Variable Expression of P-Glycoprotein in Normal, Inflamed, and Dysplastic Areas in Ulcerative Colitis T. J. Saclarides, M.D.,* S. M. Jakate, M.D.,-~ J. s. Coon, M.D., Ph.D.,-~ A. K. Bhattacharyya, M.D.,~J. M. Dominguez, M.D.,* D.J. Szeluga, Ph.D., R. D.,* R. S. Weinstein, M.D. S From the Departments ofafPathology and * General Surgery, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois, and the ~Department of Pathology, Universi[y of Arizona Health Sciences Center, Tucson, Arizona Screening programs for the detection of cancer in ulcerative colitis are inexact and not always successful in finding early, curable cancers. P-glycoprotein is a membrane-based, energy-dependent protein found in varying degrees within normal human tissue. P-glycoprotein is overexpressed in malignant tumors, particularly colorectal cancer, and is known to convey resistance to certain anticancer drugs by acting as a membrane "pump." The purpose of this study was to determine the expression of this protein in inflamed and premalignant colonic epithelium, compare its expression with normal controls, and assess its potential use as a screening tool for high-risk patients with ulcerative colitis. Using immunohistochemical techniques, the colons of 21 patients (10 with dysplasia) with ulcerative colitis were stained with monoclonal antibody C-219 (MAbC219) specific for P-glycoprotein. P-glycoprotein was expressed in 38 percent of normal areas, 71 percent of inflamed areas ( P - - 0.0156), and 70 percent of dysplastic areas. Comparing the level o f expression when progressing from normal to inflamed areas within a given patient, 11 patients (52 percent) showed increased expression, 8 (38 percent) showed equal expression, and only 2 (10 percent) showed decreased expression ( P = 0.0225). Comparing expression w h e n progressing from inflamed to dysplastic areas (10 patients), 7 showed equal expression and 3 showed increased expression ( P - 0.25). Increasing duration of disease was associated with a significant increase in Pglycoprotein expression, but only in histologically normal areas. Duration of disease had no effect on P-glycoprotein expression in inflamed or dysplastic areas. Similarly, when surgery was performed for elective reasons, there was a significant overexpression of P-glycoprotein, but only in histologically normal areas. Our findings suggest that the increase in P-glycoprotein expression from normal to inflamed and dysplastic areas reflects the premalignant nature of ulcerative colitis and occurs early in the course of the disease. Further research needs to be done to determine its role in cancer surveillance. [Key
words: P-glycoprotein; Multidrug resistance; Ulcerative colitis; Dysplasia; Colectomy] Saclarides TJ, Jakate SM, Coon JS, Bhattacharyya AK, Dominguez JM, Szeluga DJ, Weinstein RS. Variable expression of P-glycoprotein in normal, inflamed, and dysplastic areas in ulcerative colitis. Dis Colon Rectum 1992; 35:747-752.
urveillance programs for the detection of cancer in ulcerative colitis are insecure and imprecise. Because of tumor morphology and technical difficulties, periodic barium enemas or colonoscopic examinations may fail to reveal cancers, even in the hands of the most experienced examiners. In an attempt to more effectively screen highrisk patients, Morson and Pang 1 introduced in 1967 the concept of random biopsies for dysplasia as a precancerous marker. Such a means of surveillance, however, is not exact of fail-safe for several reasons. Dysplasia may not be diffuse; nor does it always precede cancer. Therefore, the absence of dysplasia can be misleading. Determination of dysplasia requires an experienced pathologist with a dedicated interest in chronic ulcerative colitis, and it may be difficult to evaluate in the face of inflammatory changes. Finally, the exact point at which to begin surveillance and the necessary frequency of examinations are unclear, and, although unlikely, cancer may develop after only a few years. 2 Several authors have investigated the expression of various tissue and serum antigens in ulcerative
S
c o l i t i s as a m e a n s o f s c r e e n i n g h i g h - r i s k p a t i e n t s for c a n c e r . I n c l u d e d h a v e b e e n c a r c i n o e m b r y o n i c a n t i g e n (CEA), 3 g a s t r o i n t e s t i n a l c a n c e r a n t i g e n (CA19-9), 4 c a r c i n o m a - a s s o c i a t e d a n t i g e n (CA-50), 5 and tumor-associated glycoprotein (TAG-72). 6 T h e s e a n t i g e n s a r e e x p r e s s e d n o t o n l y in c a n c e r ,
Presented at the meeting of the American College of Gastroenterology, Boston, Massachusetts, October 14, 1991. This work was supported in part by the Bowman Research Fund. Address reprint requests to Dr. Saclarides: Head, Section of Colon and Rectal Surgery, Department of General Surgery, Rush-Presbyterian-St. Luke's Medical Center, 1653 West Congress Parkway, Chicago, Illinois 60612. 747
748
SACLARIDES E T AL
but also in inflamed non-neoplastic colonic epithelium, thus limiting potential applications as tumor markers. Considerable research has been performed with P-glycoprotein and its role in conferring cellular resistance to anticancer drugs. 7-9 The genetic basis of multidrug resistance has been elucidated, 1~ 11 and the responsible gene in humans is the Mdrlgene.la, 13 Its encoded protein, an energy-driven 170kd membrane efflux transport, is P-glycoprotein. Normal expression of P-glycoprotein is site ~ specific 9,14,15; approximately 30 percent of patients will express P-glycoprotein in histologically normal gastrointestinal epithelium when immunostained by monoclonal antibody C-219. Cancer and the development of resistance to anticancer drugs is accompanied by an overexpression of Pglycoprotein 9, 14, 16-18; studies have shown expression in 70 percent of colorectal cancers. 19 Three patterns of P-glycoprotein immunoreactivity have been identified and described elsewhere. 2~ A supranuclear dense staining corresponding to the Golgi zone is described as the Golgi staining pattern. Immunoreactivity may also be seen throughout the cytoplasmic compartment, giving rise to a diffuse staining pattern. Luminal membrane staining may occur as well. Correlation between ABO blood type and the Golgi-type pattern of P-glycoprotein expression has also been described, a~ Although it is known that neoplasia is associated with P-glycoprotein overexpression, expression in pathologically abnormal non-neoplastic and preneoplastic tissue has not been elucidated. For this purpose, ulcerative colitis is a useful model owing to the possible sequential progression of normal mucosa through inflammation, dysplasia, and carcinoma. Carcinoma is known to occur in about 5 percent of cases of ulcerative colitis. 21 Dysplasia has been regarded as a precancerous lesion 22' 23 and may precede the development of carcinoma. 1 The purpose of this study was to determine the expression of P-glycoprotein in inflamed and dysplastic colons, compare this expression with normal controls, and assess P-glycoprotein as a potential screening device for cancer surveillance.
MATERIALS AND METHODS Twenty-eight patients with ulcerative colitis were treated by colectomy at Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois, from 1980
Dis Colon Rectum, August 1992
to 1990. Of these, tissues blocks were available in 21 instances; these cases form the basis of this study. There were 11 males and 10 females, with a mean age of 33.5 years (range, 16-70 years). The duration of disease ranged from one episode to 20 years of disease. The indications for surgery are listed in Table 1. The ABO blood types of the patients, performed by standard blood bank methods, were obtained from patient records. The colon specimens were processed for routine pathologic evaluation. Sections removed at surgery were grossly examined and fixed in 10 percent neutral buffered formalin, and representative blocks, including the resection margins, were taken for paraffin embedding. Hematoxylin and eosin stained paraffin sections from all cases were retrospectively reviewed to assess morphologic and histologic features. All cases were confirmed to be ulcerative colitis and classified as active disease (17), resolving colitis (2), or quiescent colitis (2) by standard histologic features. 24 Ten cases showed varying degrees of dysplasia, such as indefinite (2), low grade (5) or high grade (3). Dysplasia was graded according to internationally accepted criteria. 25 From each patient, representative sections of noninflamed, inflamed, and dysplastic areas were selected. In most cases (18/21), a section of noninflamed proximal colonic mucosa was available. In three cases, however, ileal mucosa from the ileal resection margin-was selected, as there was involvement of the entire large intestine by the inflammatory process. In addition, representative sections from 10 cases of dysplasia were selected. These selected sections were then processed for immunohistochemical studies. A murine monoclonal antibody, subclass IgGaA Table 1. Patient Demographics Sex: 11 males, 10 females Age: mean, 335 years (range, 16-70 years) Duration of disease before surgery One episode 1-5 years Greater than 5 years Indication for surgery Urgent Toxic colitis Obstruction Hemorrhage Elective Refractory Fear of cancer
4 10 7
6 1 2 10 2
P-GLYCOPROTEIN--ULCERATIVE COLITIS
Vol. 35, No. 8
C219, obtained from Centocor Diagnostics (Malvern, PA) was used for staining of paraffin sections. This antibody binds to a highly conserved cytoplasmic epitope of P-glycoprotein. Immunohistochemistry was performed using the Vectastain elite ABC procedure and reagents (Vector, Burlingame, CA). All steps were repeated in a negative control slide excluding the primary antibody. The extent of MAbC219 immunoreactivity was then scored. If no staining was seen in any of the three patterns, namely supranuclear, diffuse, or luminal, the specimen was scored as negative. The presence or absence of these three patterns was then individually scored as follows: zero (0) when a particular pattern was absent; 1+ when less than 5 percent of epithelial cells were immunostained with a given pattern in a section; 2+ when 5 to 25 percent of cells were immunostained with a given pattern; and 3+ when more than 25 percent of cells were immunostained with a given pattern, The sum of the three scored patterns was used to compare levels of P-glycoprotein expression in the three areas: namely normal, inflamed, and dysplastic.
TM
749
diffuse, and luminal. Figures 2A, 2B, and 2C illustrate immunostaining in normal, inflamed, and dysplastic areas. Applying the McNemar test (binomial two-tailed), a statistically significant difference was noted in P-glycoprotein expression when comparing normal with inflamed areas ( P = 0.015). Comparing normal with dysplastic areas, no statistical difference was noted owing to small sample size.
RESULTS As shown in Figure 1, 8 of 21 (38 percent) normal histologic sections expressed P-glycoprotein, whereas 15 of 21 inflamed areas (71 percent) and 7 of 10 dysplastic areas (70 percent) expressed this protein. A section was considered positive for P-glycoprotein if immunoreactivity was present in any of the three patterns, namely supranuclear,
Normal (n = 21; 38%)
A P=0.015
/ /
/
~
P=0.250 \
~
(smallsample
/ Inflame~
~
Dysplasia
(n = 21; 71%) (n = 10; 70%) Figure 1. Overallexpressionof P-glycoproteinin normal, inflamed,and dysplasticareas.
Figure2. A. SupranuclearcircumscribedGolgipattern of immunostainingin "normal"colonicmucosa. B. Inflamed area of activeulcerativecolitisshowingdiffusecytoplasmic and luminalpattern of immunostaining.C. Area of highgrade dysplasiawith diffusecytoplasmicimmunostaining.
750
SACLARIDES ET AL
The sum of scored immunoreactivity was assessed progressing from normal to inflamed and ultimately to dysplastic areas within a given patient (Table 2). Again, a binomial two-tailed analysis (sign test) was performed. When progressing from normal to inflamed areas, expression was increased in 11 patients, equal in 8, and decreased in only 2 ( P = 0.0225). When comparing dysplastic with normal areas within a given patient, expression was increased in 6, equal in 3, and decreased in 1 (P = 0.125). When progressing from inflamed to dysplastic areas, expression was increased in 3 and equal in 7 (P = 0.250). The pattern of immunostaining was assessed with respect to ABO blood type. Seven of 21 cases were blood type A, and, of thee, five exhibited predominantly the Golgi pattern of staining. Only 2 of the remaining 14 cases with non-A blood type showed the Golgi pattern. P-glycoprotein expression was assessed with respect to duration of disease (Table 3) as well as indication for surgery (Table 4). Increasing duration of disease led to a significant overexpression Table 2. Scored P-Glycoprotein Expression When Progressing from Normal to Inflamed and Dysplastic Areas Normal --, inflamed Increased expression Equal expression Decreased expression
52% 37% 10%
P = 0.02
Inflamed --, dysplastic Increased expression Equal expression
30% 70%
N8
Normal ---, dysplastic Increased expression Equal expression Decreased expression
60% 30% 10%
NS
Table 3. P-Glycoprotein Expression and Duration of Disease Normal First episode (n = 4) 5 years (n = 7)
0% 30% 71% P = 0.05
Inflamed Dysplastic 75% 60% 83% NS
100% 60% 86% NS
Table 4. P.Glycoprotein Expression and Indications for Surgery Urgent (n = 9) Elective (n = 12)
Normal
Inflamed
Dysplastic
11% 58% P = 0.027
66% 75% NS
66% 66% NS
Dis Colon Rectum, August 1992
of P-glycoprotein, but only in histologically normal areas. When surgery was performed for elective reasons, i.e., steroid dependence, intolerable side effects of medical therapy, or fear of cancer, there was a significant overexpression of P-glycoproteinl but only within normal areas. DISCUSSION There are two closely related multidrug-resistant genes in humans called MDR1 and MDR2 genes. The protein products of these genes have been called P-glycoprotein. Although the function of the MDR2 gene product is unknown, P-glycoprotein derived from the MDR1 gene has been extensively studied. It is a highly conserved pleiotropic membrane transport protein which actively transports several apparently unrelated organic compounds, including cytotoxic drugs. P-glycoprotein may serve several roles in normal physiologic processes such as cellular detoxification of lipophilic xenobiotic compounds, 8' z6, 27 intracellular transport of steroids in adrenal tissue, 28 and transport of terminal saccharides for antigen A biosynthesis across Golgi membranes. 2~ Various antibodies that can recognize P-glycoprotein at specific cytoplasmic or external domains have been described. MABC219 is the most widely used antibody which binds to highly conserved hexapeptide in the cytoplasmic domain of P-glycoprotein. 8 Normal distribution of P-glycoprotein varies among patients and organs, from nonexistent to high levels of expression. In addition, a relationship between blood type A and the Golgi pattern of immunostaining has been described. Tissues have been grouped as high (adrenal), intermediate (intestine or urinary tract), and low (lymphoreticular) expressors of P-glycoprotein. Studies have shown that approximately 30 percent of normal colons express P-glycoprotein. 19 There is a wellrecognized overexpression of P-glycoprotein in human tumors, including colon cancer, where approximately 70 percent express P-glycoprotein39 Prior to this study, the expression of P-glycoprotein in inflamed and premalignant colonic epithelium was not known. Our study showed that overexpression of P-glycoprotein is not limited to cancerous tissue. Using ulcerative colitis as our model, we found that, like noncolitic colorectal cancers, 70 percent of inflamed and dysplastic colons expressed P-glycoprotein. With histologically normal colon acting as
Vol. 35, No. 8
P-GLYCOPROTEIN--ULCERATIVE COLITIS
an internal control, the overexpression in inflamed colitic e p i t h e l i u m was statistically significant ( P = 0.0156). The p r e s e n c e of dysplasia, however, did not increase the expression of P-glycoprotein over that seen in inflamed areas. That ulcerative colitis is a premalignant condition is well d o c u m e n t e d , and o n e of the risk factors associated with cancer is disease of long duration .29 T h e exact point at which a t e n d e n c y toward cancer o n a molecular level occurs is unclear. Our data suggest that this occurs before histologic e v i d e n c e o f inflammation and dysplasia. This is s u p p o r t e d b y observing an increase in P-glycoprotein expression with disease of long duration, a finding which was statistically significant but only in histologically normal areas. In fact, the d e g r e e of P-glycoprotein overexpression in normal areas w h e n colitis was present for longer than five years app r o a c h e d that seen in noncolitic colorectal cancers. T h e overexpression in normal areas w h e n surgery was p e r f o r m e d for elective reasons probably reflects chronicity of disease. Our study lends support to the notion that ulcerative colitis has malignant potential and suggests that the neoplastic t e n d e n c y occurs early in the course of the disease. It appears that a 38 p e r c e n t i n c i d e n c e of expression in histologically normal areas might impair its use as a screening tool for high-risk patients. If, however, this subset of patients were to s u b s e q u e n t l y d e v e l o p high-grade dysplasia or even cancer, t h e n perhaps P-glycoprotein could be used to identify a high-risk group. Unfortunately, our study had a limited n u m b e r of dysplastic areas (10) and no cancers; therefore, no information can be extrapolated as to the longterm significance of P-glycoprotein expression in histologically normal or inflamed areas. A prospective surveillance study with a large n u m b e r of patients will be n e e d e d to answer this question.
REFERENCES 1. Morson BC, Pang LSC. Rectal biopsy as an aid to cancer control in ulcerative colitis. Gut 1967;8: 423-34. 2. Riddell RH. "Surveillance" in ulcerative colitis. Does it work? In Freeman HJ, ed. Inflammatory bowel disease. Boca Raton: CRC Press, 1989. 3. Greenstein AJ, Panvelliwalla DK, Katz LB, e t al. Tissue carcinoembryonic antigen, dysplasia, and disease duration in colonic inflammatory bowel disease. Am J Gastroenterol 1982;77:212-5.
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4. Allen DC, Biggart JD, Orchin JC, Foster H. An immunoperoxidase study of epithelial marker antigens in ulcerative colitis with dysplasia and carcinoma. J Clin Pathol 1985;38:18-29. 5. Frykholm G, Enblad P, Pahlman L, Busch C. Expression of the carcinoma-associated antigens CA19-9 and CA-50 in inflammatory bowel disease. Dis Colon Rectum 1987;30:545-8. 6. Thor A, Itzkowitz SH, Schlom J, Kim YS, Hanauer S. Tumor-associated glycoprotein (TAG-72) expression in ulcerative colitis. Int J Cancer 1989;43: 810-5. 7. Bradley G, Juranka PF, Ling VI Mechanisms of multidrug resistance. Biochim Biophys Acta 1988;948:87-128. 8. Jranka PF, Zastawny RL, Ling V. P-glycoprotein: multidrug resistance and superfamily of membrane associated transport proteins. FASEB J 1989;3: 2583-92. 9. Weinstein RS, Kuszak JR, Kluskens LF, Coon JS. Pglycoproteins in pathology: the multidrug resistance gene family in humans. Hum Pathol 1990;21:34-48. 10. Riordan JR, Ling V. Genetic and biochemical characterization of multidrug resistance. Pharmacol Ther 1985;28:51-75. 11. Van der Bliek AM, Bortst P. Multidrug resistance. Adv Cancer Res 1989;52:165-202. 12. Chin JE, Soffier R, Noonon KE, Choi K, Roninson IB. Structure and expression of the human MDR (Pglycoprotein) gene family. Mol Cell Biol 1989;9:3808-20. 13. Kane SE, Gottesman MM. Multidrug resistance in the laboratory and clinic. Cancer Cells 1989;1:33-6. 14. Fojo AT, Ueda K, Slaman DJ. Expression of a multidrug-resistance gene in human tumors and tissues. Proc Natl Acad Sci U S A 19878;84:265-9. 15. Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci U S A 1987;84:7735-8. 16. Juliano RL, Ling V. A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 1976;455:152-62. 17. Shen DW, Fojo A, Roninson IB, e t al. Multidrug resistance of DNA-mediated transformants is linked to transfer of the human MDR1 gene. Mol Cell Biol 1986;6:4039-44. 18. Goldstein LJ, Galski H, Fojo A, e t al. Expression of a multidrug resistance gene in human cancers. J Natl Cancer Inst 1989;81:116-24. 19. Weinstein RS, Jakate SM, Dominguez JM, e t al. Relationship of the expression of the multidrug resistance gene product (P-glycoprotein) in human colon carcinoma to local tumor aggressiveness and lymph
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SACLARIDES E T AL
node metastasis. Cancer Res 1991;51:2720-6. 20. Weinstein RS, Kuszak JR, Jakate SM, Lebovitz MD, Kluskens LF, Coon JS. ABO blood type predicts the cytolocalization of anti-P-glycoprotein monoclonal antibody reactivity in human colon and ureter. Hum Pathol 1990;21:949-58. 21. Ballantyne GH. Risk of colorectal cancer in patients with chronic ulcerative colitis and Crohn's disease. Probl Gen Surg 1987;4:154-67. 22. Lennard-Jones JE, Morson BC, Ritchie JK, Williams CB. Cancer surveillance in ulcerative colitis. Experience over 15 years. Lancet 1983;2:149-52. 23. Lennard-JonesJE, Morson BC, RitchieJK, Shove DC, Williams BM. Cancer in colitis: assessment of the individual risk by clinical and histological criteria. Gastroenterology 1977;73:1280-9. 24. Fenoglio-Preiser CM, Lantz PE, Listrom MB, Davis M, Rilke FO. Gastrointestinal pathology, an atlas and
Dis Colon Rectum, August 1992
text. New York: Raven Press, 1989. 25. Ridell RH, Goldman H, Ransohoff DF, et al. Dysplasia in inflammatory bowel disease: standardized classification with provisional clinical applications. Hum Pathol 1983;14:932-68. 26. Roninson IB. Molecular mechanisms of multidrug resistance in tumor cells. Clin Physiol Biochem 1987;5:140-51. 27. Rothenberg M, Ling V. Multidrug resistance: molecular biology and clinical relevance. JNCI 1989;81:907-10. 28. Yang C-CP, DePinho SG, Greenberger LM, Arceci RJ, Horwitz SB. Progesterone interacts with P-glycoprotein in multidrug resistant cells and in endometrium of gravid uterus. J Biol Chem 1989;264: 782-8. 29. Ohman U. Colorectal carcinoma in patients with ulcerative colitis. Am J Surg 1982;144:344-9.
words: P-glycoprotein; Multidrug resistance; Ulcerative colitis; Dysplasia; Colectomy] Saclarides TJ, Jakate SM, Coon JS, Bhattacharyya AK, Dominguez JM, Szeluga DJ, Weinstein RS. Variable expression of P-glycoprotein in normal, inflamed, and dysplastic areas in ulcerative colitis. Dis Colon Rectum 1992; 35:747-752.
urveillance programs for the detection of cancer in ulcerative colitis are insecure and imprecise. Because of tumor morphology and technical difficulties, periodic barium enemas or colonoscopic examinations may fail to reveal cancers, even in the hands of the most experienced examiners. In an attempt to more effectively screen highrisk patients, Morson and Pang 1 introduced in 1967 the concept of random biopsies for dysplasia as a precancerous marker. Such a means of surveillance, however, is not exact of fail-safe for several reasons. Dysplasia may not be diffuse; nor does it always precede cancer. Therefore, the absence of dysplasia can be misleading. Determination of dysplasia requires an experienced pathologist with a dedicated interest in chronic ulcerative colitis, and it may be difficult to evaluate in the face of inflammatory changes. Finally, the exact point at which to begin surveillance and the necessary frequency of examinations are unclear, and, although unlikely, cancer may develop after only a few years. 2 Several authors have investigated the expression of various tissue and serum antigens in ulcerative
S
c o l i t i s as a m e a n s o f s c r e e n i n g h i g h - r i s k p a t i e n t s for c a n c e r . I n c l u d e d h a v e b e e n c a r c i n o e m b r y o n i c a n t i g e n (CEA), 3 g a s t r o i n t e s t i n a l c a n c e r a n t i g e n (CA19-9), 4 c a r c i n o m a - a s s o c i a t e d a n t i g e n (CA-50), 5 and tumor-associated glycoprotein (TAG-72). 6 T h e s e a n t i g e n s a r e e x p r e s s e d n o t o n l y in c a n c e r ,
Presented at the meeting of the American College of Gastroenterology, Boston, Massachusetts, October 14, 1991. This work was supported in part by the Bowman Research Fund. Address reprint requests to Dr. Saclarides: Head, Section of Colon and Rectal Surgery, Department of General Surgery, Rush-Presbyterian-St. Luke's Medical Center, 1653 West Congress Parkway, Chicago, Illinois 60612. 747
748
SACLARIDES E T AL
but also in inflamed non-neoplastic colonic epithelium, thus limiting potential applications as tumor markers. Considerable research has been performed with P-glycoprotein and its role in conferring cellular resistance to anticancer drugs. 7-9 The genetic basis of multidrug resistance has been elucidated, 1~ 11 and the responsible gene in humans is the Mdrlgene.la, 13 Its encoded protein, an energy-driven 170kd membrane efflux transport, is P-glycoprotein. Normal expression of P-glycoprotein is site ~ specific 9,14,15; approximately 30 percent of patients will express P-glycoprotein in histologically normal gastrointestinal epithelium when immunostained by monoclonal antibody C-219. Cancer and the development of resistance to anticancer drugs is accompanied by an overexpression of Pglycoprotein 9, 14, 16-18; studies have shown expression in 70 percent of colorectal cancers. 19 Three patterns of P-glycoprotein immunoreactivity have been identified and described elsewhere. 2~ A supranuclear dense staining corresponding to the Golgi zone is described as the Golgi staining pattern. Immunoreactivity may also be seen throughout the cytoplasmic compartment, giving rise to a diffuse staining pattern. Luminal membrane staining may occur as well. Correlation between ABO blood type and the Golgi-type pattern of P-glycoprotein expression has also been described, a~ Although it is known that neoplasia is associated with P-glycoprotein overexpression, expression in pathologically abnormal non-neoplastic and preneoplastic tissue has not been elucidated. For this purpose, ulcerative colitis is a useful model owing to the possible sequential progression of normal mucosa through inflammation, dysplasia, and carcinoma. Carcinoma is known to occur in about 5 percent of cases of ulcerative colitis. 21 Dysplasia has been regarded as a precancerous lesion 22' 23 and may precede the development of carcinoma. 1 The purpose of this study was to determine the expression of P-glycoprotein in inflamed and dysplastic colons, compare this expression with normal controls, and assess P-glycoprotein as a potential screening device for cancer surveillance.
MATERIALS AND METHODS Twenty-eight patients with ulcerative colitis were treated by colectomy at Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois, from 1980
Dis Colon Rectum, August 1992
to 1990. Of these, tissues blocks were available in 21 instances; these cases form the basis of this study. There were 11 males and 10 females, with a mean age of 33.5 years (range, 16-70 years). The duration of disease ranged from one episode to 20 years of disease. The indications for surgery are listed in Table 1. The ABO blood types of the patients, performed by standard blood bank methods, were obtained from patient records. The colon specimens were processed for routine pathologic evaluation. Sections removed at surgery were grossly examined and fixed in 10 percent neutral buffered formalin, and representative blocks, including the resection margins, were taken for paraffin embedding. Hematoxylin and eosin stained paraffin sections from all cases were retrospectively reviewed to assess morphologic and histologic features. All cases were confirmed to be ulcerative colitis and classified as active disease (17), resolving colitis (2), or quiescent colitis (2) by standard histologic features. 24 Ten cases showed varying degrees of dysplasia, such as indefinite (2), low grade (5) or high grade (3). Dysplasia was graded according to internationally accepted criteria. 25 From each patient, representative sections of noninflamed, inflamed, and dysplastic areas were selected. In most cases (18/21), a section of noninflamed proximal colonic mucosa was available. In three cases, however, ileal mucosa from the ileal resection margin-was selected, as there was involvement of the entire large intestine by the inflammatory process. In addition, representative sections from 10 cases of dysplasia were selected. These selected sections were then processed for immunohistochemical studies. A murine monoclonal antibody, subclass IgGaA Table 1. Patient Demographics Sex: 11 males, 10 females Age: mean, 335 years (range, 16-70 years) Duration of disease before surgery One episode 1-5 years Greater than 5 years Indication for surgery Urgent Toxic colitis Obstruction Hemorrhage Elective Refractory Fear of cancer
4 10 7
6 1 2 10 2
P-GLYCOPROTEIN--ULCERATIVE COLITIS
Vol. 35, No. 8
C219, obtained from Centocor Diagnostics (Malvern, PA) was used for staining of paraffin sections. This antibody binds to a highly conserved cytoplasmic epitope of P-glycoprotein. Immunohistochemistry was performed using the Vectastain elite ABC procedure and reagents (Vector, Burlingame, CA). All steps were repeated in a negative control slide excluding the primary antibody. The extent of MAbC219 immunoreactivity was then scored. If no staining was seen in any of the three patterns, namely supranuclear, diffuse, or luminal, the specimen was scored as negative. The presence or absence of these three patterns was then individually scored as follows: zero (0) when a particular pattern was absent; 1+ when less than 5 percent of epithelial cells were immunostained with a given pattern in a section; 2+ when 5 to 25 percent of cells were immunostained with a given pattern; and 3+ when more than 25 percent of cells were immunostained with a given pattern, The sum of the three scored patterns was used to compare levels of P-glycoprotein expression in the three areas: namely normal, inflamed, and dysplastic.
TM
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diffuse, and luminal. Figures 2A, 2B, and 2C illustrate immunostaining in normal, inflamed, and dysplastic areas. Applying the McNemar test (binomial two-tailed), a statistically significant difference was noted in P-glycoprotein expression when comparing normal with inflamed areas ( P = 0.015). Comparing normal with dysplastic areas, no statistical difference was noted owing to small sample size.
RESULTS As shown in Figure 1, 8 of 21 (38 percent) normal histologic sections expressed P-glycoprotein, whereas 15 of 21 inflamed areas (71 percent) and 7 of 10 dysplastic areas (70 percent) expressed this protein. A section was considered positive for P-glycoprotein if immunoreactivity was present in any of the three patterns, namely supranuclear,
Normal (n = 21; 38%)
A P=0.015
/ /
/
~
P=0.250 \
~
(smallsample
/ Inflame~
~
Dysplasia
(n = 21; 71%) (n = 10; 70%) Figure 1. Overallexpressionof P-glycoproteinin normal, inflamed,and dysplasticareas.
Figure2. A. SupranuclearcircumscribedGolgipattern of immunostainingin "normal"colonicmucosa. B. Inflamed area of activeulcerativecolitisshowingdiffusecytoplasmic and luminalpattern of immunostaining.C. Area of highgrade dysplasiawith diffusecytoplasmicimmunostaining.
750
SACLARIDES ET AL
The sum of scored immunoreactivity was assessed progressing from normal to inflamed and ultimately to dysplastic areas within a given patient (Table 2). Again, a binomial two-tailed analysis (sign test) was performed. When progressing from normal to inflamed areas, expression was increased in 11 patients, equal in 8, and decreased in only 2 ( P = 0.0225). When comparing dysplastic with normal areas within a given patient, expression was increased in 6, equal in 3, and decreased in 1 (P = 0.125). When progressing from inflamed to dysplastic areas, expression was increased in 3 and equal in 7 (P = 0.250). The pattern of immunostaining was assessed with respect to ABO blood type. Seven of 21 cases were blood type A, and, of thee, five exhibited predominantly the Golgi pattern of staining. Only 2 of the remaining 14 cases with non-A blood type showed the Golgi pattern. P-glycoprotein expression was assessed with respect to duration of disease (Table 3) as well as indication for surgery (Table 4). Increasing duration of disease led to a significant overexpression Table 2. Scored P-Glycoprotein Expression When Progressing from Normal to Inflamed and Dysplastic Areas Normal --, inflamed Increased expression Equal expression Decreased expression
52% 37% 10%
P = 0.02
Inflamed --, dysplastic Increased expression Equal expression
30% 70%
N8
Normal ---, dysplastic Increased expression Equal expression Decreased expression
60% 30% 10%
NS
Table 3. P-Glycoprotein Expression and Duration of Disease Normal First episode (n = 4) 5 years (n = 7)
0% 30% 71% P = 0.05
Inflamed Dysplastic 75% 60% 83% NS
100% 60% 86% NS
Table 4. P.Glycoprotein Expression and Indications for Surgery Urgent (n = 9) Elective (n = 12)
Normal
Inflamed
Dysplastic
11% 58% P = 0.027
66% 75% NS
66% 66% NS
Dis Colon Rectum, August 1992
of P-glycoprotein, but only in histologically normal areas. When surgery was performed for elective reasons, i.e., steroid dependence, intolerable side effects of medical therapy, or fear of cancer, there was a significant overexpression of P-glycoproteinl but only within normal areas. DISCUSSION There are two closely related multidrug-resistant genes in humans called MDR1 and MDR2 genes. The protein products of these genes have been called P-glycoprotein. Although the function of the MDR2 gene product is unknown, P-glycoprotein derived from the MDR1 gene has been extensively studied. It is a highly conserved pleiotropic membrane transport protein which actively transports several apparently unrelated organic compounds, including cytotoxic drugs. P-glycoprotein may serve several roles in normal physiologic processes such as cellular detoxification of lipophilic xenobiotic compounds, 8' z6, 27 intracellular transport of steroids in adrenal tissue, 28 and transport of terminal saccharides for antigen A biosynthesis across Golgi membranes. 2~ Various antibodies that can recognize P-glycoprotein at specific cytoplasmic or external domains have been described. MABC219 is the most widely used antibody which binds to highly conserved hexapeptide in the cytoplasmic domain of P-glycoprotein. 8 Normal distribution of P-glycoprotein varies among patients and organs, from nonexistent to high levels of expression. In addition, a relationship between blood type A and the Golgi pattern of immunostaining has been described. Tissues have been grouped as high (adrenal), intermediate (intestine or urinary tract), and low (lymphoreticular) expressors of P-glycoprotein. Studies have shown that approximately 30 percent of normal colons express P-glycoprotein. 19 There is a wellrecognized overexpression of P-glycoprotein in human tumors, including colon cancer, where approximately 70 percent express P-glycoprotein39 Prior to this study, the expression of P-glycoprotein in inflamed and premalignant colonic epithelium was not known. Our study showed that overexpression of P-glycoprotein is not limited to cancerous tissue. Using ulcerative colitis as our model, we found that, like noncolitic colorectal cancers, 70 percent of inflamed and dysplastic colons expressed P-glycoprotein. With histologically normal colon acting as
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P-GLYCOPROTEIN--ULCERATIVE COLITIS
an internal control, the overexpression in inflamed colitic e p i t h e l i u m was statistically significant ( P = 0.0156). The p r e s e n c e of dysplasia, however, did not increase the expression of P-glycoprotein over that seen in inflamed areas. That ulcerative colitis is a premalignant condition is well d o c u m e n t e d , and o n e of the risk factors associated with cancer is disease of long duration .29 T h e exact point at which a t e n d e n c y toward cancer o n a molecular level occurs is unclear. Our data suggest that this occurs before histologic e v i d e n c e o f inflammation and dysplasia. This is s u p p o r t e d b y observing an increase in P-glycoprotein expression with disease of long duration, a finding which was statistically significant but only in histologically normal areas. In fact, the d e g r e e of P-glycoprotein overexpression in normal areas w h e n colitis was present for longer than five years app r o a c h e d that seen in noncolitic colorectal cancers. T h e overexpression in normal areas w h e n surgery was p e r f o r m e d for elective reasons probably reflects chronicity of disease. Our study lends support to the notion that ulcerative colitis has malignant potential and suggests that the neoplastic t e n d e n c y occurs early in the course of the disease. It appears that a 38 p e r c e n t i n c i d e n c e of expression in histologically normal areas might impair its use as a screening tool for high-risk patients. If, however, this subset of patients were to s u b s e q u e n t l y d e v e l o p high-grade dysplasia or even cancer, t h e n perhaps P-glycoprotein could be used to identify a high-risk group. Unfortunately, our study had a limited n u m b e r of dysplastic areas (10) and no cancers; therefore, no information can be extrapolated as to the longterm significance of P-glycoprotein expression in histologically normal or inflamed areas. A prospective surveillance study with a large n u m b e r of patients will be n e e d e d to answer this question.
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