Prognostic significance of lymph node metastasis in esophageal squamous cell carcinoma

Accepted Manuscript Title: Prognostic significance of lymph node metastasis in Esophageal squamous cell carcinoma Authors: Hui Wang, Feiyan Deng, Qian Liu, Yuqing Ma PII: DOI: Reference:

S0344-0338(16)30660-4 http://dx.doi.org/doi:10.1016/j.prp.2017.01.023 PRP 51738

To appear in: Received date:

14-11-2016

Please cite this article as: Hui Wang, Feiyan Deng, Qian Liu, Yuqing Ma, Prognostic significance of lymph node metastasis in Esophageal squamous cell carcinoma, Pathology - Research and Practice http://dx.doi.org/10.1016/j.prp.2017.01.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Prognostic significance of lymph node metastasis in Esophageal squamous cell

carcinoma

Hui Wang1, Feiyan Deng2, Qian Liu1, Yuqing Ma1*

1The Department of Pathology, First Teaching Hospital of Xinjiang Medical

University, Xinjiang, China

2 The Department of Pathology, the Second Hospital affiliated to Henan Medical

College of Traditional Chinese medicine, Henan, China

*Corresponding authors, [email protected]

Abstract Prediction of lymph node metastasis in esophageal squamous cell carcinoma(ESCC) is very important to have prognostic significance. The objective of this study which involved a relatively large number of ESCC patients was to investigate the correlated factors for lymph node metastasis and prognosis in ESCC. We analyzed a retrospective review of 446 patients with ESCC treated by esophagectomy between January 2010 and July 2016. The relationship between lymph node metastasis and clinicopathological parameters were analyzed. The association between overall survival and clinicopathological factors were evaluated using univariate and multivariable Cox regression models. In the present study, 36.8% esophageal squamous cell carcinoma patients were histologically shown to have lymph node metastasis. Lymph

node

metastasis

was

closely

correlated

with

tumor

differentiation(p=0.016), perineural invasion (p=0.022), advanced stage tumor(p<0.001) and venous invasion(p<0.001). Kaplan-Meier survival analysis revealed that patients with tumor size, higher T stage, perineuronal invasion, lymph node metastasis, N stage and LNR higher than 0.2 had unfavorable prognosis(p<0.05). The univariate analysis revealed for overall survival that tumor size, pathological stage, perineuronal invasion, lymph metastasis, N stage, involved LNR were relevant prognostic indicators. Furthermore, tumor size, lymph metastasis, N stage and LNR could as independent prognostic factors.

Keywords: Esophageal squamous cell carcinoma, lymph node, clinicopathological factors, prognosis, perineural invasion

Introduction Esophageal carcinoma is one of the most frequently diagnosed cancers, as well as one of the most fatal cause of cancer-related mortality worldwide[1]. In Asian,

especially in China, most esophageal carcinomas (95%) are the squamous cell histology type[2]. The overall survival of esophageal carcinoma remains poor although some improvements have been achieved in treatment [3]. It is important to investigate the initiation and progression of ESCC and to study the associated prognostic factors. The lymph node metastasis incidence of ESCC is reported to be approximately 38.2%-43% [4, 5]. Lymph node metastasis is the important prognosis factor in patients with invasive ESCC[6]. According to previous studies, ESCC patients without nodal metastases generally have a more favorable prognosis after surgical resection[7, 8]. ESCC is associated with a risk of extensive lymphatic spread, even when the cancer is confined to the submucosa. As a result, lymph node status is an important issue that has attracted considerable attention in centers dedicated to the management of this disease. The TNM staging system has become accepted worldwide as the most important reference for treatment planning in clinical oncology and prognosis prediction for esophagus cancer[9]. In the American Joint Committee on Cancer (AJCC) TNM system, N is classified according to the number of metastatic lymph nodes as N0, no lymph node metastasis; N1, 1 to 2 metastatic lymph nodes; N2, 3 to 6 metastatic lymph nodes; and N3, more than 7 metastatic lymph nodes[10]. The concept of lymph node ratio(LNR) was discussed by some reports recently. LNR has been found to have prognostic significance in several kinds of tumors, such as lung, breast, colorectal cancer[11-13].The superiority of LNR classification has been reported by some investigators, and they also showed that it is an independent prognostic indictor for Esophageal cancer [14]. China has a relatively high incidence of ESCC, but there are not sufficient data about ESCC. We began to search for tumor characteristics related to lymph node metastasis. And we assessed whether classification of lymph node metastasis enhanced of prediction of outcomes in patients with ESCC. The present study involved a relatively large number of ESCC patients and the condition of lymph node metastasis. In addition, the relation between the other clinicopathological factors and survival was also evaluated. Materials and methods

Patients In this retrospective study, data were collected from the medical records of 446 consecutive patients who underwent esophagectomy at the first teaching hospital of Xinjiang medical university, from January 2010 and July 2015. All patients were followed up every 3 months for the first years, and once a year thereafter. Overall patient survival, defined as the time from operation to death or last follow up, was used as a measure of prognosis. Clinicopathologic data included the following information: name, gender, age, tumor stage, tumor size, nodal stage, degree of tumor differentiation. To be eligible for the study, patients were required to have pathologically confirmed ESCC. Lymph nodes were identified and retrieved from formalin-fixed surgical specimens. Paraffinembedded nodes were stained with hematoxylin and eosin(HE) and were examined microscopically for metastases. And then the cytokeratin(CK) antibody was stained, in order to confirm lymph node metastasis of ESCC. The LNR was defined as the ratio of the number of metastatic lymph nodes to the total lymph nodes pathologically examined. The patients was classified into two groups(LNR1: <0.2; LNR2≥0.2). Perineural invasion (PNI) was assessed on HE-stained slides, as positive if cancer cells infiltrated into the perineurium. The CK were tested based on standard immunostaining. All specimens were evaluated by an expert pathologist in the matter of esophageal cancer. Statistical analysis All statistical analyses were performed using the SPSS 14.0 statistical program. The categorical variables were compared using the χ2 test. The overall survival(OS) was calculated from the date of surgery to the date of death or until the date of the last time the patient was known to be alive.

Survival analyses were constructed by

Kaplan–Meier curves with log-rank tests for significance. Univariate and multivariate survival analyses ware performed using cox proportional hazards model. P<0.05 was considered to be statistically significant. This study was approved by the ethics committee of this hospital. Results

The correlations between lymph node metastasis and the clinicopathological characteristics of ESCC patients There are totally 446 patients involved in this study at the first teaching hospital of Xinjiang medical university, including 310 males and 136 females. Table 1 shows the clinicopathological parameters of the studied patients. The average age was 62 years old (range, 35-84years). According to histological type, well differentiation tumors were observed in 64 (14.3%) patients, moderate in 249 (55.85%) patients, and poor in 133 (28.9%) patients. Lymph node metastasis was confirmed pathologically in 164 (36.8%) patients(Fig 1). And PNI was confirmed pathologically in 91(25.3%) patients. Of the 446 total patients, 282(63.2%) was classified as N0, 93 were classified as N1, 52 were classified as N2, and 14 were classified as N3. When classified into two groups according to LNR, Patients with a LNR less than 0.2 accounted for 84.5% of the whole cohort. In the present study, the rate of lymph node metastasis was 36.8%. The relationship between lymph node metastasis and clinicopathological findings are summarized in table 2. χ2 test analysis showed that poor differentiation, tumor with nervous invasion, advanced stage tumor and venous invasion were associated with lymph node metastasis(all p<0.05). Poor differentiation carcinomas were more likely to

have

lymph node metastases than well differentiation and

moderate

differentiation(p=0.016).There was no significant difference in gender, age, or tumor size for lymph node metastasis(Table 2).It was demonstrated that tumor differentiation, pathological stage, venous invasion and perineural invasion were predictive factors for lymph node metastasis. Prognostic significance of lymph node metastasis on patients with ESCC The Kaplan-meier method was performed to further analyze the association of lymph node metastasis with prognosis of the 446 ESCC patients. We found that the survival of patients with node metastasis positive was significantly worse than that of patients with node metastasis negative(Fig 2A). thus, lymph node metastasis could affect the prognosis of ESCC patients. next, we reported a significant correlation between the N stage subgroups and the survival. We found that the survival of

patients with N0 stage was significantly better than that of the patients with N1, N2 or N3(Fig 2B). As expected, we documented a correlation between the LNR and the overall survival. Likewise, the survival of the patients LNR<0.2 was significantly better than that of the patients with LNR≥0.2(Fig 2C). lymph node metastasis classification in the present study revealed a distinct prognostic significance. Comparison of survival according to tumor size, T stage, perineuronal invasion Survival analysis showed significant difference in overall survival among different tumor size, T stage, PNI in this entire data(Fig 3). Based on tumor size, each stage was divided into small size tumor(<4cm) and large size tumor(≥4cm). Tumors larger than 4cm were more likely to have poor prognostic than smaller tumors(Fig 3A). There were significant differences of overall survival between two group(P=0.0001). ESCC patients with T1 and T2 showed an extremely good outcome and the survival rate was significantly higher for patients with T3、T4. Survival based on the presence or absence of PNI involvement is depicted in Fig 3C. Univariate and multivariate survival analyses To identify independent prognostic factors predicting survival for ESCC, Cox regression analysis was performed. The clinicopathological variables tested in univariate analyses are shown in Table3. We found that tumor size, pathological stage, perineural invasion, lymph metastasis, N stage, involved LNR(all p<0.05) had statistically significant associations with OS of ESCC patients after curative surgery, while sex, age, tumor differentiation, venous invasion were not influential to OS(p>0.05). All of these potential important factors identified in univariate analysis were included in a multivariate Cox proportional hazard model for analysis in table 4. Tumor size, lymph node metastasis, N stage, involved LNR were found to be the significantly independent prognostic factors for OS of ESCC patients. Discussion Unfortunately, esophagus carcinoma is associated with a largely unfavorable outcome and aggressive tumor biology[15]. The identification of prognostic factors is very important for predicting ESCC patients, survival. Amongst prognostic factors predicting improved survival, lymph node metastasis is well known to be one of the

most important prognostic factors in esophageal cancer. The TNM staging system has become accepted worldwide as the most important reference for treatment planning in clinical oncology and prognosis prediction for esophagus cancer. Accurate N staging is of great importance for determining prognosis and therapy of esophageal cancer patients. In this study, we evaluated prognostic factors, particularly examining the prognostic significance of different types of LNR involvement in ESCC patients. To the best of our knowledge, this is a big retrospective study performed to date. Besides, most studies have much more cases of esophagus adenocarcinoma than esophagus squamous cell carcinoma[16, 17]. Reports based on esophagus squamous cell carcinoma predominant databases are few. Therefore, we conducted this study in 446 patients with ESCC. The lymph node metastasis incidence of all ESCC patients was 36.8% in the current study, which are similar to lower than previous results[5]. We found that lymph node metastasis was associated with poor differentiation, tumor with perineural invasion advanced stage tumor and venous invasion. Consistent with previously reported data, lymph node metastasis proved to be an important indicator of survival. According to our findings, patients with LNR≥0.2 worse than patient with LNR<0.2. Other authors examining LNR have found that less than 1.5 compared to 1.5 or more LNR to be a significant cutoff[18]. It is difficult to evaluate whether LNR is better than the number of metastasis lymph nodes. In the present study, we have figured out that both LNR and N stage were associated overall survival in ESCC. In this regard, our data clearly indicate that both the level of lymph node metastases and the N stage were independent prognostic factors, by multivariate analysis using the Cox proportional hazards model. This demonstrates that the N stage and level of lymph node metastases are complementary and are indispensable for evaluating the status of lymph node metastasis. Perineural invasion is a process by which cancer cells invade the perineurium or neural fascicles. In the 7th edition of the AJCC staging system for the classifcation of squamous cell carcinomas which published in 2010, PNI was included among the high-risk factors for patients with ESCC. PNI seemed to possess the necessary potential to provide useful information for the clinical management of patients with ESCC . It is a crucial for the local spread of tumor associated with poor prognosis.

However, there were only a few studies which had investigated on the presence and prognostic significance of perineural invasion in esophageal cancer and had not reached a consensus. We found that PNI was positive in 25.3% of patients, and the incidence of PNI increased with lymph node metastasis. We found that the PNI was accompanied by worse prognosis. Furthermore, PNI was a significant prognostic factors by using univariate analysis for overall survival. However, we did not obtain significant difference in multivariate analyses for PNI. Hence, we suggest that the PNI should be consider for precise evaluation of lymph node status. But the prognosis needs to be confirmed by future studies. Tumor size has been found to be another independent prognostic factor for ESCC. Chen et al. reported that tumor size>5cm was associated with worse 5-year survival rates(p=0.003)[19].Our results were consistent with the above studies. Based on the previous studies and our results, the tumor size was considered to have significant clinical meaning among unfavbor prognosis in ESCC. However, Wang et al. reported that tumor length in cm were not influential to overall survival (p=0.192)[14]. Then, large sample clinical analysis is required for further study. In conclusion, our study indicated that lymph node metastasis was important prognostic indictors for overall survival for patients with ESCC. Both N stage and LNR were important prognostic indictors for overall survival in ESCC cancer by both univariate and multivariate analysis. In addition, the multivariate analysis indicated that tumor size was also independent prognostic factor. PNI may be useful for evaluating the status of lymph node metastasis in ESCC. Acknowledgements The work was supported by National Natural Science Foundation of China (No. 81260308). Many thanks go to my mentor and the members in my team for the careful guidance and technical support. Compliance with ethical standards Conflict of interest: The authors declare no conflicts of interest associated with this article. Authors’ contributions

Hui Wang and Yuqing Ma designed the experiment, interpreted the data, and prepared the manuscript. Hui Wang, Feiyan Deng and Qian Liu collected the data, and helped to prepare the manuscript. All authors read and approved the final manuscript.

Reference 1.

Pisani, P., et al., Estimates of the worldwide mortality from 25 cancers in 1990. Int J Cancer, 1999. 83(1): p. 18-29.

2.

Torre, L.A., et al., Global cancer statistics, 2012. CA Cancer J Clin, 2015. 65(2): p. 87-108.

3.

Siegel, R., D. Naishadham, and A. Jemal, Cancer statistics, 2013. CA Cancer J Clin, 2013. 63(1): p. 11-30.

4.

Wang, J., et al., Evaluation of the 7th edition of the TNM classification in patients with resected esophageal squamous cell carcinoma. World J Gastroenterol, 2014. 20(48): p. 18397-403.

5.

Wu, N., et al., Prognostic significance of lymph node characteristics on survival in esophageal squamous cell carcinomas. Wien Klin Wochenschr, 2013. 125(1-2): p. 26-33.

6.

Chen, S.B., et al., Prognostic factors and outcome for patients with esophageal squamous cell carcinoma underwent surgical resection alone: evaluation of the seventh edition of the American Joint Committee on Cancer staging system for esophageal squamous cell carcinoma. J Thorac Oncol, 2013. 8(4): p. 495-501.

7.

Dhar, D.K., et al., Appraisal of a revised lymph node classification system for esophageal squamous cell cancer. Ann Thorac Surg, 2007. 83(4): p. 1265-72.

8.

Niwa, Y., et al., The Prognostic Relevance of Subcarinal Lymph Node Dissection in Esophageal Squamous Cell Carcinoma. Ann Surg Oncol, 2016. 23(2): p. 611-8.

9.

Peyre, C.G., et al., Predicting systemic disease in patients with esophageal

cancer after esophagectomy: a multinational study on the significance of the number of involved lymph nodes. Ann Surg, 2008. 248(6): p. 979-85. 10.

Yang, H.X., et al., Modification of nodal categories in the seventh american joint committee on cancer staging system for esophageal squamous cell carcinoma in Chinese patients. Ann Thorac Surg, 2011. 92(1): p. 216-24.

11.

Gajra, A., et al., Effect of number of lymph nodes sampled on outcome in patients with stage I non-small-cell lung cancer. J Clin Oncol, 2003. 21(6): p. 1029-34.

12.

Weir, L., et al., Prognostic significance of the number of axillary lymph nodes removed in patients with node-negative breast cancer. J Clin Oncol, 2002. 20(7): p. 1793-9.

13.

Qiu, H.B., et al., Ratio of metastatic to resected lymph nodes enhances to predict survival in patients with stage III colorectal cancer. Ann Surg Oncol, 2011. 18(6): p. 1568-74.

14.

Wang, N., et al., Prognostic significance of lymph node ratio in esophageal cancer. Tumour Biol, 2015. 36(4): p. 2335-41.

15.

Wilson, M., et al., Prognostic significance of lymph node metastases and ratio in esophageal cancer. J Surg Res, 2008. 146(1): p. 11-5.

16.

Rizk, N., et al., The prognostic importance of the number of involved lymph nodes in esophageal cancer: implications for revisions of the American Joint Committee on Cancer staging system. J Thorac Cardiovasc Surg, 2006. 132(6): p. 1374-81.

17.

Rice, T.W., et al., Refining esophageal cancer staging. J Thorac Cardiovasc Surg, 2003. 125(5): p. 1103-13.

18.

Huang, J., et al., Value of Positive Lymph Node Ratio for Predicting Postoperative Distant Metastasis and Prognosis in Esophageal Squamous Cell Carcinoma. Oncol Res Treat, 2015. 38(9): p. 424-8.

19.

Chen, J., et al., Number and location of positive nodes, postoperative radiotherapy, and survival after esophagectomy with three-field lymph node dissection for thoracic esophageal squamous cell carcinoma. Int J Radiat Oncol Biol Phys, 2012. 82(1): p. 475-82.

Fig1 Lymph node metastases were identified by HE staining(A) and the immunostaining marker CK( B).

Fig 2 Kaplan-Meier overall survival curves for 446 patients. The patients were stratified by lymph node metastasis(A), N stage(B), the lymph node ratio(C)LNR status.

Fig 3 kaplan- meier curves for overall survival of ESCC according to tumor size( A), pathologic T stage(B), and perineuronal invasion(C).

Fig1 Lymph node metastases were identified by HE staining(A) and the immunostaining marker CK( B).

Fig2 Kaplan-Meier overall survival curves for 446 patients. The patients were stratified by lymph node metastasis(A), N stage(B), the lymph node ratio(C)LNR status.

Fig3 kaplan- meier curves for overall survival of ESCC according to tumor size( A), pathologic T stage(B), and perineuronal invasion(C).

Table 1. Characteristics of ESCC patients. Characteristics and finding

n=446

Age(years) Range

35-84

median

62.40

Tumor size(cm) Range

0.2-9.0

median

3.6

Gender male

310(69.5%)

female

136(30.5%)

Differentiation well

64（14.3%）

moderate

249（55.8%）

poor

133(28.9%)

pathological stage T1

41 (9.2%)

T2

29(6.5%)

T3

132(29.6%)

T4

244(54.7%)

Lymph metastasis negative

282（63.2%）

positive

164（36.8%）

N stage N0

282(63.2%)

N1

98(21.9%)

N2

52(11.7%)

N3

14(3.1%)

Involved LNR

<0.2

377(84.5%)

≥0.2

69(15.5%)

Venous invasion negative

339(76.1%)

positive

107(23.9%)

Perineuronal invasion negative

333(74.7%)

positive

113(25.3%)

Table 2 The correlation between lymph node metastasis and clinicopathological characteristics. Lymph node metastasis character Negative

χ2

P value

0.243

0.671

1.806

0.405

2.983

0.084

8.299

0.016

23.771

<0.001

47.706

<0.001

5.412

0.022

positive

gender Male

193

117

female

88

48

<60

161

86

≥60

120

79

<4

170

86

≥4

111

79

well

50

14

moderate

155

94

poor

76

57

T1

37

4

T2

22

7

T3

89

43

T4

133

111

negative

248

91

positive

33

74

negative

223

110

positive

58

55

Age, y

Size

Differentiation

pathological stage

Venous invasion

Perineuronal invasion

Table 3 Univariate analysis of factors associated with overall survival(OS). character

95%CI

HR

P value

0.638-1.288

1.215

0.130

0.550-1.064

0.765

0.111

1.436-2.675

1.960

<0.001

0.945-1.562

1.215

0.130

1.155-1.753

1.423

0.001

0.800-1.805

1.202

0.376

gender Male/female Age,y ≥60/<60 Size ≥4/<4 Differentiation Poor/moderate /well pathological stage T1/T2/T3/T4 Venous invasion Present/absent Perineuronal invasion Present/absent

1.098-2.354

1.608

0.015

1.424-2.703

1.962

<0.001

1.257-1.767

1.490

<0.001

1.482-3.146

2.159

<0.001

Lymph metastasis Present/absent N stage N0/N1/N2/N3 Involved LNR ≥0.2/<0.2

Table 4 Multivariate analysis of factors associated with overall survival. character

95%CI

HR

P value

1.319-2.795

1.920

0.001

0.939-1.554

1.208

0.141

Size ≥4/<4 pathological stage T1/T2/T3/T4 Perineuronal invasion Present/absent

0.863-1.952

1.298

0.210

1.239-2.559

1.780

0.002

1.131-1.676

1.377

0.001

1.168-2.897

1.839

0.009

Lymph metastasis Present/absent N stage N0/N1/N2/N3 Involved LNR ≥0.2/<0.2