Expression of oncogene products and growth factors in early gallbladder cancer, advanced gallbladder cancer, and chronic cholecystitis

Expression of oncogene products and growth factors in early gallbladder cancer, advanced gallbladder cancer, and chronic cholecystitis

Expression of Oncogene Products and Growth Factors in Early Gallbladder Cancer, Advanced Gallbladder Cancer, and Chronic Cholecystitis MASAHIKO YUKAWA...

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Expression of Oncogene Products and Growth Factors in Early Gallbladder Cancer, Advanced Gallbladder Cancer, and Chronic Cholecystitis MASAHIKO YUKAWA, DAISUKE HIRAYAMA, KEISUKE KAWAI, MD, SAKAN MAEDA, MD,

MD, TAKAHIRO FUJIMORI, MD, MD, YUKA IDEI, MD, TETSUO AJIKI, MD, REIKO SUGIURA, MD, AND KOU NAGASAKO, MD

The expression of oncogene products and growth factors (epidermal growth factor, transforming growth factor-& erbB-4, ~(LF p 21, and c-mye)in gallbladder cancer and chronic cholecystitiswas measured by immunohistochemical staining on paraffin-embedded serial sections. Expression of these products was graded according to staining intensity in an area of positively stained cells. This study reports the detection of oncogene products and growth factors in cholecystitis as well as in early and late gallbladder cancer. The multiexpression of oncogene products and growth factors was greater for both gallbladder cancer groups as compared with the cholecystitis group. The percentage of epidennal growth factor positivity diminished with increased proportion of interstitial tissue and, conversely, the percentage of transforming growth factor positivity increased with increased proportion of interstitial tissue. The proportion of rat positivity was significantly greater in both early and advanced cholecystic cancer as compared with cholecystitis, but also was considerable even for cholecystitis. These results suggest that various oncogenes may have significant roles in gallbladder cancer and that collagen synthesis is reduced by epidermal growth factor and enhanced by transforming growth factor-@. HUMPATHOL 24:X’-40. Copyright 0 1993 by W.B. Saunders Company

of oncogenes to cholecystic carcinoma. Accordingly, we have conducted an immunohistochemical study on oncogene products and growth factors with particular reference to chronic cholecystitis and to early and advanced cholecystic cancer. MATERIALS

AND METHODS

Nine chronic cholecystitis specimens, 13 early cholecystic cancer specimens, and nine advanced cholecystic cancer specimens were used for immunohistochemical assay. Cancers limited to the mucosa or invading as far as the muscular layer, regardless of any involvement of Rokitansky-Aschoff sinuses, were designated as early cholecystic cancer.g The expression of oncogene products and growth factors (epidermal growth factor [EGF], transforming growth factor/3 [TGF-81, erbB-2, ~(1sp 21, and c-myc) was determined by immunohistochemical staining on paraffin-embedded serial sections. After deparaffinization, the sections were immersed for 13 minutes in methanol solution containing 0.3% H202. They were then covered with normal goat or horse serum and reacted at room temperature for 20 minutes. Finally, they were incubated for 30 minutes with the primary antibodies. The primary antibodies used were anti-h-EGF rabbit IgG (1:20 dilution; Wakunaga, Hiroshima, Japan), anti-TGF-fl rabbit IgG (1:750 dilution; R&D System, Minneapolis, MN), c-erbB-2 rabbit antibody (1:30 dilution; Triton Biosciences Inc, Alameda, CA), anti-ra p2 1 mouse antibody RAP-5 (1:50,000 dilution; provided by Dr Schlom, National Institutes of Health, Bethesda, MD), and anti-c-myc mouse antibody MYC-1 (1: 100 dilution; provided by Dr Suku, Nagasaki University, Nagasaki,

The rapid progress of molecular pathologic research in recent years has revealed that various oncogenes and cancer inhibitor genes are involved in all stages of the carcinogenic process, from initiation to growth and progression.’ Oncogenes include many encoding growth factors and their associated receptors, as well as other proteins collectively constituting an information transmission system centered on cell membranes. Oncogenes include chromosomal translocations and deficiencies, DNA recombinations, gene amplification, etc, which are all under investigation by various methods of detection. Molecular pathologic research on the adenoma-carcinoma sequence with reference to colorectal cancer has been especially prolific, and mutations in genes such as ras, ~53, and DCC are believed to be involved in this carcinogenic process.2-8 However, few specific reports have appeared detailing the relation

Japan).

Anti-rabbit and anti-mouse IgG was used as the second antibody, and the reaction was performed at room temperature for 30 minutes. Reaction with the avidin-biotin complex reagent (Vector Laboratories Inc, Burlingame, CA) was performed at room temperature for 30 minutes. After color development with 0.03% diaminobenzidine (including 0.01% H202), nuclear staining was performed with hematoxylin. Both the distribution (the percentage of positive cells) and the intensity of staining were assessed in a semiquantitative fashion. The following system was used to score the distribution of positive cells: none (not stained), 0; focal (
From the Department of Pathology, Kobe University School of Medicine, Kobe, Japan; and the Institute of Gastroenterology, Tokyo Women’s Medical College, Tokyo, Japan. Accepted for publication April 7, 1992. Key words: gallbladder cancer, oncogene product, growth factor. Address correspondence and reprint requests to Masahiko Yukawa, MD, Department of Pathology, Kobe University School of Medicine, 7-5-l Kusunoki-cho, Chuo-ku Kobe City, Hyogo, 650, Japan. Copyright 0 1993 by W.B. Saunders Company 0046-8177/93/2401-0007$5.00/O

37

HUMAN PATHOLOGY

Volume 24, No. 1 (January

1993)

FIGURE 1. Early gallbladder cancer. (Stained with Rapd; magnification X50.)

FIGURE 2. Early gallbladder cancer. (Stained with Rap-5; magnification X100.)

FIGURE 3. Advanced gallbladder cancer, (Stained with Rap-5; magnification x100.)

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ONCOGENE

PRODUCTS

IN GALLBLADDER

CANCER

(Yukawaet al)

TABLE 1. Positive Rates for Early Cancer, Advanced Cancer, and Cholecystitis as Detected by Various Oncogene Products and Growth Factors RAP-5 (96) Early cancer

(92)

Advanced cancer

6/9

(67)

Cholecystitis

3/9

(33) -J

21/31

+

2/9

(22) 1

l/9

(11) I

12/31

(68)

g/13 WY++

+

o/9

(0)--J

l/9

(11) I

lo/31

(39)

TGF (%)

EGF (96)

erbB-2 (96)

9/13(W7 L-l

1

12/H

Total

MYC-1 (96)

+

(32)

6/13

(46)

8/13

(62)

5/9

(56)1

3/9

(33)

l/9

(ll)j+

O/9

(0) I

11/31

(35)

12/31

(39)

1

+

Abbreviations: EGF, epiderrnal growth factor; TGF, transforming growth factor. Symbols: +, difference is significant (I’ < .Ol); ++, difference is significant (P < .05).

Proportions of positive staining reactions in relation to invasive growth patterns are presented in Table 3. In the advanced cancer group ru.sp 2 1, myc, EGF, and TGF were detected in lower proportions among cases displaying pronounced invasive tendencies. Percentages of positive staining reactions in relation to relative proportions of parenchymal and interstitial tissue in tumors are presented in Table 4. Percentages of ra.s p 2 1 and EGF positivity diminished with increased proportion of interstitial tissue and percentage of TGF positivity increased with increased proportion of interstitial tissue. Table 5 shows the number of cases positive for one or several oncogene products. The percentage of cases simultaneously displaying positive reactions for more than one oncogene product was greater for both gallbladder cancer groups than for the cholecystitis group. Early cancer cases were simultaneously positive for several oncogene products with greater frequency as compared with advanced cancer cases. In the cholecystitis group, although one case was positive for two different oncogene products, none was simultaneously positive for three or more.

parisons of immunohistological results were made with a twotailed, unpaired Student’s t test.

RESULTS Reactivity with anti-ras p 21, EGF,and TGF was confined to the cytoplasm (Figs 1 to 3). Reactivity with anti-myc and erbB-2 was confined to the cell nucleus. There was no staining within any of the stromal tissues for either of those products. Proportions of positive staining reactions for various oncogene products and growth factors in cholecystitis and cholecystic cancer are presented in Table 1. The respective percentages of positivity decreased in the following order: ra.s p 21 (33%), myc (1 l%), EGF (1 l%), TGF (O%), and erbB-2 (11%) in cholecystitis; ra.s p 21 (92%), myc (69%), EGF (46%), TGF (62%), and e&B-2 (69%) in early cancer; and ru..sp 21 (67%), myc (22%), EGF (56%), TGF (33%), and e&B-P (0%) in advanced cancer. The proportion of EGF positivity was greatest among advanced cancer patients, whereas the other substances were most frequently detected in the early cancer group. Except for e&B-2, the proportions of positivity for the various oncogene products were consistently high in both cancer groups as compared with the cholecystitis group. e&B-2 was detected with the lowest frequency among the advanced cancer cases. Proportions of positive staining reactions in relation to histopathologic type are presented in Table 2. No significant correlations were recognized between histopathologic type and proportion of positivity stained reactions.

DISCUSSION Numerous reports have appeared concerning the detection of oncogene products in various forms of

TABLE 3. Positive Rates for Intramural and Extramural Growth Patterns as Detected by Various Oncogene Products and Growth Factors

TABLE 2. Positive Rates for Histologic Type as Detected by Various Oncogene Products and Growth Factors

Early Tub P0r Advanced Tub P0r

erbB-2

RAP-5

MYC-1

12/13 (92) 0

9/13 (69) 0

i/13

l/3 l/6

O/3 O/6

2/3 4/6

(67) (67)

(33) (17)

(69)l ++ (0) --J (0)

TGF (%)

MYC-1 (0)

e&B-2 (W

4/5 (80) 8/S (100) 0

3/5 (60) 6/8 (75) 0

2/5 (40) 7/a (88) 0

2/5 (40) 4/a (50) 0

3/5 (60) 5/B (63) 0

0 3/4 (75) 3/5 (60)

0 114 (25) l/5 (20)

0 O/4 (0) O/5 (0)

:,4 (100) l/5 (20)

i/4 (50) l/5 (20)

Early

EGF

TGF

6/13 (46) 0

8/13 (62)

2/3 3/6

O l/3 2/3

(67) (50)

EGF (%6)

RAP-5 (W

; Y Advanced a* Y

(33) (33)

Abbreviations: 0, the cancerous lesion shows expansive growth and exhibits a distinct border from the surrounding tissue; 8, the growth pattern of the cancerous lesion is intermediate between a and y; y, the cancerous lesion shows infiltrative growth with an ill-defined border; EGF, epidermal growth factor; TGF, transforming growth factor.

Abbreviations: EGF, epidermal growth factor; TGF, transforming growth factor; Tub, tubular adenocarcinoma; Par, poorly differentiated adenocarcinoma. Symbol: ++, difference is significant (I’ < .05).

39

HUMAN PATHOLOGY

Volume 24, No. 1 (January 1993) TABLE 5. Numbers of Cases Positive for One or Several Oncogene Products and Growth Factors

cancer, but reports of this type relating to gallbladder cancer have been few and have been confined to assessment of the overexpression of individual oncogene products. Virtually no reports have appeared on the overexpression of several oncogene products in gallbladder cancer as investigated by serial sections. The purpose of the present study was to comparatively assess the various oncogene products in cholecystitis as well as in early and late cholecystic cancer by the application of immunohistochemical techniques that permitted a simultaneous comparison of the expression of several oncogene products and growth factors. The interaction of a number of different genes is believed to be involved throughout the complex process of carcinogenesis, from initiation to subsequent progression.’ The coexistence of several oncogene products in the same tissue, as observed in the present study, constitutes an interesting form of supporting evidence for this view. In the present study the percentage of EGF positivity diminished with increasing proportion of interstitial tissue, whereas TGF-fi displayed the opposite trend. These findings corroborate other reports that collagen synthesis is reduced by EGF and enhanced by TGF-/3. Mutations of the r~ls gene are said to occur in the early stage of the adenoma-carcinoma sequence characterizing the development of colorectal cancer.8 Transformation of the stomach mucosa from benign to malignant phenotype is associated with an increase in c-Ha-ru.s p2 1 expression. l1 In the present study the proportion of ras positives was significantly greater in both early and advanced cholecystic cancer as compared with cholecystitis, but also was considerable (33%) in cholecystitis. Moreover, although isolated dysplasia in the gallbladder is rare, dysplasia adjacent to cholecystic carcinomatous lesions is detected with a high prevalence.‘* These two facts suggest that a dysplasia-carcinoma sequence or intestinal metaplasia-carcinoma sequence may be involved in the pathogenesis of cholecystic cancer,

1

MYG1 (96)

erbB-2 (‘R)

EGF (96)

0 12/13 (92) 0 l/l 2/3 3/5

(100) (67) (60)

i/l3 0

(69)

O/l l/3 l/5

(0) (33) (20)

&3 0 O/l O/3 O/5

i/13 0

(46)

0 a/l3 0

(62)

(0) (0) (0)

l/l 2/3 2/5

(100) (67) (50)

O/l l/3 v5

(0) e-w (40)

5

REFERENCES 1. Kitagawa T: Differential diagnosis of early cancer and oncogenes. Jpn J Cancer Clin 34:1259-1263, 1988 2. Bodmer WF, Bailey CJ, Bodmer J, et al: Localization of the gene for familial adenomatous polyposis on chromosome 5. Nature 328:614-616, 1987 3. Leppert M, Dobbs M, Scambler P, et al: The gene for familial polyposis coli maps to the long arm of chromosome 5. Science 238: 1411-1413,1987 4. Law DJ, Olschwang S, Monpezat J, et al: Concerted nonsynthetic ahelic loss in human colorectal carcinoma. Science 241:961965, 1988 5. Baker SJ, Fearon ER, Nigro JM, et al: Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science 244: 217-221, 1989 6. Fearon ER, Cho KR, Nigro JM, et al: Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 247: 49-56, 1990 7. Kinzler KW, Nilbert MC, Vogelstein B, et al: Identification of a gene located at chromosome 5q21 that is mutated in colorectal cancers. Science 251:1366-1369, 1991 8. Eric RF, Vogelstein B: A genetic model for tumorigenesis. Cell 61:759-767, 1990 9. Watanabe H, Shirai Y, Rijima H, et al: Characteristics of early carcinoma of the gallbladder. Ran Tan Sui 10:527-534, 1985 10. Shimizu M, Saitoh Y, Itoh H: Immunohistochemical staining of Ha-ru.r oncogene product in normal, benign, and malignant human pancreatic tissues. Hum Path01 21:607-612, 1990 11. Ohuchi N, Hand PH, Merloo G, et al: Enhanced expression of c-Ha-ru.r p21 in human stomach adenocarcinomas defined by immunoassays using monoclonal antibodies in situ. Cancer Res 47: 14 131420,1987 12. Yamagiwa H: Mucosal dysplasia of gallbladder: Isolated and adjacent lesions to carcinoma. Jpn J Cancer Res 80:238-243, 1989

TGF (96)

(92)

4

analogous to the adenoma-carcinoma sequence characteristic of colorectal cancer. The question of whether dysplasia or intestinal metaplasia in the gallbladder should be regarded as precancerous can only be answered by further research; however, the above-mentioned findings appear to constitute reasonable grounds for conjecturing that the ras gene is initiating the process of transition from cholecystitis to gallbladder cancer, analogous to the role of the gene in colorectal cancer. In the present study activation of oncogenes diminished in the progression from early to advanced cancer (Table 1) and from less invasive to invasive cancer (Table 3). We suspect that cancer cells might change their clones in their progression or different oncogenes might be related to early cancer and advanced cancer. However, our immunohistochemical study did not provide definitive results.

Early Med Int Sci Advanced Med Int Sci

3

13/13 (100) 11/13 (76) 7/13 (54) 7/13 (54) 5/13 (38) Early cancer 5/9 (56) 3/9 (33) l/9 (11) O/9 (0) Advancedcancer 8/9 (89) Cholecystitis 6/g (67) l/9 (11) O/9 (0) O/9 (0) O/9 (0)

TABLE 4. Positive Rates for Amount of Interstitial Connective Tissue as Detected by Various Oncogene Products and Growth Factors RAP-5 (%)

2

Abbreviations: EGF, epidemxd growth factor; TGF, transforming growth factor; med, medullary type: the amount of interstitial tissue is scanty; int, intermediate type: the amount of interstitial tissue is intermediate between tbe medullary and scirrbous types; sci, scirrhous type:the amount of interstitial tissue is abundant.

40