Detection and Clinical Significance of Lymph Node Micrometastasis in Gastric Cardia Adenocarcinoma

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1 The Journal of International Medical Research 2012; 40: [first published online ahead of print as 40(1) 3] Detection and Clinical Significance of Lymph Node Micrometastasis in Gastric Cardia Adenocarcinoma Y RU, L ZHANG, Q CHEN, SG GAO, GP WANG, ZF QU, TY SHAN, N QIAN AND XS FENG Department of Oncology, Cancer Institute, First Affiliated Hospital of Henan University of Science and Technology, Luoyang, China OBJECTIVES: Lymph node micro metastasis was investigated in gastric cardia adenocarcinoma (GCA) patients without lymph node metastasis on routine pathological examination. The relationship between micrometastasis and clinicopathological features was also evaluated. METHODS: A total of 349 lymph nodes were obtained from 45 patients with GCA. Micrometastases were detected by immunohistochemical staining for the markers cytokeratin 19 (CK19) and CD44 variant 6 (CD44v6). RESULTS: A total of 33 lymph nodes (9.5%) from 15 patients (33.3%) were positive for CK19. Of these, 27 lymph nodes (7.7%) from 12 patients (26.7%) were also positive for CD44v6. Micrometastasis was significantly related to depth of tumour invasion and Lauren classification (intestinal or diffuse). The recurrence rate was significantly higher and 2-year survival rate significantly lower in patients with than in those without lymph node micrometastasis, showing the necessity of detecting micrometastasis in GCA patients who test negative for lymph node metastasis on routine examination. CONCLUSION: CK19 and CD44v6 were shown to be good markers for micrometastasis detection. KEY WORDS: GASTRIC CARDIA CARCINOMA; PROGNOSIS; LYMPH NODE MICROMETASTASIS; DETECTION; CYTOKERATIN 19; CD44 VARIANT 6; IMMUNOHISTOCHEMISTRY Introduction Metastasis is one of the fundamental biological characteristics of malignant tumours and is the leading cause of death in cancer patients. It is the result of a series of multiple intrinsically linked steps. Tumour micrometastasis, in which single, scattered or clustered cancer cells exist in tissues or organs outside those of the primary tumour, is an early stage in the development of metastatic spread. These transferred cells are usually not detectable by haematoxylin and eosin (H&E) staining or other routinely used methods. One of the most common malignancies in China is gastric carcinoma, for which the most important prognostic factor is lymph node metastasis. 1 3 Recent advances in immunohistochemical and molecular biology techniques have made it possible to detect lymph node micrometastasis not found on routine H&E evaluation. 4 8 Lymph node micrometastasis has an impact on the staging of gastric carcinoma and is a 293

2 significant risk factor in determining the prognosis of these patients. 9 In recent years the incidence of gastric cardia adenocarcinoma (GCA) has risen rapidly, whereas that of distal gastric adenocarcinoma has steadily decreased. These trends suggest that GCA is different in nature from distal gastric adenocarcinoma. There are a number of reports on lymph node micrometastasis in distal gastric adenocarcinoma 10,11 but little has been published on such micrometastases in GCA. Cytokeratin 19 (CK19), which originates from epithelial cells, is a good tissue-specific marker for epithelial tumour micro - metastasis and is highly expressed in the intestinal tract but not in normal lymph node tissue or blood. In addition, the cell surface adhesion molecule, CD44, has been gaining interest: CD44 variant 6 (CD44v6), a CD44 splicing variant, can influence tumour cell invasion and metastatic behaviour and shows tumour specificity. 12 In the present study, immunohisto - chemical staining with anti-ck19 and anti- CD44v6 monoclonal antibodies was performed to detect lymph node micrometastasis in GCA patients testing negative for lymph node metastasis on routine pathological examination. Possible correlated factors were investigated and the clinical significance of lymph node micrometastasis is discussed. Patients and methods PATIENTS Patients with no lymph node metastases were recruited from those with pathologically confirmed GCA undergoing gastrectomy and lymphadenectomy at the First Affiliated Hospital of Henan University of Science and Technology, Luoyang, China, between January 2004 and December None of the patients had received preoperative chemotherapy or radiotherapy. Patients were followed for up to 41 months; recurrence in the stomach and mortality rates during the follow-up period were recorded. All patients provided written informed consent for samples to be taken and the study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Henan University of Science and Technology, Luoyang, China. TUMOUR EVALUATION Tumour size (defined as the maximum diameter of the surface of the primary tumour), the depth of invasion and the degree of differentiation, were recorded. Tumours were staged using the tumour, node, metastasis (TNM) classification 13 and histological type was determined, according to the Lauren classification of gastric adenocarcinomas, into intestinal and diffuse types. 14 HISTOPATHOLOGY Lymph nodes were collected from each patient, fixed in formalin, paraffinembedded and sliced (4 µm thick) for histopathological examination. One slice from each lymph node biopsy was then selected at random for H&E staining and examined under a microscope independently by two experts in biopsy film reading. Samples obtained during the surgical removal of benign gastric lesions were used as negative controls and those from GCA patients showing metastases on H&E examination were used as positive controls. IMMUNOHISTOCHEMISTRY Three slices (4 µm thick) cut from the formalin-fixed, paraffin-embedded lymph node specimens were used for immunohistochemical testing, using mouse 294

3 antihuman CD44v6 and CK19 monoclonal antibodies, with a streptavidin peroxidase kit (Fuzhou Maixin Biotechnology Development, Fuzhou, China) according to the manufacturer s instructions. The results were visualized using a diaminobenzidine chromogenic enzyme substrate kit (Fuzhou Maixin Biotechnology Development); CK19- positive and CD44v6-positive staining appeared as a brown/yellow coloration in the cell membrane and/or cytoplasm and was regarded as a definite positive result when found in two adjacent biopsies from the same specimen. A double-blind method was used to observe the results. Specimens staining positive for CK19 or CD44v6 were examined using a high-power ( 400) microscope to confirm the presence of micrometastases. STATISTICAL ANALYSES Calculation of the sample size required was determined using Power and Precision software (Biostat, Englewood, NJ, USA). Relationships between lymph node micrometastasis and clinicopathological parameters were investigated using the χ 2 - test. Survival of patients with GCA was evaluated using Kaplan Meier survival analysis and the log-rank test. A P-value < 0.05 was considered to be statistically significant. Data were analysed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). The study was designed with the help of an expert statistician. Results A total of 45 patients with pathologically confirmed GCA with no lymph node metastases on routine pathological examination were included in the study. They comprised 36 men and nine women, ranging in age from 41 to 77 years, with a mean age of 62.6 years. Tumour diameter was 2 cm in 11 patients and > 2 cm in 34 patients. Tumours were staged as T 1 (n = 6), T 2 (n = 15), T 3 (n = 20) or T 4 (n = 4). On histopathological examination there were nine cases of welldifferentiated, 17 cases of moderately differentiated and 19 cases of poorly differentiated adenocarcinoma. A total of 349 lymph nodes were collected, giving a mean of 7.8 per patient. Ten lymph nodes from patients with benign gastric conditions (seven cases of gastric ulcer and three of leiomyoma) were used as negative controls. Two metastatic lymph nodes from GCA patients with positive H&E staining were used as positive controls. All 349 lymph node specimens were confirmed negative for metastases on H&E staining. A total of 33 lymph nodes (9.5%) from 15 patients (33.3%) were positive for CK19. Of these, 27 lymph nodes (7.7%) from 12 patients (26.7%) were also positive for CD44v6 (Fig. 1). Positive cells were either scattered singly or gathered in small groups of two, three or more cells in the lymph sinuses, mainly the marginal sinus. 15 All 10 control lymph nodes from patients with benign conditions were negative on H&E, CK19 and CD44v6 staining. The two control lymph nodes showing positive H&E staining were also positive for both CK19 and CD44v6. The relationships between lymph node micrometastasis and various clinicopathological features are shown in Table 1. The presence of CK19-positive or CD44v6-positive cells was significantly related to depth of invasion (P = and P = 0.046, respectively) and Lauren classification (P = and P = 0.007, respectively), but not to age, sex, tumour size or degree of differentiation. Three patients were lost to follow-up. The remaining 42 patients were followed up for 6 295

4 A B 25 µm 25 µm FIGURE 1: Lymph node sections from a patient with gastric cardia adenocarcinoma showing small clusters of cells immunohistochemically positive for (A) cytokeratin 19 or (B) CD44 variant 6 TABLE 1: Relationships between lymph node micrometastases, as indicated by the presence of cells positive for cytokeratin-19 (CK19) and CD44 variant 6 (CD44v6), and clinicopathological parameters in patients with gastric cardia adenocarcinoma CK19-positive CD44v6-positive Total No. of Statistical Statistical Parameter patients n significance n significance Sex NS NS Male Female Age NS NS < 50 years years Tumour size NS NS 2 cm > 2 cm Depth of invasion P = P = Mucosa Lamina muscularis mucosae Serous membrane Degree of differentiation NS NS Well differentiated Moderately differentiated Poorly differentiated Lauren classification P = P = Intestinal type Diffuse type NS, no statistically significant difference (P > 0.05; χ 2 -test). 41 months. Among the 15 patients with lymph node micrometastasis, recurrence occurred in seven (46.7%) and four died 6 25 months after surgery. Among the

5 patients without lymph node micrometastasis, recurrence occurred in one (3.7%); this patient died 14 months after surgery. The recurrence rate was significantly higher in the micrometastasis group than in the nonmicrometastasis group (χ 2 = 8.925, P = 0.003). In addition, the 2-year survival rate (63.6%) in the micrometastasis group was significantly lower than that in the nonmicrometastasis group (95.6%) (P = 0.011; Fig. 2). Discussion The occurrence of GCA and its evolution is a very complex process. Lymph node metastasis is the most important prognostic factor for GCA. 16 Determining the presence of lymph node metastasis is also important for postsurgical treatment staging. Lymph node micrometastasis, where tumour diameter is < 2 mm, is difficult to detect on routine pathological examination. Cytokeratins are components of epithelial cells and the intermediate filaments of epithelial cancer cells. They occur widely in epithelial cells but not in mesenchymal tissues. In the process of cell malignant transformation and tumorigenesis, keratin continues to exist in epithelial tumours, however mesenchymal cells fail to express keratin even after malignant transformation. This provides a theoretical basis for the idea that the presence of keratin in the digestive tract indicates that metastasis to the digestive tract has occurred. More than 20 kinds of cytokeratins have been discovered to date. CK19 is highly expressed in the digestive tract, but not in normal lymph node tissue or blood, and is a good tissue-specific marker for epithelial tumour micrometastasis. In recent years CD44, which is a typical cell surface adhesion molecule, has gained increasing interest. Its structure and function 100 Positive for LNM 80 Survival (%) Negative for LNM Survival time (months) FIGURE 2: Kaplan Meier survival curve for the patients with gastric cardia adenocarcinoma according to whether they were positive (n = 15) or negative (n = 27) for lymph node micrometastasis (LNM); the between-group 2-year survival rate differed significantly (63.6% versus 95.6%, P = 0.011) 297

6 undergo significant changes during the process of malignant transformation, affecting tumour cell invasion and transfer behaviour. CD44v6, a splicing variant of CD44, belongs to a class of single-chain membrane glycoproteins. It is involved in lymphocyte homing, cell adhesion and migration, as well as a number of other functional activities. Protein expression of CD44v6 has been shown to be related to disease progression, lymph node metastasis and prognosis in gastric carcinoma. 17,18 In the present study, micrometastasis indicated by the presence of CK19 with or without CD44v6 was seen in 33 lymph nodes (9.5%) from 15 patients (33.3%); this is consistent with the results of Yun et al. 10 We found that lymph node micrometastasis was correlated with the depth of invasion and Lauren classification: the micrometastasis rate was much higher in diffuse than in intestinal cancers. This finding may be explained by the association of an E- cadherin gene mutation with diffuse-type gastric cancer, which results in decreased cell adhesion and easily separated and aggressive tumour cells. 19 As in previous reports, 20,21 in the present study there was no significant correlation between lymph node micrometastasis and age, sex, tumour size or degree of tumour differentiation. Lee et al. 4 and Yasuda et al. 5 reported that the 5-year survival rate was lower in gastric cancer patients with lymph node micrometastasis than in those without. Other studies, however, have reported that lymph node micrometastasis is not associated with prognosis. 20 In the present study, the recurrence rate was higher and the 2-year survival rate lower in the micrometastasis group than in the nonmicrometastasis group. This suggests that the detection of lymph node micrometastasis enables accurate prognostic and recurrence predictions. It has been shown that lymph node micrometastasis can be seen even in the early stages of gastric cancer. 22 Endoscopic resection for early-stage gastric cancer must, therefore, be performed with utmost precision. Even though minimally invasive endoscopic treatment had been selected to treat early gastric cancer in the patients included the present study, it was possible that micrometastases were present. In conclusion, the results of the present study have demonstrated the need to test for micrometastasis in GCA patients who are negative for lymph node metastasis on routine examination, because the presence of micrometastasis is an important prognostic indicator. CK19 and CD44v6 were shown to be good markers for micrometastasis detection. Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 15 June 2011 Accepted subject to revision 4 July 2011 Revised accepted 21 December 2011 Copyright 2012 Field House Publishing LLP References 1 Saito H, Fukumoto Y, Osaki T, et al: Prognostic significance of level and number of lymph node metastases in patients with gastric cancer. Ann Surg Oncol 2007; 14: Barbour AP, Rizk NP, Gonen M, et al: Lymphadenectomy for adenocarcinoma of the gastroesophageal junction (GEJ): impact of adequate staging on outcome. Ann Surg Oncol 2007; 14: Al-Moundhri MS, Al-Bahrani B, Burney IA, et al: The prognostic determinants of gastric cancer treatment outcome in Omani Arab patients. Oncology 2006; 70: Lee E, Chae Y, Kim I, et al: Prognostic relevance of immunohistochemically detected lymph 298

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