Changes of Tgfb1 and Tgfbrii Expression in Esophageal Precancerous and Cancerous Lesions: a Study of a High-Risk Population in Henan, Northern China

Diseases of the Esophagus (2002) 15, 74–79 Ó 2002 ISDE/Blackwell Publishing Asia http://www.paper.edu.cn

Original article

Changes of TGFb1 and TGFbRII expression in esophageal precancerous and cancerous lesions: a study of a high-risk population in Henan, northern China

Q. Zhou,1 L. Dong Wang,1,2 F. Du,1 Y. Zhou,3 Y. Rui Zhang,4 B. Liu,5 C. Wei Feng,6 S. Shan Gao,1 Z. Min Fan,1 C. S. Yang,7 S. Zheng2 1Laboratory for Cancer Research, College of Medicine, Zhengzhou University, Zhengzhou, Henan; 2Cancer Institute, Zhejing University, Hangzhou, China; 3Department of Oncology, The First Aﬃliated Hospital of Zhengzhou University and 4Department of Gastroenterology, Henan Province Hospital, Zhengzhou, Henan; 5Department of Gastroenterology, Tong Ren Hospital, Capital Medical University, Beijing; 6Department of Gastroenterology, The Second Aﬃliated Hospital of Zhengzhou University, Zhengzhou, Henan, China; 7Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, NJ, USA

SUMMARY. The level of transforming growth factor b1 (TGFb1) and transforming growth factor bII receptor (TGFbRII) was determined immunohistochemically in normal tissues and tissues with different severities of lesions (basal cell hyperplasia, BCH; dysplasia, DYS; carcinoma in situ, CIS; and squamous cell carcinoma, SCC) from surgically resected human esophagi and esophageal biopsies of symptom-free subjects. The samples were from an area with high esophageal cancer incidence in northern China (Linzhou, formerly Linxian, and nearby county Huixian in Henan Province). Peroxidase immunostain (ABC) and conventional hematoxylin and eosin stain were used. The tissue sections were incubated with antibodies of TGFb1 and TGFbRII overnight. The immunoreactivity was observed in cytoplasm of the esophageal specimen. From normal to BCH to DYS to CIS and to SCC, the positive immunostaining rates for TGFb1 increased significantly (P < 0.05). A linear correlation between the positive immunostaining rates of TGFb1 and the different lesions was observed (P < 0.05). From well- to moderately- and poorly differentiated SCC, the positive immunostaining rates for TGFb1 decreased gradually, but the difference was not significant (P > 0.05). In contrast, with the lesions progressing from normal to BCH to DYS to CIS and to SCC, the positive immunostaining rates for TGFbRII decreased significantly (P < 0.05). From well- to moderately- and poorly differentiated SCC, the positive immunostaining rates for TGFbRII decreased significantly (P < 0.05). There was a linear correlation between the positive rates of TGFbRII and different lesions and SCC differentiation (P < 0.05). The present results indicated that the alterations of TGFb1 and TGFbRII is a frequent event in esophageal multistage carcinogenesis, the absent or lower expression of TGFbRII may lead to the loss of cell proliferation control by TGFb1 and the overexpression of TGFb1 may be a negative feedback response caused by the lower expression of TGFbRII protein.

INTRODUCTION EC incidence between the high- and low-incidence area could be ashigh as500:1. 1–4 Linzhou city Esophageal carcinoma (EC) is one of the most (formerly Linxian) and nearby county Huixian in common malignant diseases in the world with Henan province have been well recognized ashigh- remarkable geographic distribution; the ratio for incidence area for EC; the average incidence rates for men and women are 161 and 103 per 100 000, respectively. EC remains the leading cause of cancer- related death in these areas.5 Esophageal carcino- Address correspondence to: Dr Li Dong Wang, Laboratory for genesis is considered as a multistage progressive Cancer Research, College of Medicine, Zhengzhou University, Zhengzhou, Henan, 450052, People’sRepublic of China. process. The early indicator for the subject predis- Tel. ⁄ Fax: (+86) 371 6970165; E-mail: [email protected] posed to EC is abnormal hyperproliferation of

74 中国科技论文在线 http://www.paper.edu.cn esophageal epithelial cells, morphologically manifes- China. No selection process was involved. Out of the ted as basal cell hyperplasia (BCH) and dysplasia 32 subjects examined, there were 20 men (28–70 years (DYS), which have been considered as precancerous of age with a mean ± SD of 50 ± 7) and 12 women lesions of EC.5–9 However, the information for the (25–69 yearsof age with a mean ± SD of 46 ± 17). mechanism of esophageal epithelial cell proliferation The biopsies were taken each from the middle-third regulation isvery limited. of the esophagus (30–32 cm from the incisor teeth) Recent studies indicate that some polypeptides from each subject. All the tissues were fixed with 85% may be involved in epithelial cell proliferation and alcohol, embedded with paraffin, and serially sec- differentiation, which may play an important role in tioned at 5 lm. The sections were mounted onto the malignant transformation of the epithelial cells. histostick-coated slides. Four or five adjacent ribbons Transforming growth factor b1 (TGFb1) issucha were collected for histopathologic analysis (hematox- polypeptide, inhibiting cell proliferation and inducing ylin and eosin stain) and for immunohistochemical cell differentiation.10–14 TGFb1 could induce the staining. formation of tissue matrix. It has been frequently observed that many different kinds of human cancer Histopathologic analysis cells lost their response to TGFb1, which hasbeen considered as one of the vital stages in carcinogenesis. Histopathologic diagnosis for esophageal epithelia The inhibition effect of TGFb1 on cell proliferation wasmade according to cellular morphologic changes dependson a compound of two related serine ⁄threon- and tissue architecture using previously established ine kinase trans-membrane proteinscalled receptor I criteria.26 In brief, the normal esophageal epithelium (TGFbRI) and II (TGFbRII). The inactivation of contained one to three proliferating basal cell layers; each receptor will lead to TGFb1 resistance.15–25 So the papillae were confined to the lower half of the far, the information for the alterationsof TGF b1 and epithelium. In BCH, the thickness of the proliferating TGFbRII expression in multistage progress of eso- basal zone surpassed 15% of the total epithelial phageal carcinogenesis is very limited. thickness. DYS was characterized by nuclear atypia To characterize the changesof TGF b1and (enlargement, pleomorphism, and hyperchromasia), TGFbRII expression in human esophageal carcino- loss of normal cell polarity, and abnormal tissue genesis, the present study was undertaken to deter- maturation. SCC wascharacterized by confluent and mine the protein level of TGFb1 and TGFbRII in invasive sheets of cohesive, polymorphous cells with normal tissues and tissues with different severities of hyperchromatic nuclei. lesions of BCH, DYS, carcinoma in situ (CIS), and squamous cell carcinoma (SCC) from surgically Immunohistochemical staining resected human esophagi and esophageal biopsies of symptom-free subjects. The subjects were from Linz- Anti-TGFb1 antibody isa polyclonal rabbit antiserum hou and nearby county Huixian in Henan province, against the C-terminal domain of TGFb1 of human high-incidence areasfor EC in northern China. origin (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-TGFbRII antibody isa polyclonal rabbit antiserum against the C-terminal domain of TGFbRII of MATERIALS AND METHODS human origin (Santa Cruz Biotechnology, Santa Cruz, CA). The avidin–biotin–peroxidase complex method Tissue collection and processing was used for the immunostaining of TGFb1 and A total of 74 surgically resected primary EC speci- TGFbRII. In brief, after de-waxing, inactivating menswere collected from the patientsin Linzhou and endogenous peroxidase activity and blocking cross- Huixian. Out of the 74 specimens examined, 44 were reactivity with normal serum (Vectastain Elite Kit; from men (40–74 yearsof age with a mean ± SD of Vector, Burlingame, CA), the sections were incubated 56 ± 9) and 30 were from women (37–69 yearsof overnight at 4°C with a diluted solution of the primary age with a mean ± SD of 54 ± 7). All the patients antibodies(1:100 for TGF b1 and TGFbRII). Location had not received either chemotherapy or radiother- of the primary antibodies was achieved by subsequent apy before surgery. The resected esophageal speci- application of a biotinylated antiprimary antibody, an menswere cut longitudinally and flattened. Iodine avidin–biotin complex conjugated to horseradish per- staining with Lugo’s solution was applied to the oxidase, and diaminobenzidine (Vectastain Elite Kit). whole mucosa. Based on the iodine stain results, all Normal serum blocking and omission of the primary the unstained mucosa and part of the stained mucosa antibodies were used as negative controls. Clear were cut and recorded. A total of 175 samples were cytoplasm and cell membrane staining was the cri- collected from the esophageal mucosa-adjacent can- teria for a positive reaction. Several terms were used cer. Esophageal biopsies were taken from 32 symp- to describe immunostaining patterns as previously tom-free subjects who volunteered to participate in a established, including ÔdiffuseÕ, ÔfocalÕ and Ôscat- routine endoscopic screening for EC in Huixian, teredÕ.8,9 中国科技论文在线 http://www.paper.edu.cn

Table 1. Immunoreactivity of TGFb1 and TGFbRII in esophageal normal epithelia and the epithelia with different severities of lesions TGFbRII TGFb1 No. of positive No. of positive Histologic No. of samples stains stains types examined (n,%) (n,%) Normal 43 16 (37)* 42 (98)* BCH 46 21 (46) 46 (100) DYS 31 25 (81) 25 (81) CIS 13 12 (92) 6 (46) SCC 74 67 (91) 28 (38) *Significantly different from adjacent low-grade lesions (P < 0.05 using). The changes of positive immunostaining rates of TGFb1 and TGFbRII were correlated with lesion progression using the lineal tendency test (v2 ¼ 60.62, P < 0.05). BCH, basal cell hyperplasia; CIS, carcinoma in situ; DYS, dysplasia; SCC, squamous cell carcinoma.

observed as isolated lesions in the surgically resected esophageal specimens. Out of the 175 samples adjacent to cancer tissues examined, 33 were diagnosed as normal, 31 asBCH, 27 asDYS, and 10 asCIS. All the 74 cancer specimens were confirmed as SCC. Out of the 32 biopsies examined, 10 were diagnosed as normal, 15 asBCH, four asDYS, and three asCIS. The positive immunostaining for TGF1 and TGFbRII wasobservedwith different ratesin normal esophageal tissue and the lesions of BCH, DYS, CIS, and SCC. Figure 1 shows the representative of TGFb1 and TGFbRII immunostaining in BCH (Fig. 1A) and SCC (Fig. 1B). In BCH, DYS and CIS, the positive cells were invariably associated with cell proliferative activity. The positive cells were distributed in the proliferative basal cell zone and along papillae. The positive immunostaining rate for TGFb1 was low in normal tissue, increased in BCH, and further increased in DYS, CIS, and SCC (Table 1). The significant difference for TGFb1 immunostaining rate was observed among the normal epithelia with DYS, CIS and SCC, and BCH with DYS, CIS and SCC (P < 0.05); there wasa good linear correlation between the immunostaining positive ratesof TGFb1 and the different lesions’ Fig. 1 Immunostaining of TGFb1 and TGFbII in basal cell progression (P < 0.05). The positive immunostain- hyperplasia (BCH) (A ; TGFb1, magnification 40·) and well ing ratesfor TGF b1 wasvery high in well differ- differentiated SCC (B ; TGFbRII, magnification 20·). Immunore- activity is located in the cytoplasm and cell membrane. The positive entiated SCC (98%, Table 2) and decreased in cells were invariably associated with cell proliferative activity. moderately- and poorly differentiated SCC. The difference, however, wasnot significant ( P > 0.05, Statistical analysis Table 2). The v2-test was used for the percentage of samples In contrast with TGFb1, an interesting result was with positive stain. Spearman correlation test and that the positive immunostaining rates for TGFbRII linear tendency test were used for the correlation wasvery high in the normal epithelia and BCH, and between positive rates and different severities of decreased apparently with the lesion progression lesions (P < 0.05 was considered significant). from BCH to DYS to CIS and to SCC (Table 1). The significant difference between each two adjacent groupswasobservedexcept normal and BCH, CIS RESULTS and SCC (P < 0.05). There wasa linear correlation between the positive rates of TGFbRII and the Histopathologically, CIS was more frequently found lesions’ progression from normal to BCH to DYS to adjacent to the SCC, and BCH and DYS were usually CIS, and to SCC. From well- to moderately- and 中国科技论文在线 http://www.paper.edu.cn

Table 2. Immunoreactivity of TGFb1 and TGFbRII in SCC and itscorrelation with different degreesof differentiation TGFb1 TGFbRII Differentiation No. of samples No. of positive stains No. of positive stains degreesof SCC examined (n,%) (n,%) High-differentiated 46 45 (98) 26 (57)* Moderate-differentiated 20 16 (80) 2 (10) Low-differentiated 8 6 (75) 0 (0) *The changes of positive immunostaining rates of TGFbRII were correlated with differentiation progression using the lineal tendency test (v2 ¼ 21.917, P < 0.05). SCC, squamous cell carcinoma.

Table 3. Immunostaining patterns of TGFb1 in esophageal normal epithelia and the epithelia with different severities of lesions TGFb1 Diffuse pattern Focal pattern Scattered pattern Histologic No. of No. of positive stains No. of positive stains No. of positive stains type positive stains (n,%) (n,%) (n,%) Normal 16 13 (81)* 3 (18)* 0 (0) BCH 21 16 (76) 5 (24) 0 (0) DYS 25 18 (72) 6 (24) 1 (4) CIS 12 4 (33) 8 (67) 0 (0) SCC 67 13 (19) 44 (66) 10 (15) *Spearman correlation test showed good correlation between the changes of ÔdiffuseÕ and ÔfocalÕ patterns and lesions’ progression (r ¼ 0.53893, P < 0.05). BCH, basal cell hyperplasia; CIS, carcinoma in situ; DYS, dysplasia; SCC, squamous cell carcinoma.

Table 4. Immunostaining pattern of TGFbRII in esophageal normal epithelia and the epithelia with different severities of lesions* TGFbRII Diffuse pattern Focal pattern Scattered pattern Histologic No. of No. of positive stains No. of positive stains No. of positive stains type positive stains (n,%) (n,%) (n,%) Normal 42 41 (98) 1 (2) 0 (0) BCH 46 42 (91) 4 (9) 9 (0) DYS 25 18 (72) 5 (20) 20 (2) CIS 6 3 (50) 2 (33) 33 (1) SCC 28 5 (18) 19 (68) 68 (4) *Spearman correlation test showed good correlation between the changes of ÔdiffuseÕ and ÔfocalÕ patterns and lesions’ progression (r ¼ 0.60542, P < 0.05). BCH, basal cell hyperplasia; CIS, carcinoma in situ; DYS, dysplasia; SCC, squamous cell carcinoma.

poorly differentiated SCC, the positive immunostain- ples from symptom-free subjects (Fig. 2). The biopsy ing ratesfor TGF bRII decreased (Table 2), and the samples usually contained only epithelial cells. Im- differenceswere significant between well- and mod- munostain-positive cells were observed in normal erately-differentiated SCC group (P < 0.05). epithelia, the lesions of BCH, DYS and CIS, and The ÔdiffuseÕ immunostaining pattern for TGFb1 along the longitudinally sectioned papillae or around wasobservedmore frequently in normal epithelia and the cross-sectioned papillae (Fig. 2). BCH, but the ÔfocalÕ pattern wasfrequently observed in CIS and SCC (Table 3). Similar results were observed in TGFbRII (Table 4). DISCUSSION Correlation analysis for TGFb1 and TGFbRII immunostaining showed that the coincidental posit- An interesting observation in this study was that the ive and negative ratesfor TGF b1 and TGFbRII in immunoreactivity of TGFb1 occurred in the early the same slide were 35% (26 out of 74) and 7% (5 out stage of esophageal carcinogenesis. The positive of 74), respectively. The correlations were significant immunostaining cells were distributed in the prolif- (P < 0.05). erative basal cell zone and along papillae in early A similar pattern of TGFb1 and TGFbRII protein lesions and were invariably associated with cell immunostaining was also observed in biopsies sam- proliferative activity. With the lesions progressed 中国科技论文在线 http://www.paper.edu.cn

The third interesting observation was that the positive immunostaining rates for TGFb1 and TGFbRII showed a significant correlation with SCC differentiation. The ÔfocalÕ immunostaining pattern wasmore frequently observedin well differentiated SCC, which may be used as a prognosis maker. Finally, a high coincidental positive and negative immunostaining rates for TGFb1 and TGFbRII (40%) wasobserved.Considering that with the lesions’ progression from BCH to DYS to CIS and to SCC, TGFb1-positive immunostaining rate increased significantly, whereas TGFbRII-positive immunostaining rate decreased significantly, we con- cluded that the absent or lower expression of TGFbRII may lead to the loss of cell proliferation control by TGFb1. The over-expression of TGFb1 observed in this study may be a negative feedback response caused by the lower expression of TGFbRII protein. The molecular basis for the observed overexpression of TGFb1 and low-expression of TGFbRII and role of unbalance in expression of TGFb1 and TGFbRII in the progression of esophageal precancerouslesionsrequire to be carefully investigated in laboratory and follow-up studies. The good correlation of TGFb1 and TGFbRII immunostaining rates with precancerous lesion progression suggest that these two parameters may be Fig. 2 Immunostaining of TGFb1 in biopsy sample with basal useful biomarkers to assess risk for the development cell hyperplasia (BCH). Immunoreactivity is located in the cyto- of EC. plasm and cell membrane. The positive cells were distributed in the proliferative basal cell zone and along papillae (magnification 20·). Acknowledgments from normal to BCH to DYS to CIS, and to SCC, the This study was supported in part by the National positive immunostaining rates for TGFb1 increased Outstanding Young Scientist Award of China significantly, the increasing tendency for TGFb1 had 30025016 (China), State Key Project for Basic a good linear correlation with lesions’ progression. Research G1998051206 (China) and NCI CA65871 Immunostaining positive rates for TGFb1 wasthe (USA). lowest in normal epithelia and reached the highest in CIS and SCC (2.5-fold). The present results indicated References that overexpression of TGFb1 may be one of the important molecular eventsinvolved in the early 1. Yang C S. Research on esophageal cancer in China: a review. Cancer Res1980; 40: 2633–2644. stage of esophageal carcinogenesis, and may play an 2. Lu J B, Yang W X, Zu S K et al. Cancer mortality and important role in the esophageal epithelial cell mortality trendsin Henan, China, 1974–85. Cancer Detect hyperproliferation. Prev 1988; 13: 167–173. 3. Warwick G P. Some aspects of the epidemiology and etiology Another interesting observation was that, in con- of esophageal cancer with particular emphasis on the Transkei, trast with TGFb1, the TGFbRII immunostaining South Africa. Cancer Res1973; 17: 81–229. positive rates was the highest in normal epithelia and 4. Cook-Mozaffari P. The epidemiology of cancer of the esophagus. Nutr Cancer 1979; 1: 51–60. reached the lowest in CIS and SCC (2.6-fold). With 5. Qiu S L, Yang G R. Precursor lesions of esophageal cancer in the lesions progressed from normal to BCH to DYS high-risk populations in Henan province. Cancer 1988; 62: to CIS, and to SCC, the TGFbRII positive immu- 551–557. 6. Wang L D, Lipkin M, Qiu S L et al. Labeling index and nostaining rates decreased significantly, the decreas- labeling distribution of cells in esophageal epithelium of ing tendency for TGFbRII had a good linear individuals at increased risk for esophageal cancer in Huixian, correlation with lesions’ progression. The present China. Cancer Res1990; 50: 2651–2653. 7. Correa P. Precursors of gastric and esophageal cancer. Cancer results indicated that the lower expression of 1982; 50: 1554–2565. TGFbRII may be one of the important mechanisms 8. Wang L D, Hong J Y, Qiu S L et al. Accumulation of P53 leading to the loss of cell proliferation control and protein in human esophageal precancerous lesions: a possible early biomarker for carcinogenesis. Cancer Res 1993; 53: 1783. esophageal carcinogenesis. 中国科技论文在线 http://www.paper.edu.cn

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