EC than precancerous lesion samples (OR = 2.89; 95% CI, 2.20--3.79; Figure [2B](#F2){ref-type="fig"}). The results of a sensitivity analysis indicated the authenticity of our results (Figure [3B](#F3){ref-type="fig"}) and a Begg\'s test for publication bias was not statistically significant (P = 0.74; Figure [4B](#F4){ref-type="fig"})

Comparison of the frequency of methylation of the *CDKN2A* promoter between precancerous esophageal lesions and healthy controls {#s2_4}
--------------------------------------------------------------------------------------------------------------------------------

The analysis of the association between methylated *CDKN2A* and esophageal precancerous lesions included 612 precancerosis samples and 381 healthy controls from 14 studies. As demonstrated in Figure [2C](#F2){ref-type="fig"}, the methylation frequency of *CDKN2A* was significantly elevated in precancerous lesions compared with controls (OR = 7.38; 95% CI, 4.31--12.66). Sensitivity analysis confirmed the robustness of our results (Figure [3C](#F3){ref-type="fig"}) and a Begg\'s test revealed no publication bias (P \> 0.05, Figure [4C](#F4){ref-type="fig"}).

Association of methylation of the *CDKN2A* promoter with clinicopathological features of EC {#s2_5}
-------------------------------------------------------------------------------------------

A total of 1313 ECs from 16 studies were used to evaluate the relationships between *CDKN2A* methylation and clinicopathological features, including age, gender, smoking behavior, alcohol consumption, tumor location, tumor diameter, differentiation grade, tumor stage, clinical stage, and lymph node metastasis (Table [4](#T4){ref-type="table"}). Our findings demonstrated that *CDKN2A* methylation was significantly associated with differentiation grade (poor vs. well and moderate: OR = 1.79; 95% CI, 1.20--2.67; P \< 0.01) and clinical stage (advanced vs. early: OR = 2.56; 95% CI, 1.33--4.92; P \< 0.01). However, there were no associations between other clinicopathological characteristics and *CDKN2A* promoter methylation in EC.

###### The association of *CDKN2A* promoter methylation with clinicopathological features of esophageal cancer patients

  Characteristic          No.       Case type/control type   Cases/controls   OR (95% CI)         *P*    Heterogeneity   
  ----------------------- --------- ------------------------ ---------------- ------------------- ------ --------------- ---------
  ***I*^2^ (%)**          ***P***                                                                                        
  Age                     8         Older/younger            259/230          0.98 (0.65--1.47)   0.91   0               0.45
  Gender                  12        Male/female              568/259          0.97 (0.68--1.38)   0.84   0               0.83
  Smoking behavior        8         Yes/no                   409/354          1.10 (0.54--2.28)   0.79   71.2            0
  Alcohol consumption     5         Yes/no                   282/322          0.92 (0.47--1.82)   0.82   59.1            0.04
  Differentiation grade   11        Poor/well and moderate   144/614          1.79 (1.20--2.67)   0      25.2            0.2
  T stage                 6         T~3+4~/T~1+2~            409/230          0.88 (0.42--1.82)   0.73   68.8            0.01
  Clinical stage          9         III + IV/I+II            307/288          2.56 (1.33--4.92)   0.01   55.9            0.02
  Lymph node metastasis   8         Yes/no                   379/358          1.69 (0.76--3.75)   0.2    77.4            \< 0.01
  Diameter                4         \> 5 cm/\< 5 cm          100/98           1.37 (0.74--2.52)   0.32   0               0.91
  Location                7         Up and middle/down       456/237          0.79 (0.54--1.14)   0.2    0               0.6

No., number of studies.

The accuracy of testing for *CDKN2A* methylation for EC diagnosis {#s2_6}
-----------------------------------------------------------------

In the current analysis, the diagnostic value of methylated *CDKN2A* for EC diagnosis was assessed using data from 41 eligible case-control studies. The pooled sensitivity and specificity values were 0.52 (95% CI, 0.44--0.59) and 0.96 (95% CI, 0.93--0.98), respectively. The area under the summary receiver operating characteristic (SROC) curve (AUC) based on the specificity and sensitivity was 0.83 (95% CI, 0.79--0.86) (Figure [5A](#F5){ref-type="fig"}). The AUCs based on subgroups where methylation was tested in tissue and blood samples were 0.82 (95% CI, 0.79--0.85) (Figure [5B](#F5){ref-type="fig"}) and 0.90 (95% CI, 0.87--0.92) (Figure [5C](#F5){ref-type="fig"}), respectively. As shown in the Figure [6](#F6){ref-type="fig"}, Fagan plot analysis demonstrated that the probabilities of a patient being diagnosed with EC were 82%, 93%, and 98% following a positive result for methylation of *CDKN2A*, where the pretest probabilities of being diagnosed with EC were set to 25%, 50%, and 75%, respectively; when the test was negative, the probabilities of diagnosis with EC reduced to 14%, 33%, and 60%, respectively.

![Summary of receiver operating characteristic (SROC) plots of methylated *CDKN2A* for the diagnosis of esophageal cancer based on all samples, tissue samples, and blood samples\
**(A)** All samples: sensitivity, 0.52 (95% CI, 0.44--0.59); specificity, 0.96 (95% CI, 0.93--0.98); AUC, 0.83 (95% CI, 0.79--0.86). **(B)** Tissue samples: sensitivity, 0.53 (95% CI, 0.45--0.61); specificity, 0.95 (95% CI, 0.91--0.98); AUC, 0.82 (95% CI, 0.79--0.85). **(C)** Blood samples: sensitivity, 0.42 (95% CI, 0.23--0.63); specificity, 1.00 (95% CI, 0.46--1.00); AUC, 0.90 (95% CI, 0.87--0.92).](oncotarget-08-50071-g005){#F5}

![Fagan plot analysis to evaluate the clinical applicability of screening for methylated *CDKN2A* in esophageal cancer diagnosis\
**(A)** The post-test probability was 82% at a pretest probability of 25%. **(B)** The post-test probability was 93% at a pretest probability of 50%. **(C)** The post-test probability was 98% at a pretest probability of 75%.](oncotarget-08-50071-g006){#F6}

DISCUSSION {#s3}
==========

EC is a complex, progressive disease with multiple stages. Barrett\'s esophagus (BE), a metaplastic condition where the squamous-lined esophageal mucosa is replaced by specialized intestinal mucosa, and dysplasia, are precursor lesions of EC \[[@R2], [@R22]\].

Epigenetic modifications, including DNA methylation, contribute significantly to the pathogenesis of EC. DNA methylation is a relatively early molecular change that is a candidate biomarker in several carcinomas \[[@R23]\]. Increasing evidence demonstrates that methylation of the promoter of *CDKN2A*, an important negative regulator of cell growth and proliferation \[[@R24]\], is involved in the tumorigenesis and progression of various tumors, including pancreatic \[[@R25]\], gastric \[[@R26]\], and prostate \[[@R27]\] cancers. Although there have been several reports that the frequency of *CDKN2A* promoter methylation is higher in EC samples than those from cancer-free controls, the association and the role of *CDKN2A* promoter methylation in EC remain controversial. We performed this meta-analysis to address these issues, achieve insights into the role of *CDKN2A* promoter methylation in EC carcinogenesis, and evaluate its diagnostic value in screening for early stage EC.

The current study incorporated a total of 41 case-control studies, including 2487 ECs, 618 precancerosis (including dysplasia and BE), and 2367 control samples. Our results indicate that the frequency of methylation of the *CDKN2A* promoter was significantly higher in EC than in healthy controls and precancerous lesions. Moreover, the level of *CDKN2A* methylation was also remarkably higher in precancerous lesions than in healthy controls. This evidence supports the association of methylated *CDKN2A* with EC carcinogenesis. Recent research has revealed that ESCC and EAC have different molecular features \[[@R28]\]. In our results, the OR of the association with *CDKN2A* methylation for the EAC subgroup was greater than that for the ESCC subgroup, indicating that this molecular feature may be more relevant in EAC. The subgroup analysis by ethnicity showed that *CDKN2A* promoter methylation was associated with an increased risk of EC in Asian and Caucasian, as well as African population, with the Caucasian population showing a higher OR than the Asian and African population, suggesting that the Caucasian population may be more susceptible to *CDKN2A* promoter methylation. The subgroup analysis of sample source presented a significant correlation between hypermethylated *CDKN2A* and EC in both tissue and blood samples, and the pooled OR of blood samples was remarkably higher than that of tissue samples, which suggested that hypermethylated *CDKN2A* may be a useful noninvasive biomarker for blood detection.

Additionally, we evaluated the relationship between *CDKN2A* methylation and the clinicopathological characteristics of EC. A previous systematic review reported that there was a trend towards increased frequency of *CDKN2A* methylation according to EC differentiation grade; however, the association was not statistically significant \[[@R29]\]. In contrast, our results indicate that methylation of the *CDKN2A* promoter is significantly more common among patients with poorly differentiated tumors and advanced clinical stage, compared with those with well/moderately differentiated tumors and early stage disease. Together, these results support the hypothesis that hypermethylation of the *CDKN2A* promoter is involved in the progression of EC.

We further assessed the diagnostic potential of *CDKN2A* promoter methylation for EC, based on data from 41 eligible case-control studies. The pooled sensitivity and specificity for all included studies were 0.52 and 0.96, respectively, indicating that *CDKN2A* promoter methylation could be a useful biomarker for diagnosis of EC. In the current study, we also analyzed SROC and AUC statistics. SROC is a comprehensive index synthesizing sensitivity and specificity, which can be used to evaluate the accuracy of diagnostic tests. The SROC curve and AUC can also be used to evaluate diagnostic power, where an AUC closer to 1.0 signifies that the test has better discrimination. The AUC calculated from all studies included in the current analysis was 0.83, indicating that *CDKN2A* promoter methylation is an extremely useful biomarker for EC diagnosis. In addition, there is increasing evidence that abnormal DNA methylation in body fluid samples is a promising biomarker for cancer screening and diagnosis \[[@R30], [@R31]\]. As a relatively noninvasive test, blood sample collection is generally acceptable to patients and is a promising route for clinical application of methylation screening. Previous studies have reported that abnormal DNA methylation markers in blood samples can be used to diagnose various cancers \[[@R32], [@R33]\]. Our subgroup analysis showed that the AUC value for the blood sample group was 0.9, higher than that for the tissue group (0.82), which are in accordance with numerous previous studies \[[@R34]\]. The existence of cell-free circulating tumor DNA (ctDNA) in blood sample could be responsible for this phenomenon. This form of circulating DNA is presumably shed from tumors, either through necrosis or apoptosis \[[@R35]\]. It has been reported that tumor-derived somatic alterations in DNA can be detected in ctDNA with broad clinical application, high sensitivity, and specificity \[[@R36]--[@R38]\]. All these above indicated that testing for *CDKN2A* methylation in blood samples has potential as a noninvasive tool for the diagnosis of EC. Future rigorous clinical research studies with larger sample sizes will be essential to validate our findings.

The clinical utility of *CDKN2A* methylation was also evaluated by Fagan plot analysis \[[@R39], [@R40]\]. The results demonstrated that, when the pre-test probabilities were assumed to be 25%, 50%, and 75%, a corresponding 82%, 93%, and 98% of patients would be correctly diagnosed with EC following positive *CDKN2A* methylation tests; moreover, a diagnosis of EC could be ruled out for 86%, 67%, and 40% of patients following negative results. These analyses suggest that methylated *CDKN2A* has good diagnostic power to discriminate patients with EC from healthy individuals.

The present meta-analysis had several limitations. First, only articles published in English and Chinese were included in the study. Second, significant heterogeneity was observed in the current analysis, and its source was not definitively identified. Third, we did not specifically investigate the association of *CDKN2A* methylation and different EC histology subtypes; therefore, an updated meta-analysis, including more rigorous studies with large sample sizes in the future would support, and could add to the findings of the present study.

In conclusion, our findings provide strong evidence that *CDKN2A* methylation is involved in the carcinogenesis and progression of EC and that it is a promising biomarker for the diagnosis of EC, especially by screening of blood samples. Future large-scale studies are necessary to support or add to our findings, especially regarding the role of *CDKN2A* methylation in the prediction of prognosis, and to support the clinical application of our findings for the benefit of patients with EC.

MATERIALS AND METHODS {#s4}
=====================

Literature search {#s4_1}
-----------------

Five electronic databases (including PubMed, Google Scholar, Web of Science, Embase, and China National Knowledge Infrastructure) were searched for eligible studies until October 20, 2016. The following search terms, and various combinations of them, were used: "*p16*," "*p16^INK4a^*," "*p14^arf^", "p14"*, "*CDKN2A (p16 and p14)*," "cyclin-dependent kinase inhibitor 2A," "methylation," "DNA methylation," "promoter methylation," "esophageal carcinoma," "esophagus cancer," "esophageal tumor," and "esophageal malignancy." In addition, we performed a manual search of references to find potentially relevant articles.

Selection criteria {#s4_2}
------------------

We collected all eligible articles that addressed the association between *CDKN2A* methylation and EC. Articles with data meeting the following criteria were included: (1) all samples confirmed by pathology, including ECs, esophageal precancerous lesions (dysplasia or Barrett\'s esophagus), and healthy controls; (2) case-control or cohort studies of *CDKN2A* promoter methylation in EC; (3) included sufficient data regarding the methylation frequency of the *CDKN2A* promoter to enable the calculation of odds ratios and 95% confidence intervals; and (4) where authors published several reports using the same population or overlapping data, only the most complete study with the most information was selected.

Data quality assessment {#s4_3}
-----------------------

The quality of studies was assessed according to Newcastle--Ottawa Scale (NOS) criteria \[[@R41]\]. The NOS evaluation system includes three aspects: (1) subject selection: 0--4 points; (2) comparability of subjects: 0--2 points; and (3) clinical outcome: 0--3 points. NOS scores range from 0 to 9; and a score ≥ 7 indicates good quality. Only studies with scores ≥ 7 were included in the analysis.

Data extraction {#s4_4}
---------------

Three authors (QL, JL, and CZ) independently reviewed all the available articles and extracted relevant data from eligible articles. The following information was recorded: the first author\'s name; the country, ethnicity of subjects, year of publication, sample type, number of cases, control type, and detection method of methylation of the study; and the methylation frequency of *CDKN2A*, and clinicopathological parameters (including age, gender, smoking behavior, alcohol consumption, tumor location, tumor diameter, differentiation grade, tumor stage, clinical stage and lymph node metastasis) of the subjects. Inconsistent information was discussed by all authors and a consensus reached on its use.

Statistical analyses {#s4_5}
--------------------

Data were analyzed using Stata statistical software, version 12.0 (Stata Corporation, College Station, TX, USA). Overall odds ORs and corresponding 95% CIs were calculated to evaluate the strengths of the associations between *CDKN2A* methylation and different EC pathological processes (cancer vs. healthy control, cancer vs. precancerous lesion, precancerous lesion vs. healthy control), as well as clinicopathological features. Potential heterogeneity was quantified using Cochran\'s Q statistic and *I^2^* tests \[[@R42]\]. When significant heterogeneity was observed among studies, a random-effects model (DerSimonian-Laird method) \[[@R43]\] was applied to calculate a pooled OR (*I^2^* \> 50% or *P* \< 0.05), otherwise, a fixed-effects model (Mantel--Haenszel method) was used \[[@R44]\]. Meta-regression and subgroup analyses were performed to explore the sources of heterogeneity. Sensitivity analyses were conducted to determine the influence of individual studies on the pooled results, through the omission of single studies \[[@R45]\]. Publication bias was quantitatively assessed using Begg\'s linear regression tests \[[@R46]\]. Pooled sensitivity and specificity were used to estimate the diagnostic efficacy of testing for *CDKN2A* promoter methylation. Two indicators, the SROC and AUC, were also calculated to evaluate the stability and accuracy of the diagnostic capacity of *CDKN2A* promoter methylation for EC \[[@R47]\]. Fagan plot analysis was performed with 25%, 50%, and 75% pre-test probability to assess the diagnostic power of *CDKN2A* promoter methylation in clinical practice \[[@R39]\]. All *P* values are two-sided and *P* values \< 0.05 were considered statistically significant.

This research was supported by grants from the Medical and Health Research Project of Zhejiang Province (No. 2013KYB231; No. 2014KYB229), Medical Association Clinical Research Project Funding of Zhejiang Province (No. 2015ZYC-A49), and Ningbo Natural Science Foundation (No. 2015A610221).

**Author contributions**

Conceived and designed the experiments: JL and QL. Performed the experiments: JL and CZ. Analyzed the data: JL, CZ, and QL. Contributed analysis tools: JL and CZ. Wrote the manuscript: JL and CZ.

**CONFLICTS OF INTEREST**

None of the authors has any commercial or other associations that might pose a conflicts of interest.

EC

:   esophageal cancer

EAC

:   esophageal adenocarcinoma

ESCC

:   esophageal squamous cell carcinoma

BE

:   Barrett\'s esophagus

*CDKN2A*

:   cyclin-dependent kinase inhibitor 2A

SROC

:   summary receiver operating characteristic

AUC

:   the area under the SROC

NOS

:   the Newcastle--Ottawa Scale

OR

:   odds ratio

CI

:   confidence interval
