Carcinoembryonic antigen (CEA) level, CEA ratio, and treatment outcome of rectal cancer patients receiving pre-operative chemoradiation and surgery
- Kai-Lin Yang1,
- Shung-Haur Yang2, 5,
- Wen-Yih Liang3, 5,
- Ying-Ju Kuo3,
- Jen-Kou Lin2, 5,
- Tzu-Chen Lin2,
- Wei-Shone Chen2, 5,
- Jeng-Kae Jiang2, 5,
- Huann-Sheng Wang2, 5,
- Shih-Ching Chang2, 5,
- Lee-Shing Chu4, 5 and
- Ling-Wei Wang1, 5Email author
© Yang et al; licensee BioMed Central Ltd. 2013
Received: 1 September 2012
Accepted: 26 February 2013
Published: 1 March 2013
To investigate serum carcinoembryonic antigen (CEA) as a prognostic factor for rectal cancer patients receiving pre-operative chemoradiotherapy (CRT).
Between 2000 and 2009, 138 patients with advanced rectal cancer receiving CRT before surgery at our hospital were retrospectively classified into 3 groups: pre-CRT CEA <6 ng/ml (group L; n = 87); pre-CRT CEA ≥ 6 ng/ml and post-CRT CEA <6 ng/ml (group H-L; n = 32); and both pre- and post-CRT CEA ≥ 6 ng/ml (group H-H; n = 19). CEA ratio (defined as post-CRT CEA divided by pre-CRT CEA), post-CRT CEA level and other factors were reviewed for prediction of pathologic complete response (pCR).
Five-year disease-free survival (DFS) was better in groups L (69.0%) and H-L (74.5%) than in group H-H (44.9%) (p = 0.024). Pathologic complete response was observed in 19.5%, 21.9% and 5.3% of groups L, H-L and H-H respectively (p = 0.281). Multivariate analysis showed that ypN stage and pCR were independent prognostic factors for DFS and that post-CRT CEA level was independently predictive of pCR. As a whole, post-CRT CEA <2.61 ng/ml predicted pCR (sensitivity 76.0%; specificity 58.4%). For those with pre-CRT CEA ≥6 ng/ml, post-CRT CEA and CEA ratio both predicted pCR (sensitivity 87.5%, specificity 76.7%).
In patients with pre-CRT serum CEA ≥6 ng/ml, those with “normalized” CEA levels after CRT may have similar DFS to those with “normal” (<6 ng/ml) pre-CRT values. Post-CRT CEA level is a predictor for pCR, especially in those with pre-CRT CEA ≥6 ng/ml.
KeywordsRectal cancer Pre-operative chemoradiation Carcinoembryonic antigen CEA ratio Tumor response
Pathologic complete response
Tumor regression grade
American Joint Committee on Cancer
Eastern Cooperative Oncology Group
Magnetic resonance imaging
Lower anterior resection
Statistical Package for Social Sciences software
Analysis of variance
Receiver operating characteristic curve
Area under the curve
Deleted in colon cancer
Rectal cancer is one of the leading causes of cancer deaths in the world . For locally advanced rectal cancer, pre-operative chemoradiotherapy (CRT) followed by radical surgery is a standard treatment [2–4]. Pre-operative CRT can improve locoregional tumor control, downstage the tumor and increase the probability of sphincter-sparing surgery [2–6]. The response of tumors to CRT varies between different patients. Tumor regression grade (TRG) is widely used to determine the tumor response to CRT in pathology [7–10]. Pathologic complete response (pCR) could be observed in 8%–25% of certain patients after regular doses of pre-operative CRT [2, 3, 5, 6]. However, TRG and pCR can only be determined microscopically after surgery. A useful predictive model for the response of rectal cancer to pre-operative CRT has not yet been well established.
Serum carcinoembryonic antigen (CEA) is commonly measured in pre-treatment workups for rectal cancer patients . The prognostic value of serum CEA levels has been widely discussed in the relevant literature: poor tumor response to CRT and an increased risk of recurrence have been observed in patients with elevated CEA levels before or after CRT [12–15]. It has also been reported that the reduction ratio of pre- to post-CRT serum CEA levels may be a prognostic factor for disease-free survival in rectal cancer patients with a pre-CRT CEA of more than 6 ng/ml . However, it is rarely reported whether the clinically derived CEA parameters (including CEA reduction ratio) are correlated to pCR obtained after surgery. The purpose of this study is to evaluate the significance of serum CEA levels before treatment and their subsequent changes in predicting clinical outcomes and pathologic tumor responses for patients with rectal cancer receiving pre-operative CRT.
This study was approved by the institutional review board of Taipei Veterans General Hospital (No. 2011-05-0041C). Between May 2000 and July 2009, 191 patients with histologically confirmed rectal adenocarcinomas, either locally advanced disease (clinical T3, T4 or node-positive disease by AJCC staging system) or low seated primary T2 disease (<6 cm from anal verge), were treated with pre-operative CRT followed by radical surgery at Taipei Veterans General Hospital. The Eastern Cooperative Oncology Group (ECOG) performance score of all the patients was 0–2.
Among the 191 patients, 30 were excluded because of missing CEA levels after CRT; 20 were excluded due to CEA levels before or after CRT measured by enzyme immunoassay (EIA); and a further 3 receiving transanal excision instead of radical proctectomy were also excluded. The remaining 138 patients undergoing radical proctectomy with serum CEA levels measured both before and after CRT by means of radioimmunoassay (RIA) (CEA-RIACT®; CIS bio international, Gif-sur-Yvette, France), were included in this study.
Before CRT, computed tomography (CT) scans or magnetic resonance imaging (MRI, 1.5-T Siemens Vision scanner with pelvic array coil and intrarectal tube) and proctoscopy were used to evaluate the primary disease and clinical lymph node status; chest X-rays and abdominal ultrasonography were used for systemic evaluation. Among the 138 patients included in this study, pelvic CTs were done before CRT in 61 patients, and pelvic MRI s were done on the other 77 patients.
The detail of CRT in the protocol was described in our previous publication . The prescription dose to whole pelvis was 45 Gy in 20 fractions over 4 weeks. For primary T4 disease only, a boost of 5.4 Gy in 3 fractions to the gross rectal tumors with a 1.5 cm margin followed pelvic irradiation. The median RT duration was 26 days. Oral chemotherapy agents, tegafur-uracil (UFUR; TTY Biopharm, Taipei, Taiwan) 200 mg/m2/day and leucovorin (Wyeth Lederle Laboratories, Taipei, Taiwan) 45 mg/day, were administered concurrently with RT (days 1–28) and after completion of RT (days 36–63; dose of tegafur-uracil adjusted to 250 mg/m2/day). The total daily doses of both drugs were divided into three doses per day.
At a median interval of 6.3 weeks (range, 3.4–12.4 weeks) after completion of RT, radical proctectomy with total mesorectal excision (TME) for rectal cancer was implemented. Lower anterior resection (LAR) was performed in 114 patients (82.6%) and abdominoperineal resection (APR) in 24 (17.4%), as indicated.
According to physicians’ suggestions and patients’ decisions, post-operative 5-fluorouracil based chemotherapy was implemented in 71.8% of the patients with pathologic stage III-IV and in 23.2% of the patients with pathologic stage 0-II.
Carcinoembryonic antigen (CEA)
Serum CEA levels before CRT (pre-CRT CEA) were measured around one week before CRT, and Serum CEA levels after CRT (post-CRT CEA) were measured within one week prior to surgery. In our hospital, the normal limit of serum CEA measured by RIA was set as <6 ng/ml. According to this cutoff value, all patients were classified into 3 CEA change groups: pre-CRT CEA <6 ng/ml (group L); pre-CRT CEA ≥6 ng/ml and post-CRT CEA <6 ng/ml (group H-L); and both pre- and post-CRT CEA ≥6 ng/ml (group H-H). The extent of CEA reduction was evaluated by CEA ratio (defined as post-CRT CEA divided by pre-CRT CEA).
After the completion of combined treatments, the patients were regularly followed up with physical examinations and measurement of serum CEA levels every 3–6 months for the first 2 years. Follow-up colonoscopies, pelvic CT scans, and chest radiography were also performed every 6–12 months for at least 5 years. In this study, recurrences were diagnosed either pathologically or radiologically.
Statistical Package for Social Sciences software (SPSS version 17.0, Chicago, IL) was used. Chi-square test, Fisher’s exact test, independent t-test or one-way analysis of variance (ANOVA) was implemented to analyze variables. Local control (LC) and disease-free survival (DFS) from the time of surgery were calculated by Kaplan-Meier method, using log-rank tests for comparison. Univariate and multivariate analysis by Cox proportional hazards model or logistic regression were performed. Receiver operating characteristic curves (ROC), involving the Youden index (maximum [sensitivity + specificity - 1]), were used to determine optimal cutoff values. A p-values <0.05 (two-sided test) was considered significant.
Overall characteristics of the patients
Of the 138 patients, 98 (71%) were males. The mean age was 63 (range 33–83 years), and mean pre- and post-CRT CEA levels (ng/mL) were 13.2 (range, 1.3–400.0) and 3.7 (range, 0.4–39.5) respectively. Free resection margin were found in all surgical specimens. Twenty-five patients (18%) achieved pCR after CRT. The median follow-up time from the start of RT was 59 months (range, 3–141 months).
Clinicopathologic features, local control and disease-free survival of patients with reference to CEA change groups
Clinicopathologic features between group L, group H-L and group H-H
Group L (n = 87)
Group H-L (n = 32)
Group H-H (n = 19)
Age (years), mean (range)
Clinical T stage
Clinical N stage
Clinical Stage Grouping
Stage III-IV †
Pathologic T stage
ypT0, Tis, T1-2
Pathologic N stage
Pathologic Stage Grouping
yp stage 0 *
yp stage I-II
yp stage III-IV §
Distance from anal verge (cm), mean (range)
Univariate and multivariate analysis of predictive factors for DFS (n = 138)
No. of patients
5-year DFS (%)
Clinical T stage
Clinical N stage
Pathologic T stage
ypT0, Tis, T1-2
Pathologic N stage
Distance from anal verge
CEA change groups
Pathologic N stage
Clinical predictors of pCR after chemoradiation
Univariate and multivariate analysis of predictive factors for pCR (n = 138)
Clinical T stage
Clinical N stage
Clinical stage grouping
Stage III-IV †
Distance from anal verge
Pre-CRT CEA level
Post-CRT CEA level
CEA change groups
Post-CRT CEA level
For those with pre-CRT CEA <6 ng/ml (n = 87; Figure 3B), none of the ROC curves of various CEA parameters was significantly predictive of pCR. For those with pre-CRT CEA ≥6 ng/ml (n = 51; Figure 3C), the ROC curves of post-CRT CEA level and CEA ratio were both significantly predictive of pCR with AUCs of 0.783 (p = 0.012) and 0.733 (p = 0.038), respectively.
Serum CEA concentrations are usually measured for rectal cancer patients. Measurement of serum CEA levels is inexpensive, standardized for normal limit, widely used and easily performed, as compared with other potential prognostic markers for rectal cancer patients, such as CA 19–9, p53, ras expression, thymidine synthase, dihydropyrimidine dehydrogenase, 18q loss of heterozygosity, deleted in colon cancer (DCC) protein, DNA ploidy or micro-RNA signature [11, 17, 18]. The micro-RNA signature may be of value in predicting pCR , but will need further validation. Several studies had focused on the predictive value of pre- and post-CRT CEA levels in patients with rectal cancer receiving pre-operative CRT [12–15, 19, 20]. In this study, we found not only pre-CRT CEA levels had prognostic significance, “normalization” of these values and CEA ratio also predicted tumor response and may be helpful in the design of individualized treatment for rectal cancer with high CEA levels before treatment.
There is some controversy as to the role of pre-CRT CEA in rectal cancer patients. It was reported that pre-CRT CEA levels >2.5 or >5 ng/ml were associated with poor pCR rates and poor disease-free survival on univariate analysis, but not for both on multivariate analysis [12, 13]. However, in other studies, pre-CRT CEA levels were a common predictor of downstaging, pCR and tumor response on multivariate analysis [14, 15]. As the pre-CRT CEA levels increase, the rates of good response might decrease. In a recent study, for pre-CRT CEA <3, 3–6, 6–9 and >9 ng/ml, the rates of good response were 36%, 24%, 16% and 8%, respectively . On the other hand, post-CRT CEA level >5 ng/ml was associated with decreased pCR rates and disease-free survival on univariate and multivariate analysis [12, 19]. The literature mentioned above suggests that pre- or post-CRT CEA level 3–6 ng/ml may serve as a valuable threshold for prognosis and prediction of pathologic tumor response. The change of CEA levels before and after CRT were also investigated in some Korean studies [16, 20]. Park et al. categorized locally advanced rectal cancer patients into 3 groups according to pre- and post-CRT CEA levels (> or ≦3 ng/ml). They concluded that these groupings could be of clinical value as a predictor of response (TRG) to preoperative CRT and as an independent prognostic factor. However, the idea of CEA reduction ratio (like our CEA ratio) had not been previously mentioned until Kim’s study. They also classified rectal cancer patients into 3 groups according to pre- and post-CRT CEA levels, though with different cut off values (> or ≦6 ng/ml), For patients with pre-CRT CEA >6 mg/ml, they were further divided by whether post-CRT CEA was ≥70% lower than pre-CRT CEA. Similarly, they had a better 5-year DFS for the lower pre-CRT group and higher pre-CRT group with a CEA reduction ratio ≥ 70% than the other group. However, they could not definitely explain the pathological basis behind the CEA reduction ratio; and it was unknown whether pCR were related to this ratio.
Our study implied that pre-CRT CEA levels may be prognostic of local tumor control but may not be predictive of pCR. Higher pre-CRT CEA levels could be related to more advanced locoregional spread and thus associated with poorer local control, but not necessarily reflect sensitivity to CRT. The 5-year DFS rates of groups L, H-L and H-H among our patients were compatible with the two Korean studies mentioned above. Group H-H carried significantly higher risks of ypN1-2 disease and pathologic stage III-IV than groups L and H-L. This could explain why group H-H had the worst 5-year DFS rate of the three groups. Both ypN stage and pCR were parameters representing tumor response to CRT and, in our multivariate analysis, were independent prognostic factors for DFS. Though CEA change groups were not, this grouping made before surgery was obviously related to these two parameters (Table 1). Perhaps, the small number of patients may also make CEA change group statistically less prognostic. Furthermore, both ypN stage and pCR are pathologic features, limiting their prognostic value before operation.
We tried to find a clinical parameter predicting the pathologic response. CEA change groups were relevant to pCR, but may not be significant enough. Interestingly, we found male gender was a negative predictor for pCR in univariate analysis, although not anymore in multivariate analysis. Post-CRT CEA level was the only independent predictor for pCR in our data. By using ROC curves, post-CRT CEA level was a better predictor than pre-CRT CEA level or CEA ratio (Figure 3A), and the optimal cutoff value of post-CRT CEA was 2.61 ng/ml. However, when restricting to those with pre-CRT CEA <6 ng/ml, this observation was weakened and not statistically valuable (Figure 3B). On the other hand, for those with pre-CRT CEA ≥6 ng/ml, the AUC was even larger for post-CRT CEA level to predict pCR, and CEA ratio had value as good as post-CRT CEA level did (Figure 3C). This suggested that the pathologic tumor response was related to post-CRT CEA levels rather than to pre-CRT CEA levels, especially for those with high pre-CRT CEA levels.
RT-surgery interval is a well-known predictor for tumor response to pre-operative CRT, but we did not identify this even in univariate analysis, which could be due to less variance of the interval in our patients. The mean RT-surgery intervals of the patients with post-CRT CEA <2.61 ng/ml and those with post-CRT CEA ≥2.61 ng/ml were 44.6 days (range, 27–87) and 44.8 days (range, 24–79), respectively (p = 0.946). The comparable results supported that post-CRT CEA groups could predicted pCR independently. Besides, when dividing all patients into two groups according to the post-CRT CEA cutoff value 2.61 mentioned above, lower post-CRT CEA group correlated with earlier pathologic stages (p = 0.031; not shown in the result section) and better overall survival (p = 0.005).
The change of serum CEA levels before and after CRT seems to be more obvious in patients with pre-CRT serum CEA ≥6 ng/ml than those with normal levels (mean CEA ratio ± standard deviation, 0.36 ± 0.25 vs. 0.87 ± 0.30; p < 0.001); accordingly, a low post-CRT CEA level or CEA ratio may represent a marked reduction of tumor burden after CRT, especially when pre-CRT CEA levels are higher than the normal limit. We observed pCR may be even more predictable (larger AUC) when using post-CRT CEA level or CEA ratio in those with pre-CRT CEA levels ≥6 ng/ml; the optimal cutoff values of post-CRT CEA level and CEA ratio were 2.87 ng/ml and 0.22, respectively (not shown in the result section), with the same sensitivity 87.5%, specificity 76.7% and maximum Youden index (0.642). But, it was still not reasonable to conclude that surgery could be totally omitted for those with elevated pre-CRT CEA and low post-CRT CEA levels (i.e. low CEA ratios). We hypothesized that significant CEA reduction may play a role in supporting significant tumor regression for patients with both high pre-CRT CEA and clinical good response after CRT. For some highly selected cases like these, more conservative surgery may be used to preserve the sphincter without compromising local control .
In this study, all the patients were treated according to the same treatment protocol of pre-operative CRT, which had been proved to be effective and tolerable . However, several limitations to this study exist, including retrospective design, relatively small patient numbers, imbalance of case numbers in different groups, variation in RT-surgery intervals, and inconsistent principle and regimen for adjuvant chemotherapy. Many patients were excluded due to absence of serum CEA levels or due to different laboratory techniques for measurement.
In conclusion, for locally advanced rectal cancer patients with pre-treatment CEA levels ≥6 ng/ml, “normalization” of these values after CRT may predict similar tumor response and DFS to that of those patients with pre-CRT CEA <6 ng/ml. Those patients with persistent high CEA level after CRT would have the poorest response and worst DFS. We hypothesize that low post-CRT CEA levels obtained before surgery may predict pCR in rectal cancer patients receiving pre-operative CRT, while CEA ratios may also predict pCR only in patients with pre-CRT serum CEA ≥6 ng/ml.
Authors would like to thank Dr. Chin-Fu Hsiao for the professional advice about biostatistics.
- Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D: Global cancer statistics. CA Cancer J Clin 2011, 61:69–90.PubMedView Article
- Sauer R, Becker H, Hohenberger W, Rodel C, Wittekind C, Fietkau R, Martus P, Tschmelitsch J, Hager E, Hess CF, Karstens JH, Liersch T, Schmidberger H, Raab R: Preoperative versus postoperative chemoradiotherapy for rectal cancer. N Engl J Med 2004, 351:1731–1740.PubMedView Article
- Gerard JP, Conroy T, Bonnetain F, Bouche O, Chapet O, Closon-Dejardin MT, Untereiner M, Leduc B, Francois E, Maurel J, Seitz JF, Buecher B, Mackiewicz R, Ducreux M, Bedenne L: Preoperative radiotherapy with or without concurrent fluorouracil and leucovorin in T3–4 rectal cancers: results of FFCD 9203. J Clin Oncol 2006, 24:4620–4625.PubMedView Article
- Bosset JF, Collette L, Calais G, Mineur L, Maingon P, Radosevic-Jelic L, Daban A, Bardet E, Beny A, Ollier JC: Chemotherapy with preoperative radiotherapy in rectal cancer. N Engl J Med 2006, 355:1114–1123.PubMedView Article
- Kao PS, Chang SC, Wang LW, Lee RC, Liang WY, Lin TC, Chen WS, Jiang JK, Yang SH, Wang HS, Lin JK: The impact of preoperative chemoradiotherapy on advanced low rectal cancer. J Surg Oncol 2010, 102:771–777.PubMedView Article
- Wang LW, Yang SH, Lin JK, Lin TC, Chan WK, Chen WS, Wang HS, Jiang JK, Lee RC, Li AF, Chao Y, Chi KH, Yen SH: Pre-operative chemoradiotherapy with oral tegafur-uracil and leucovorin for rectal cancer. J Surg Oncol 2005, 89:256–263. discussion 263–254PubMedView Article
- Ryan R, Gibbons D, Hyland JM, Treanor D, White A, Mulcahy HE, O'Donoghue DP, Moriarty M, Fennelly D, Sheahan K: Pathological response following long-course neoadjuvant chemoradiotherapy for locally advanced rectal cancer. Histopathology 2005, 47:141–146.PubMedView Article
- Bouzourene H, Bosman FT, Seelentag W, Matter M, Coucke P: Importance of tumor regression assessment in predicting the outcome in patients with locally advanced rectal carcinoma who are treated with preoperative radiotherapy. Cancer 2002, 94:1121–1130.PubMedView Article
- Bozzetti F, Andreola S, Bertario L: Pathological features of rectal cancer after preoperative radiochemotherapy. Int J Colorectal Dis 1998, 13:54–55.PubMedView Article
- Dworak O, Keilholz L, Hoffmann A: Pathological features of rectal cancer after preoperative radiochemotherapy. Int J Colorectal Dis 1997, 12:19–23.PubMedView Article
- Locker GY, Hamilton S, Harris J, Jessup JM, Kemeny N, Macdonald JS, Somerfield MR, Hayes DF, Bast RC Jr: ASCO 2006 update of recommendations for the use of tumor markers in gastrointestinal cancer. J Clin Oncol 2006, 24:5313–5327.PubMedView Article
- Ishihara S, Watanabe T, Kiyomatsu T, Yasuda K, Nagawa H: Prognostic significance of response to preoperative radiotherapy, lymph node metastasis, and CEA level in patients undergoing total mesorectal excision of rectal cancer. Int J Colorectal Dis 2010, 25:1417–1425.PubMedView Article
- Das P, Skibber JM, Rodriguez-Bigas MA, Feig BW, Chang GJ, Wolff RA, Eng C, Krishnan S, Janjan NA, Crane CH: Predictors of tumor response and downstaging in patients who receive preoperative chemoradiation for rectal cancer. Cancer 2007, 109:1750–1755.PubMedView Article
- Park YA, Sohn SK, Seong J, Baik SH, Lee KY, Kim NK, Cho CW: Serum CEA as a predictor for the response to preoperative chemoradiation in rectal cancer. J Surg Oncol 2006, 93:145–150.PubMedView Article
- Yoon SM, Kim DY, Kim TH, Jung KH, Chang HJ, Koom WS, Lim SB, Choi HS, Jeong SY, Park JG: Clinical parameters predicting pathologic tumor response after preoperative chemoradiotherapy for rectal cancer. Int J Radiat Oncol Biol Phys 2007, 69:1167–1172.PubMedView Article
- Kim CW, Yu CS, Yang SS, Kim KH, Yoon YS, Yoon SN, Lim SB, Kim JC: Clinical significance of pre- to post-chemoradiotherapy s-CEA reduction ratio in rectal cancer patients treated with preoperative chemoradiotherapy and curative resection. Ann Surg Oncol 2011, 18:3271–3277.PubMedView Article
- Kouri M, Pyrhonen S, Mecklin JP, Jarvinen H, Laasonen A, Franssila K, Nordling S: The prognostic value of DNA-ploidy in colorectal carcinoma: a prospective study. Br J Cancer 1990, 62:976–981.PubMedView Article
- Della Vittoria Scarpati G, Falcetta F, Carlomagno C, Ubezio P, Marchini S, De Stefano A, Singh VK, D'Incalci M, De Placido S, Pepe S: A specific miRNA signature correlates with complete pathological response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer. Int J Radiat Oncol Biol Phys 2012, 83:1113–1119.PubMedView Article
- Perez RO, Sao Juliao GP, Habr-Gama A, Kiss D, Proscurshim I, Campos FG, Gama-Rodrigues JJ, Cecconello I: The role of carcinoembriogenic antigen in predicting response and survival to neoadjuvant chemoradiotherapy for distal rectal cancer. Dis Colon Rectum 2009, 52:1137–1143.PubMedView Article
- Park JW, Lim SB, Kim DY, Jung KH, Hong YS, Chang HJ, Choi HS, Jeong SY: Carcinoembryonic antigen as a predictor of pathologic response and a prognostic factor in locally advanced rectal cancer patients treated with preoperative chemoradiotherapy and surgery. Int J Radiat Oncol Biol Phys 2009, 74:810–817.PubMedView Article
- Bonnen M, Crane C, Vauthey JN, Skibber J, Delclos ME, Rodriguez-Bigas M, Hoff PM, Lin E, Eng C, Wong A, Janjan NA, Feig BW: Long-term results using local excision after preoperative chemoradiation among selected T3 rectal cancer patients. Int J Radiat Oncol Biol Phys 2004, 60:1098–1105.PubMedView Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.