CASE REPORTS. Human Aberrant Crypt Foci With Carcinoma In Situ From a Patient With Sporadic Colon Cancer. Case Report

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1 GASTROENTEROLOGY 1996;111: CASE REPORTS Human Aberrant Crypt Foci With Carcinoma In Situ From a Patient With Sporadic Colon Cancer ANASTASIOS K. KONSTANTAKOS,* I MEI SIU, THOMAS G. PRETLOW, THOMAS A. STELLATO,* and THERESA P. PRETLOW *Department of Surgery and Institute of Pathology, Case Western Reserve University Medical Center, Cleveland, Ohio by Pretlow et al., 6 Pretlow, 7 Bird, 8 and Pretlow and Pret- low 9 ), suggests that these aberrant crypt foci are putative precursors of colon cancer. The demonstration of invasive cancer in aberrant crypt foci in rodents 6,7 lends support to this hypothesis. Although dysplasia has been reported previously in aberrant crypt foci in humans, 3 5,10,12 this report provides the first detailed description of aberrant crypt foci with carcinoma in situ from a patient with sporadic colon cancer. This patient had two primary co- lon cancers 11 years apart, multiple aberrant crypt foci with carcinoma in situ, and pseudomelanosis coli at the time of resection of his second colon cancer. Aberrant crypt foci are putative preneoplastic lesions found in the colons of carcinogen-treated rodents and at an increased frequency in humans at increased risk for colon cancer. There is a strong association between aberrant crypt foci and colon cancer, including many shared phenotypic and genetic alterations. The aim of this study is to present further evidence of a relation- ship between aberrant crypt foci and colon cancer in humans. Multiple aberrant crypt foci from a single patient were identified in unembedded colonic mucosa. Histological sections of the aberrant crypt foci and adja- cent mucosa were evaluated for dysplasia, proliferative activity, and pigment-laden macrophages that were characterized with histochemical techniques. The first patient with sporadic colon cancer identified with aberrant crypt foci with carcinoma in situ is described. It is interesting that this 99-year-old patient had multiple carcinomas in situ, pseudomelanosis coli, and two metachronous colon cancers. These data lend support to the hypothesis that aberrant crypt foci are precursors of some colon cancers. A Case Report The occurrence of carcinoma in situ in two of the first three aberrant crypt foci evaluated in the grossly normal mu- cosa from a patient with sporadic colon cancer led us to search for unusual circumstances or family history that would provide further insights into aberrant crypt foci and/or their relationship to colon cancer. In 1984, when this white man was 88 years old, a moderately well-differentiated adenocarcinoma that infiltrated the serosa was removed from the sigmoid colon. Two of eight lymph nodes examined had carcinoma. Several postoperative follow-up colonoscopies failed to show any recur- rence of malignancy. In 1990, four sessile polyps were noted. Biopsies were performed on three of these polyps, measuring between 0.5 and 1 cm, and found to be tubulovillous adeno- mas; a biopsy was not performed on the fourth polyp, measur- ing õ0.5 cm. Yearly rectal examinations and guaiac tests on his stool specimens were negative. In 1995, at the age of 99 years, the patient presented with a 2 3-month history of constipation and mild, intermittent, crampy abdominal discomfort that occasionally localized to the right lower quadrant. He reported a decrease in the caliber of his stools and a 5-lb weight loss during this time. A variety of laxatives were used by this patient, but none of them appeared to contain anthraquinone compounds. A chest x-ray showed at least one, possibly two, indeterminate nodules in berrant crypt foci were first described 1 in the colons of carcinogen-treated rodents and later 2 5 at higher incidence in humans at increased risk for colon cancer. These lesions (Figure 1) are identified microscopically in whole-mount segments of colonic mucosa that have been stained, usually with methylene blue, to allow rapid identification of individual crypts from the mucosal sur- face. The crypts in aberrant crypt foci are two to three times larger than normal crypts, have a thickened layer of epithelial cells that generally stains darker than normal crypts, are microscopically elevated, usually have slitshaped lumina, and have an increased pericryptal space between them and the surrounding normal crypts (re- viewed by Pretlow et al., 6 Pretlow, 7 Bird, 8 and Pretlow and Pretlow 9 ). The strong association between aberrant crypt foci and Abbreviation used in this paper: PAS, periodic acid Schiff. colon cancer, including shared phenotypic 3,10 and ge by the American Gastroenterological Association netic 11 alterations in both rodents and humans (reviewed /96/$3.00

2 September 1996 CARCINOMA IN SITU IN HUMAN ABERRANT CRYPTS 773 from 115 cm 2 of colonic mucosa from this patient. Serial, 5- mm sections were mounted two per slide on Superfrost Plus treated slides (Fisher Scientific Co., Pittsburgh, PA). Every tenth slide was stained with H&E for histopathologic evaluation. Three or more slides from different regions of each focus were stained immunohistochemically with monoclonal antibody clone COL-1 (Zymed, San Francisco, CA) for the demonstration of carcinoembryonic antigen to demarcate clearly in histological sections the aberrant crypt foci that had been identified microscopically in whole-mount mucosa before embedding the tissue in paraffin. 10 Representative histological sections from three aberrant crypt foci were immunohistochemically stained 13 with MIB-1 (Biogenex, San Ramon, CA) monoclonal antibody for the demonstration of Ki-67 antigen, an antigen that is associated with cells in the proliferative phase. Representative histological sections were incubated with Figure 1. An aberrant crypt focus from this patient contains many Perls stain for the demonstration of iron, 14 with periodic acid large crypts with elongated and/or serrated lumina surrounded by Schiff (PAS) reagent 15 and with the Nile blue sulfate method small normal crypts with circular lumina in unembedded colonic mu- for the demonstration of lipofuscin. 16 cosa stained with methylene blue. Histological sections of this focus show moderate dysplasia (original magnificatio 251). Results Three aberrant crypt foci, two of which had multithe right upper lobe. The patient was well nourished; a ple 5-mm sections with carcinoma in situ, were identified strongly positive guaiac test on his stool was the only notable in a single 19-cm 2 piece of grossly normal colonic mucosa finding at the time of admission. His hematocrit was 33, and located between 5 and 10 cm from the colon cancer in a double-contrast barium enema showed a 5-cm apple-core lesion in the middle of the ascending colon. A right hemicolecdysplasia. Because this is the first identification of carci- this patient. The third aberrant crypt focus had moderate tomy was performed; there was no evidence of gross intranoma in situ in aberrant crypt foci from humans with abdominal metastases at the time of surgery. The patient s postoperative course was uneventful, and he was able to return sporadic colon cancer, we evaluated an additional 96 cm 2 to his previous level of activity. of grossly normal colonic mucosa from this patient. Only Pathological findings included a 4.5-cm, infiltrating, mod- three additional aberrant crypt foci were identified: one erately differentiated adenocarcinoma that extended into the with moderate dysplasia 2 5 cm from the cancer, one pericolonic fat adjacent to but not including the serosal surface. with carcinoma in situ (Figure 2A C) cm from Vascular invasion was observed. Multiple tubular adenomas the cancer, and another with moderate dysplasia were noted; the surgical margins were free of tumor. Metastatic cm from the cancer (Table 1). This gave an overall frecarcinoma was identified in two of 15 lymph nodes examined. quency of 0.05 aberrant crypt foci/cm 2 with 0.17% of Materials and Methods the mucosal surface covered with aberrant crypt foci. It is of interest that all six aberrant crypt foci identified The colectomy specimen in 1995 was obtained in saline at 4 C from the operating room by the Western Division of the from this patient showed some degree of dysplasia. In- Cooperative Human Tissue Network located at Case Western creased expression of carcinoembryonic antigen was ob- Reserve University. The studies were approved by the Univertions of some of the aberrant crypt foci were evaluated served in all of the aberrant crypt foci. Histological secsity Hospitals of Cleveland Institutional Review Board for Human Investigations in accordance with assurance filed with immunohistochemically for the expression of Ki-67 antiand approved by the U.S. Department of Health and Human gen, an antigen that is expressed in proliferating cells. Services. Strips of grossly normal colonic mucosa, approxi- Three of the three aberrant crypt foci evaluated for Kimately cm, were removed from the submucosa, snap- 67 antigen (Table 1) showed proliferating cells in the frozen flat, and stored over liquid nitrogen at 0195 C. Eight upper portions of the crypts. pieces of mucosa were thawed at 4 C with agitation in 1% Histological sections of these aberrant crypt foci and paraformaldehyde in 0.1 mol/l sodium phosphate buffer (ph 7.4), fixed flat for 1 hour at room temperature in 10% formalin, adjacent normal mucosa also showed large mononuclear and stained in 0.2% methylene blue. Aberrant crypt foci were cells laden with yellow-brown pigment in the lamina identified and marked with permanent ink under 301 magnihuman aberrant crypt foci from more than 60 pa- propria (Figure 3A). In our histological evaluation of fication; they were embedded in paraffin as described previously. 3,10 We identified and evaluated six aberrant crypt foci tients, 3,10,17 this is the first patient in whom we have

3 774 KONSTANTAKOS ET AL. GASTROENTEROLOGY Vol. 111, No. 3 Figure 2. Histological section of an aberrant crypt focus with carcinoma in situ stained with H&E. (A) A low-power view of the focus, marked with yellow ink, with surrounding normal colonic mucosa. (B) Figure 3. Histological section of normal colonic mucosa from this A higher magnificatio of some of the crypts in this aberrant crypt patient. (A) Note the mononuclear cells laden with yellow-brown pigfocus. Note the marked loss of mucin and the nuclear pleomorphism, ment in the lamina propria (H&E). (B) An adjacent section in which stratification and loss of basal orientation in the top two glands the pigment in the mononuclear cells stains blue even after incubation compared with the more normal-appearing glands below. (C ) Higher in H 2 O 2 (Nile blue sulfate). (C ) An adjacent section in which the pigment magnificatio of crypts with carcinoma in situ in this aberrant crypt in the mononuclear cells stains magenta (PAS; original magnifi focus (original magnification A, 301; B, 2001; C, 4001). cation: A C, 2501).

4 September 1996 CARCINOMA IN SITU IN HUMAN ABERRANT CRYPTS 775 Table 1. Characterization of Aberrant Crypt Foci Distance to Degree of No. of crypts ACF size Crypt size in Ki-67 ACF no. tumor (cm) dysplasia per focus (mm 2 ) ACF (mm 2 ) expression Moderate ND 2 a 5 10 Moderate Shifted upwards Severe Shifted upwards Severe Shifted upwards 5 b Severe ND Moderate ND Mean { SD 56 { { { 0.02 ACF, aberrant crypt foci; ND, not done. a Figure 1. b Figure 2. noted pigment-laden macrophages. Histochemical analy- previous studies (0.03 { 0.01 mm 2 [unreported data, 21 ses of these pigmented cells showed lipofuscins with Nile December 1995] and 0.04 { 0.02 mm 2 [unreported blue sulfate reagent (Figure 3B), PAS-reactive material data, 17 March 1996]). (Figure 3C), and a lack of detectable iron with Perls It is noteworthy that the aberrant crypt foci in this stain. The magenta staining of pigment in the mononu- patient showed proliferating cells in the upper regions clear cells with PAS reagent 18 and the blue color retained of the crypts. In normal crypts and in aberrant crypt foci by the pigment after reaction with Nile blue sulfate even evaluated to date in both humans 22 and rats, 23 the zone after a 24-hour incubation in 10% H 2 O 16 2 are indicative of proliferation has been limited to the lower portions of lipofuscin rather than melanin. The presence of large of the crypts. The finding of proliferating cells near the mononuclear cells or macrophages with lipofuscin gran- surfaces of the crypts mimics that observed in adenomas ules in colonic lamina propria is consistent with pseudo- (discussed in article by Pretlow et al. 23 ). The most outmelanosis coli that is associated with the use of anthra- standing feature of the aberrant crypt foci in this patient quinone laxatives. 19,20 was the severity of the dysplasia observed in multiple Discussion aberrant crypt foci. Roncucci et al. 4 classified their aberrant crypt foci into only two grades of dysplasia: low and The presence of carcinoma in situ in three of six high grade. The aberrant crypt focus with high-grade aberrant crypt foci, the first carcinoma in situ to be idennot dysplasia in the photomicrograph of Roncucci et al. 4 does tified in humans with sporadic colon cancer, in a patient appear to be as advanced as some of the aberrant with two metachronous colon cancers and pseudomelanoin crypt foci that we observed in this patient, i.e., the cells sis coli suggests that these three phenomena may be their focus showed less disruption of orientation, less related. Although the frequency and area of mucosa occupied nuclear stratification, and the retention of more mucin. by the aberrant crypt foci in this patient were greater Melanosis coli or pseudomelanosis coli has been recog- than in many patients, the values reported (0.05 aberrant nized for more than 150 years (reviewed by Speare 19 ). crypt foci/cm 2 and 0.17% of mucosa with aberrant crypt Speare 19 defined melanosis coli as the pigmentation of foci) are within the ranges reported previously (0.023 { the colonic mucosa from the ileocecal valve to the anorec aberrant crypt foci/cm 2 and 0.063% { 0.14% of tal line. The gross appearance of the colonic mucosa mucosa with aberrant crypt foci) for the right colons of varies widely from buff color to black depending on patients with colon cancer on the right side. 3,6 The num- the amount of pigment present. Several investigators, ber of crypts per aberrant crypt focus (56 { 22 crypts/ including Speare, 19 have induced the accumulation and focus; Table 1) was larger in foci from this patient than elimination of pigment in humans by the administration in 27 aberrant crypt foci (29 { 29 crypts/focus; unreported and withdrawal of anthracene laxatives. Speare 19 and ear- data, December 1995) from 20 patients in a recent lier investigators (reviewed by Speare 19 ) speculated that study 21 but was very similar to that (55 { 56 crypts/ the pigment in the mononuclear cells was derived from focus; unreported data, March 1996) observed in another the pigment in the laxatives with anthracene or anthraquinone study of 50 aberrant crypt foci from 28 patients. 17 In compounds. Later experimental studies 24 showed contrast, the average sizes of the crypts within most of that the anthraquinone compounds induce apoptosis of the aberrant crypt foci from this patient (0.06 { 0.02 the colonic surface epithelium. The resulting apoptotic mm 2 ; Table 1) are much larger than those observed in bodies appear to be phagocytosed by intraepithelial mac-

5 776 KONSTANTAKOS ET AL. GASTROENTEROLOGY Vol. 111, No. 3 rophages that carry them to the lamina propria where hypotheses that pseudomelanosis coli increases the risk the apoptotic bodies are converted to lipofuscin pigment for colon cancer and that aberrant crypt foci are precursors in the lysosomes of the macrophages. 24 Although this of some colon cancers. patient reported using a variety of laxatives, none of them appeared to contain anthraquinone compounds. It is not References known whether other agents or conditions, such as other 1. Bird RP. Observation and quantificatio of aberrant crypts in the harsh laxatives, chronic constipation, or even very adings. murine colon treated with a colon carcinogen: preliminary find Cancer Lett 1987; 37: vanced age, can induce apoptosis of the colonic surface 2. O Riordan MA, Barrow BJ, Jurcisek JA, Stellato TA, Pretlow TP. epithelium with a similar accumulation of lipofuscin pig- Aberrant crypts in the colons of humans and carcinogen-treated ment. rats are enzyme-altered (abstr). Proc Am Assoc Cancer Res 1990; 31:85. The incidence of pseudomelanosis coli has been re- 3. Pretlow TP, Barrow BJ, Ashton WS, O Riordan MA, Pretlow TG, ported variously as 1% 4% (reviewed by Morgenstern Jurcisek JA, Stellato TA. Aberrant crypts: putative preneoplastic et al. 25 ), 5.9% in a selected series of colons with neo- foci in human colonic mucosa. Cancer Res 1991; 51: plasms, % in a retrospective study of more than 4. Roncucci L, Stamp D, Medline A, Cullen JB, Bruce WR. Identifica 3000 patients, 20 and 7% in a prospective study of 1095 tion and quantificatio of aberrant crypt foci and microadenomas patients. 20 In the prospective study, patients with adeno- in the human colon. Hum Pathol 1991; 22: mas had a 9.8% incidence of pseudomelanosis coli (P Å 5. Roncucci L, Medline A, Bruce WR. Classificatio of aberrant crypt foci and microadenomas in human colon. Cancer Epidemiol Bio ) compared with a 6.9% incidence among those markers Prev 1991; 1: without any abnormality; patients with carcinomas had 6. Pretlow TP, O Riordan MA, Pretlow TG, Stellato TA. Aberrant an 18.6% incidence of pseudomelanosis coli (P õ crypts in human colonic mucosa: putative preneoplastic lesions. J Cell Biochem Suppl 1992; 16G: ). 20 From these data, it was calculated that, for 7. Pretlow TP. Alterations associated with early neoplasia in the patients who misuse these anthraquinone laxatives, the colon. In: Pretlow TG, Pretlow TP, eds. Biochemical and molecular relative risk for colon cancer increases to A previ- aspects of selected cancers. Volume 2. San Diego: Academic, 1994: ous report of an 18-year-old woman who was exposed to 8. Bird RP. Role of aberrant crypt foci in understanding the pathoa high dose of danthron, an anthraquinone laxative, from genesis of colon cancer. Cancer Lett 1995; 93: age 14 months to 5 or 6 years and who died of a leiomyotines. 9. Pretlow TP, Pretlow TG. Neoplasia and preneoplasia of the intessarcoma In: Bannasch P, ed. Pathology of neoplasia and preneopla- of the small intestine suggested the possibility sia in rodents, EULEP color atlas of pathology. Volume 2. Stuttof a connection between this class of laxatives and can- gart, Germany: Schattauer (in press). cer. 26 There are several in vivo 27 and in vitro 28,29 studies 10. Pretlow TP, Roukhadze E, O Riordan MA, Chan JC, Amini SB, that confirm the tumorigenic potential of some anthracrypt Stellato TA. Carcinoembryonic antigen in human colonic aberrant foci. Gastroenterology 1994; 107: quinones found in some laxatives. In addition, the oral 11. Pretlow TP, Brasitus TA, Fulton NC, Cheyer C, Kaplan EL. K-ras administration of laxatives with anthraquinones results mutations in putative preneoplastic lesions in human colon. J in very high colonic cell proliferation 24 hours later. 30 Natl Cancer Inst 1993; 85: Otori K, Sugiyama K, Hasebe T, Fukushima S, Esumi H. Emer- Morgenstern et al. 25 made the interesting observation gence of adenomatous aberrant crypt foci (ACF) from hyperplastic that both benign and malignant neoplasms in pseudo- ACF with concomitant increase in cell proliferation. Cancer Res melanosis coli colons lack the pigmented macrophages 1995; 55: Shi S-R, Imam SA, Young L, Cote RJ, Taylor CR. Antigen retrieval found in the lamina propria of the surrounding normal immunohistochemistry under the influenc of ph using monomucosa. Although the aberrant crypt foci from our pa- clonal antibodies. J Histochem Cytochem 1995; 43: tient with pseudomelanosis coli were not totally devoid 14. Johnson FB. Perls iron stain. In: Prophet EB, Mills B, Arrington of pigmented macrophages, many histological sections JB, Sobin LH, eds. Armed Forces Institute of Pathology, laboratory methods in histotechnology. Washington, DC: American Registry of these lesions appeared to have less pigment than the of Pathology, 1992:195. surrounding mucosa. An increased incidence (17%) of 15. Gaffney E. Periodic acid Schiff (PAS) procedure. In: Prophet EB, synchronous invasive cancers has also been reported in Mills B, Arrington JB, Sobin LH, eds. Armed Forces Institute of patients with pseudomelanosis coli. 25 This is in contrast Pathology, laboratory methods in histotechnology. Washington, DC: American Registry of Pathology, 1992:151. to a 2.8% 31,32 to 3.5% 33 rate of occurrence for synchro- 16. Pearse AGE. Pigments and pigment precursors. In: Histochemistry: nous colon cancers and a 1.6% rate for metachronous theoretical and applied. 3rd ed. Volume 2. Appendix 26. Baltimore: Williams & Wilkins, 1972: colon cancers in a large series of patients with sporadic 17. Siu I, Pretlow TG, Pretlow TP. Dysplasia in human aberrant crypt colon cancer. It is interesting that the first patient with foci, putative precursors of colon cancer. Proc Am Assoc Cancer sporadic colon cancer to be identified with aberrant crypt Res 1996; 37: Johnson FB. Pigments and minerals. In: Prophet EB, Mills B, foci with carcinoma in situ has pseudomelanosis coli and Arrington JB, Sobin LH, eds. Armed Forces Institute of Pathology, two metachronous colon cancers that were diagnosed 11 laboratory methods in histotechnology. Washington, DC: American Registry of Pathology, 1992:183 years apart. The data from this patient support the 196.

6 September 1996 CARCINOMA IN SITU IN HUMAN ABERRANT CRYPTS Speare GS. Melanosis coli: experimental observations on its pro- 28. Wolfl D, Schmutte C, Westendorf J, Marquardt H. Hydroxyanthraduction and elimination in 23 cases. Am J Surg 1951; 82:631 quinones as tumor promoters: enhancement of malignant trans formation of C3H mouse fibroblast and growth stimulation of 20. Siegers C-P, von Hertzberg-Lottin E, Otte M, Schneider B. Anthra- primary rat hepatocytes. Cancer Res 1990; 50: noid laxative abuse a risk for colorectal cancer? Gut 1993; 34: 29. Westendorf J, Marquardt H, Poginsky B, Dominiak M, Schmidt J, Marquardt H. Genotoxicity of naturally occurring hydroxyanthra- 21. Augenlicht LH, Richards C, Corner G, Pretlow TP. Evidence for quinones. Mutat Res 1990; 240:1 12. genomic instability in human colonic aberrant crypt foci. Onco- 30. Kleibeuker JH, Cats A, Zwart N, Mulder NH, Hardonk MJ, devries gene 1996; 12: EGE. Excessively high cell proliferation in sigmoid colon after an 22. Roncucci L, Pedroni M, Fante R, Di Gregorio C, Ponz de Leon M. oral purge with anthraquinone glycosides. J Natl Cancer Inst Cell kinetic evaluation of human colonic aberrant crypts. Cancer 1995; 87: Res 1993; 53: Moertel CG, Bargen JA, Dockerty MB. Multiple carcinomas of the 23. Pretlow TP, Cheyer C, O Riordan MA. Aberrant crypt foci and colon large intestine. A review of the literature and a study of 261 tumors in F344 rats have similar increases in proliferative activ- cases. Gastroenterology 1958; 34: ity. Int J Cancer 1994; 56: Moertel CG. Multicentric adenocarcinomas of the colon and rec- 24. Walker NI, Bennett RE, Axelsen RA. Melanosis coli: a conse- tum. Recent Results Cancer Res 1966; 7: quence of anthraquinone-induced apoptosis of colonic epithelial 33. Heald RJ, Bussey HJR. Clinical experiences at St. Mark s hospital cells. Am J Pathol 1988; 131: with multiple synchronous cancers of the colon and rectum. Dis 25. Morgenstern L, Shemen L, Allen W, Amodeo P, Michel SL. Mela- Colon Rectum 1975; 18:6 10. nosis coli: changes in appearance when associated with colonic neoplasia. Arch Surg 1983; 118: Patel PM, Selby PJ, Deacon J, Chilvers C, McElwain TJ. Anthraquinone laxatives and human cancer: an association in one case. Received January 15, Accepted April 8, Postgrad Med J 1989; 65: Address requests for reprints to: Theresa P. Pretlow, Ph.D., Insti- 27. Mori H, Yoshimi N, Iwata H, Mori Y, Hara A, Tanaka T, Kawai K. tute of Pathology, Case Western Reserve University, 2085 Adelbert Carcinogenicity of naturally occurring 1-hydroxyanthraquinone in Road, Cleveland, Ohio Fax: (216) rats: induction of large bowel, liver and stomach neoplasms. Supported by grants CA48032, CA66725, CA54031, DK51347, Carcinogenesis 1990; 11: and P30CA43703 from the National Institutes of Health.