ANATOMIC PATHOLOGY Original Article
Small Cell Carcinomas of the Large Intestine ALLEN B. BURKE, M.D. ( M A J , USAF, MC), KRIS M. SHEKITKA, M.D. (LTCOL, USAF, MC), AND LESLIE H. SOBIN, M.D.
differentiation (1 of 15) and fewer liver metastases (4 of IS) than did small cell carcinomas. Among all 59 tumors studied, small cell histologic characteristics correlated better with liver involvement than did endocrine markers or other histologic features. Small cell carcinomas of the large intestine are aggressive tumors with a propensity for early liver involvement. Although there is a spectrum of squamous, endocrine, and glandular features in large bowel tumors of low degrees of differentiation, the identification of a small cell component appears to be most clinically relevant. (Key words: Colon; Rectum; Small cell carcinoma) Am J Clin Pathol 1991;95:315-321
Small cell carcinomas of the colon and rectum are highly malignant tumors similar in histologic characteristics, behavior, and histochemistry to small cell carcinoma of the lung.1 They show at least some evidence of endocrine differentiation2"4 and may contain glandular and squamous foci,5"8 making classification difficult. Endocrine granules are present in small numbers of cells in typical adenocarcinomas of the gastrointestinal tract. 910 The endocrine content of poorly differentiated adenocarcinomas and undifferentiated non-small cell carcinomas, however, has not been well characterized. The frequency of such endocrine differentiation could be of diagnostic" and conceptual interest in terms of the
relation between small cell, poorly differentiated adenocarcinomas and undifferentiated carcinomas. We studied a group of small cell colorectal carcinomas and compared their clinical, histologic, and immunohistochemical features with those of a group of poorly differentiated adenocarcinomas and undifferentiated carcinomas. MATERIALS AND METHODS Criteria for Selection
All malignant epithelial neoplasms of the large intestine with blocks or tissue accessioned to the Armed Forces Institute of Pathology between 1986 and 1988 were prospectively studied. Typical carcinoids and well- or modFrom the Department of Gastrointestinal Pathology. Armed Forces erately differentiated adenocarcinomas forming glandular Institute of Pathology. Washington, D.C. structures were excluded, because these have been well Received April 12, 1990; received revised manuscript and accepted studied and pose few difficulties in diagnosis. "Typical" for publication June 28, 1990. carcinoids indicate tumors with characteristic growth Presented at the Fall Meeting, American Society of Clinical Pathologists, November 1989, Washington, D.C. patterns, fewer than 1 mitosis/high-power field (HPF), and The opinions or assertions contained herein are the private views of the lack of necrosis; we have reported these in a previous the authors and are not to be construed as official or reflecting the views publication.12 These restrictions resulted in 59 epithelial of the Department of the Army, the Department of the Air Force, or the Department of Defense. neoplasms that had large areas of solid growth that did Address reprint requests to Dr. Burke: Department of Gastrointestinal not show glandular differentiation by hematoxylin and Pathology, Armed Forces Institute of Pathology, Washington, D.C. eosin stains. Tumors were retained for study even if ad20306-6000. 315
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The authors studied the clinical and pathologic features of 38 small cell carcinomas of the large intestine. Most were located in the right colon. Overlying adenomas were present in 45% and squamous differentiation in 21% of tumors. Endocrine differentiation was present in all tumors by at least one method; neuronspecific enolase, dense-core granules, and synaptophysin were present in most cases. Seventy-one percent of tumors metastasized to the liver; 64% of patients were dead at five months followup. Twenty-one poorly differentiated adenocarcinomas and undifferentiated carcinomas of the large intestine accessioned during the same period showed less endocrine (7 of 21) and squamous
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ANATOMIC PATHOLOGY Article
jacent areas consisted of adenoma or gland-forming adenocarcinoma or if there were foci of squamous differentiation. In two cases in which the epithelial nature of the tumor was in question, a cytokeratin stain was performed.
TABLE 1. Antiserum
Source
Dilution
NSE Chromogranin Leu-7 Serotonin Carcinoembryonic antigen
DAKO Hybritech DAKO Immunonuclear DAKO (polyclonal, unabsorbed) Hybritech Boehringer Mannheim
1:400 1:200 1:400 1:20 (purified) 1:160
Topographic Distribution For tabulation purposes, tumors from the cecum to the splenic flexure were considered right-sided and those in the descending colon to the rectum, left sided. Histologic Study
Histochemistry Argyrophilia was assessed by the Churukian-Schenk method 14 and mucin by the PAS methods with diastase digestion.
1:160 1:50
Electron Microscopic Examination Tissue for electron microscopic examination was obtained by trimming selected areas of 45 tumors from paraffin blocks. Pieces of tissue that were 1 mm 2 in size were placed in xylene overnight, fixed in 2.5-3% phosphatebuffered glutaraldehyde, postfixed in 1% phosphate-buffered osmium tetroxide, dehydrated in graded ethanols, and embedded in Effapoxy® (Ernest E. Fullam, Inc., Latham, NY). One-micron-thick sections were stained with toluidine blue O stain. Thin sections were stained with 4% uranyl acetate and lead citrate, mounted on carboncoated copper grids, and examined with a Zeiss EM 109® electron microscope (Carl Zeiss, Inc., Thornwood, NY). The presence of dense-core granules, as defined by membrane-bound round structures measuring 10-50 nm in diameter, was evaluated by one of us (K.M.S.) without knowledge of immunohistochemical findings or clinical data. Follow-up Information Follow-up information (Table 2) was based on questionnaires and clinical data sent with the consultation material. The liver was considered involved by tumor if there was a positive biopsy specimen, positive computed tomography (CT) scan, or liver involvement at autopsy; the liver was considered uninvolved if there was specific mention of a normal-appearing liver with negative biopsy results at surgery, negative CT scan, or no involvement at autopsy.
Immunohistochemistry
RESULTS
Immunostains for neuron-specific enolase (NSE), chromogranin, Leu-7, serotonin, synaptophysin, cytokeratin, and carcinoembryonic antigen were performed according to the peroxidase-antiperoxidase technique of Sternberger,15 with the use of diaminobenzidine as chromogen. Dilutions and sources of antisera are listed in Table 1. One representative section from the primary tumor was stained in each case and was graded as positive if more than 10% of the tumor cells stained. Positivity was assessed by two of us (A.P.B., K.M.S.) without knowledge of clinical history or the presence of metastases.
A.J.C.P. •
Histologic Features Small Cell Carcinomas (38 tumors). These were composed of cells with minimal nuclear clearing, inconspicuous nucleoli, and dispersed chromatin. Cellular cohesion with identifiable cytoplasm was present throughout 12 tumors, yielding the appearance of intermediate small cell carcinoma (Figs. 1 and 2). Six tumors had an oat-cell appearance throughout; in contrast to intermediate small cell carcinoma, these lacked distinct cytoplasm and had fusiform nuclei (Fig. 3). Twenty tumors had admixtures
rch 1991
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Two of us (A.P.B., K.M.S.) independently classified the tumors into three categories based on hematoxylin and eosin and periodic acid-Schiff(PAS) -stained slides: small cell carcinoma, poorly differentiated adenocarcinoma, and undifferentiated carcinoma. For small cell carcinoma, we used criteria similar to those published for small cell carcinomas of the lung and colon. 113 Poorly differentiated adenocarcinomas were classified on the basis of intracytoplasmic mucin vacuoles present on PAS stain, in areas where glandular differentiation was not appreciated on hematoxylin and eosin stain. Undifferentiated carcinomas lacked both glands and intracytoplasmic mucin.1 Categorization was done without knowledge of clinical findings and immunohistochemical results. Mitotic counts were performed by one of us (A.P.B.) on 5-fim sections with the use of a X40 power objective (490-jim field diameter), without knowledge of clinical or other pathologic data. The highest count in ten consecutive fields in up to 50 viewed (depending on the size of tumor) was recorded; the count was expressed in mitoses/HPF.
Cytokeratin AE1/3 Synaptophysin
BURKE, SHEKITKA, AND SOBIN
317
Small Cell Colorectal Carcinomas TABLE 2. CLINICOPATHOLOGIC FEATURES: SMALL CELL, POORLY DIFFERENTIATED ADENOCARCINOMAS AND UNDIFFERENTIATED CARCINOMAS Poorly Differentiated Adenocarcinoma (15 tumors)
Undifferen tiated Carcinoma (6 tumors)
63 years 22:16 22R, 7L, 8Re 45% 26% 24%
69 years 7:8 12R, 3L 27% 33% 0
56 years 5:1 4R, 1L, IRe 33% 17% 17%
31% 37% 84% 18% 56% 16% 81% 89% 89% 71% 36%
0 0 20% 0 13% 0 31% 100% 73% 7% 7/9
40% 50% 83% 17% 67% 0 50% 100% 83% 50% 2/4
Re = rectum; R = right colon; L - left colon. • Well- or moderately differentiated adjacent areas.
t At the time of surgery. t Cases for which five-month follow-up was available.
of intermediate small cell and oat-cell carcinoma. Intracytoplasmic mucin was invariably absent, although intraluminal mucin was present in four cases that had acinarlike structures. Overlying adenomas were present in 17 cases (Fig. 4) and squamous differentiation in 9 (Fig. 5). Poorly Differentiated Adenocarcinomas (15 tumors). These tumors did not show gland formation on routine stains but demonstrated PAS-positive cytoplasmic mucin vacuoles (Fig. 6). In contrast to small cell carcinomas, there were prominent nucleoli and nuclear clearing. None of these tumors had squamous areas; in eight cases wellor moderately differentiated areas were present and four had overlying adenomas. Undifferentiated Carcinomas (six cases). These tumors had nuclear features similar to those of poorly differentiated adenocarcinomas but lacked intracytoplasmic mucin (Fig. 7). Two had overlying adenomas. Four cases resembled large cell carcinoma of the lung. Two cases lacked epithelial characteristics and resembled lymphoma (Fig. 8); a positive stain for cytokeratin was necessary to establish a diagnosis of carcinoma. One of these tumors had numerous argyrophil cells by the Churukian-Schenk method and was positive for chromogranin. Results of Tests for Endocrine
Differentiation
More than 80% of the small cell carcinomas were positive for NSE and dense-core granules (Table 2). Positivity for chromogranin, Leu-7, and argyrophilia was often focal or patchy. Synaptophysin positivity was usually more diffuse (Fig. 9). The squamous areas were negative for en-
docrine markers with the exception of occasional weak NSE positivity; overlying adenomas and adenocarcinomas were generally negative for all endocrine markers, with the exception of rare positive cells. The undifferentiated carcinomas showed endocrine features in three of six cases. Four of 15 poorly differentiated adenocarcinomas had dense-core granules by electron microscopic examination but were usually negative for other endocrine markers. Frequency of Liver Metastases:
Follow-up
The frequency of liver metastases was higher at the time of surgery for the patients with small cell carcinoma than for those with poorly differentiated adenocarcinoma. The patients with small cell carcinomas received a variety of treatment regimens, including radiation, Cytoxan® (cyclophosphamide; Bristol-Myers/Mead Johnson Oncology, Evansville, IN), vincristine, Adriamycin® (doxorubicin HCI; Adria Laboratories, Dublin, OH), dacarbazine, 5fluorouracil, lomustine, and streptozocin. The variety of treatment regimens and the small numbers of cases make statements regarding efficacy of treatment impossible, but most patients did not respond to therapy. A single patient received combination anti-oat cell therapy (Cytoxan, vincristine, Adriamycin, D T I Q a n d is alive at 12 months despite liver metastases. Two-thirds of patients with small cell carcinomas were dead at five months. Histology of Metastases Histologic material was available for 21 small cell tumors. All metastases had the appearance of small cell car-
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Mean age M:F Location Adenoma Adenocarcinoma* Squamous change Positivity for Churukian-Schenk Chromogranin Neuron-specific enolase Leu-7 Synaptophysin Serotonin Dense-core granules Transmural extension Lymph node metastases Liver metastasesf Alive at 5-month follow-up:):
Small Cell Carcinoma (38 tumors)
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FIG. 5 (upper, left). Small cell carcinoma. The small cell carcinoma merges with squamous cell elements. Hematoxylin and eosin (X250). FIG. 6 (upper, right). Poorly differentiated adenocarcinoma. Unlike small cell carcinoma, there is nuclear clearing and prominent nucleoli. Periodic acid-Schiff-positive mucin vacuole (arrow). Hematoxylin and eosin (X400). FIG. 7 (lower, left). Undifferentiated carcinoma. Large cells, prominent nucleoli (not features of small cell carcinoma); mucin stains were negative (not shown). Hematoxylin and eosin (X400). FIG. 8 (upper, right). Undifferentiated carcinoma. Tumor composed of sheets of cells with prominent nucleoli and a lack of nuclear molding, resembling lymphoma. Tumor stained diffusely positive for cytokeratin, chromogranin, synaptophysin, and Leu-7 (not shown). Hematoxylin and eosin (XI50).
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ANATOMIC PATHOLOGY Original Article
FIG. 9. Small cell carcinoma. Cells positive in a patchy distribution for synaptophysin. Peroxidase-antiperoxidase (X250).
Statistical Analysis The effect of individual variables on liver metastasis at the time of surgery was studied for 34 transmural small cell carcinomas, 15 transmural poorly differentiated adenocarcinomas, and 6 transmural undifferentiated carcinomas. Liver involvement versus no liver involvement was compared by the chi-squared statistic (Fisher's exact test) with each histologic, immunohistochemical, and ultrastructural variable (Table 3). Small cell histologic characteristics were more significant in predicting liver metastases than other variables, although chromogranin and NSE were also significant. To assess independence of variables, a step-wise mulTABLE 3. VARIABLES ASSOCIATED WITH LIVER METASTASIS: SMALL CELL, POORLY DIFFERENTIATED ADENOCARCINOMAS AND UNDIFFERENTIATED CARCINOMAS Variable
P Value
Small cell histologic characteristics Neuron-specific enolase* Chromograninf Squamous differentiationf Dense-core granules*
0.0001 0.002 0.005 0.006 0.04
* Not independently significant by step-wise regression. t Minimally independently significant by step-wise regression (see text).
tiple regression was performed. Small cell histologic characteristics correlated with liver metastasis (r = 0.70); combined with small cell histologic characteristics, chromogranin positivity (r = 0.74) and squamous change (r = 0.79) increased the correlation. Other immunohistochemical variables did not show an independent correlation with liver metastasis. DISCUSSION The clinical course of the patients with small cell carcinomas in our study was marked by a high frequency of liver metastasis and early death. These findings agree with other reports of small cell carcinoma of the colon.2"4 One of our patients with liver metastases did well for 12 months with oat-cell chemotherapy; responses of small cell colorectal carcinomas to oat-cell treatment have been reported anecdotally.16 In our series, a variety of chemotherapeutic regimens were used, possibly because the tumors, despite similar histologic characteristics, were given different diagnoses by contributing pathologists. Uniform diagnostic criteria should be applied to obtain meaningful assessments of treatment responses. All of the small cell tumors in our series demonstrated endocrine differentiation by at least one special technique. The endocrine nature of small cell carcinomas of the lung and colorectum is well documented with the use of ultrastructure and immunohistochemical stains for NSE, chromogranin, synaptophysin, and Leu-7.17"21 We have found that many markers lack sensitivity, and the most sensitive marker, NSE, is known to lack specificity.19 The
A.J.C.P. • March 1991
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cinoma, with the exception of one case: one liver metastasis from a mixed small cell carcinoma-adenocarcinoma showed only moderately differentiated adenocarcinoma.
BURKE, SHEKITKA, AND SOBIN
32]
Small Cell Colorectal Carcinomas lack of sensitivity necessitates the use of multiple markers in some cases to demonstrate endocrine differentiation. In addition, four of six undifferentiated carcinomas and one-third of the poorly differentiated adenocarcinomas also reacted for endocrine markers, indicating that endocrine differentiation is not specific for any one histologic type of carcinoma. Because of this lack of specificity, tests for endocrine differentiation are not always reliable in distinguishing small cell carcinoma and undifferentiated carcinoma. Not all of the small cell tumors had densecore granules by electron microscopic examination, but this may be a result of sampling and the small numbers of granules present.
REFERENCES I. JassJR, Sobin LH. Histological typing of intestinal tumors. WHO International Histological Classification of Tumors, 2nd ed. Berlin: Springer-Verlag, 1989;33.
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Many small cell carcinomas in our series expressed divergent differentiation, namely glandular and squamous. However, virtually all metastatic lesions from small cell carcinomas were of small cell type. Several tumors had an overlying adenoma with little or no transition to the small cell carcinoma, leading to the initial diagnosis of metastatic small cell carcinoma. The association between colonic adenomas and endocrine tumors has been documented previously.2,22'23 We could not meaningfully correlate pathologic parameters with survival data because of differences in treatment. Early liver metastasis is a relatively good indicator of tumor aggressiveness, however. Our data indicate that, although some tests of endocrine differentiation are highly associated with liver metastases, small cell histologic characteristics are the best single predictor of liver metastasis among colorectal carcinomas of low differentiation. Other variables were independently not significant, with the exception of chromogranin and squamous change, which were minimally so. For these reasons, we believe that the histologic appearance is the simplest and most reliable method for the classification of colorectal carcinomas. However, because all small cell tumors had evidence of endocrine differentiation, these markers may be helpful in diagnosis and chromogranin positivity may prove to be of prognostic significance in itself. In conclusion, small cell carcinomas of the large intestine have endocrine features that may require multiple techniques for demonstration. They often show coexistent squamous and glandular differentiation. Their behavior is aggressive, marked by early liver metastases. Additional studies to assess treatment response depend on uniform, reproducible classification of these lesions. Although endocrine markers may be helpful in diagnosis, they are not specific; the routine light microscopic appearance is sufficient for clinically relevant classification.
2. Mills SE, Allen JS. Cohen AR. Small-cell undifferentiated carcinoma of the colon. A clinicopathological study of five cases and their association with colonic adenomas. Am J Surg Pathol 1983;7: 643-651. 3. Schwartz AM, Orenstein JM. Small cell undifferentiated carcinoma of the rectosigmoid colon. Arch Pathol Lab Med 1985;I09:629632. 4. Shirouzu K, Morodomi T, Isomoto H, Yamauchi Y, Kakegawa T, Morimatsu M. Small-cell carcinoma of the rectum. Clinicopathologic study. Dis Colon Rectum 1985;28:434-39. 5. Damjanov I, Amenta PS, Bosman FT. Undifferentiated carcinoma of the colon containing exocrine, neuroendocrine and squamous cells. Virchows Arch [A] 1983;401:57-66. 6. Kalomiris D, Geary K.R, Ordonez NG, El-Naggar A, Robey-Caffey S. Small cell carcinoma of the colon and rectum. Mod Pathol 1990;3:50A. 7. Palvio DHB, Sorensen FB, KJove-Magensen M. Stem cell carcinoma of the colon and rectum. Report of two cases and review of the literature. Dis Colon Rectum 1985;28:440-445. 8. Petrelli M, Tetangco E, Reid JD. Carcinoma of the colon with undifferentiated, carcinoid, and squamous features. Am J Clin Pathol 1981;75:581-584. 9. Gibbs NM. Incidence and significance of argentaffin and Paneth cells in some tumours of the large intestine. J Clin Pathol 1967;20: 826-831. 10. Smith DM, Haggitt RC. The prevalence and prognostic significance of argyrophil cells in colorectal carcinomas. Am J Surg Pathol 1984;8:123-128. 11. Wick MR, Weatherby RP, Weiland LH. Small cell neuroendocrine carcinoma of the colon and rectum: clinical, histologic, and ultrastructural study and immunohistochemical comparison with cloacogenic carcinoma. Hum Pathol 1987;18:9-21. 12. Federspiel BH, Burke AP, Sobin LH, Shekitka KM. Rectal and colonic carcinoids. A clinicopathologic study of 84 cases. Cancer 1990;65:135-140. 13. Shimosato Y, Sobin L, Spencer H, Yesner R. Histological typing of lung tumors. WHO International Histological Classification of Tumors, 2nd ed. Geneva: World Health Organization, 1981 ;23— 24. 14. Churukian CJ, Schenk EA. A modification of Pascual's argyrophil method. Journal of Histotechnology 1979;2:102-103. 15. Sternberger LA, Hardt PH, Cuculis JJ. The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 1970;18:315-333. 16. Redman BG, Pazdur R. Colonic small cell undifferentiated carcinoma: a distinct pathological diagnosis with therapeutic implications. Am J Gastroenterol 1987;82:382-385. 17. Gould VE, Chejfec G. Neuroendocrine carcinomas of the colon. Ultrastructural and biochemical evidence of their secretory function. Am J Surg Pathol 1978;2:31-38. 18. Gould VE, Lee I, Wiedenmann B, Moll R, Chejfec G, Franke WW. Synaptophysin: a novel marker for neurons, certain neuroendocrine cells, and their neoplasms. Hum Pathol 1986; 17:979— 983. 19. Leader M, Collins M, Patel J, Henry K. Antineuron specific enolase staining reactions in sarcomas and carcinomas: its lack of neuroendocrine specificity. J Clin Pathol 1986;39:1186-1192. 20. Lloyd RV, Cano M, Rosa P, Hille A, Huttner WB. Distribution of chromogranin A and secretogranin I (chromogranin B) in neuroendocrine cells and tumors. Am J Pathol 1988;130:296-304. 21. Michels S, Swanson PE, Robb JA, Wick MR. Leu-7 in small cell neoplasms. An immunohistochemical study with ultrastructural correlations. Cancer 1987;60:2958-2964. 22. Mori K., Shinya H, Kalisman M. A composite tumor in tubulovillous adenoma of the rectum. Dis Colon Rectum 1978:21:506-509. 23. Moyana TN, Qizilbash AH, Murphy F. Composite glandular carcinoid tumors of the colon and rectum, report of two cases. Am J Surg Pathol 1988;12:607-611.
Small Cell Carcinomas of the Large Intestine ALLEN B. BURKE, M.D. ( M A J , USAF, MC), KRIS M. SHEKITKA, M.D. (LTCOL, USAF, MC), AND LESLIE H. SOBIN, M.D.
differentiation (1 of 15) and fewer liver metastases (4 of IS) than did small cell carcinomas. Among all 59 tumors studied, small cell histologic characteristics correlated better with liver involvement than did endocrine markers or other histologic features. Small cell carcinomas of the large intestine are aggressive tumors with a propensity for early liver involvement. Although there is a spectrum of squamous, endocrine, and glandular features in large bowel tumors of low degrees of differentiation, the identification of a small cell component appears to be most clinically relevant. (Key words: Colon; Rectum; Small cell carcinoma) Am J Clin Pathol 1991;95:315-321
Small cell carcinomas of the colon and rectum are highly malignant tumors similar in histologic characteristics, behavior, and histochemistry to small cell carcinoma of the lung.1 They show at least some evidence of endocrine differentiation2"4 and may contain glandular and squamous foci,5"8 making classification difficult. Endocrine granules are present in small numbers of cells in typical adenocarcinomas of the gastrointestinal tract. 910 The endocrine content of poorly differentiated adenocarcinomas and undifferentiated non-small cell carcinomas, however, has not been well characterized. The frequency of such endocrine differentiation could be of diagnostic" and conceptual interest in terms of the
relation between small cell, poorly differentiated adenocarcinomas and undifferentiated carcinomas. We studied a group of small cell colorectal carcinomas and compared their clinical, histologic, and immunohistochemical features with those of a group of poorly differentiated adenocarcinomas and undifferentiated carcinomas. MATERIALS AND METHODS Criteria for Selection
All malignant epithelial neoplasms of the large intestine with blocks or tissue accessioned to the Armed Forces Institute of Pathology between 1986 and 1988 were prospectively studied. Typical carcinoids and well- or modFrom the Department of Gastrointestinal Pathology. Armed Forces erately differentiated adenocarcinomas forming glandular Institute of Pathology. Washington, D.C. structures were excluded, because these have been well Received April 12, 1990; received revised manuscript and accepted studied and pose few difficulties in diagnosis. "Typical" for publication June 28, 1990. carcinoids indicate tumors with characteristic growth Presented at the Fall Meeting, American Society of Clinical Pathologists, November 1989, Washington, D.C. patterns, fewer than 1 mitosis/high-power field (HPF), and The opinions or assertions contained herein are the private views of the lack of necrosis; we have reported these in a previous the authors and are not to be construed as official or reflecting the views publication.12 These restrictions resulted in 59 epithelial of the Department of the Army, the Department of the Air Force, or the Department of Defense. neoplasms that had large areas of solid growth that did Address reprint requests to Dr. Burke: Department of Gastrointestinal not show glandular differentiation by hematoxylin and Pathology, Armed Forces Institute of Pathology, Washington, D.C. eosin stains. Tumors were retained for study even if ad20306-6000. 315
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The authors studied the clinical and pathologic features of 38 small cell carcinomas of the large intestine. Most were located in the right colon. Overlying adenomas were present in 45% and squamous differentiation in 21% of tumors. Endocrine differentiation was present in all tumors by at least one method; neuronspecific enolase, dense-core granules, and synaptophysin were present in most cases. Seventy-one percent of tumors metastasized to the liver; 64% of patients were dead at five months followup. Twenty-one poorly differentiated adenocarcinomas and undifferentiated carcinomas of the large intestine accessioned during the same period showed less endocrine (7 of 21) and squamous
316
ANATOMIC PATHOLOGY Article
jacent areas consisted of adenoma or gland-forming adenocarcinoma or if there were foci of squamous differentiation. In two cases in which the epithelial nature of the tumor was in question, a cytokeratin stain was performed.
TABLE 1. Antiserum
Source
Dilution
NSE Chromogranin Leu-7 Serotonin Carcinoembryonic antigen
DAKO Hybritech DAKO Immunonuclear DAKO (polyclonal, unabsorbed) Hybritech Boehringer Mannheim
1:400 1:200 1:400 1:20 (purified) 1:160
Topographic Distribution For tabulation purposes, tumors from the cecum to the splenic flexure were considered right-sided and those in the descending colon to the rectum, left sided. Histologic Study
Histochemistry Argyrophilia was assessed by the Churukian-Schenk method 14 and mucin by the PAS methods with diastase digestion.
1:160 1:50
Electron Microscopic Examination Tissue for electron microscopic examination was obtained by trimming selected areas of 45 tumors from paraffin blocks. Pieces of tissue that were 1 mm 2 in size were placed in xylene overnight, fixed in 2.5-3% phosphatebuffered glutaraldehyde, postfixed in 1% phosphate-buffered osmium tetroxide, dehydrated in graded ethanols, and embedded in Effapoxy® (Ernest E. Fullam, Inc., Latham, NY). One-micron-thick sections were stained with toluidine blue O stain. Thin sections were stained with 4% uranyl acetate and lead citrate, mounted on carboncoated copper grids, and examined with a Zeiss EM 109® electron microscope (Carl Zeiss, Inc., Thornwood, NY). The presence of dense-core granules, as defined by membrane-bound round structures measuring 10-50 nm in diameter, was evaluated by one of us (K.M.S.) without knowledge of immunohistochemical findings or clinical data. Follow-up Information Follow-up information (Table 2) was based on questionnaires and clinical data sent with the consultation material. The liver was considered involved by tumor if there was a positive biopsy specimen, positive computed tomography (CT) scan, or liver involvement at autopsy; the liver was considered uninvolved if there was specific mention of a normal-appearing liver with negative biopsy results at surgery, negative CT scan, or no involvement at autopsy.
Immunohistochemistry
RESULTS
Immunostains for neuron-specific enolase (NSE), chromogranin, Leu-7, serotonin, synaptophysin, cytokeratin, and carcinoembryonic antigen were performed according to the peroxidase-antiperoxidase technique of Sternberger,15 with the use of diaminobenzidine as chromogen. Dilutions and sources of antisera are listed in Table 1. One representative section from the primary tumor was stained in each case and was graded as positive if more than 10% of the tumor cells stained. Positivity was assessed by two of us (A.P.B., K.M.S.) without knowledge of clinical history or the presence of metastases.
A.J.C.P. •
Histologic Features Small Cell Carcinomas (38 tumors). These were composed of cells with minimal nuclear clearing, inconspicuous nucleoli, and dispersed chromatin. Cellular cohesion with identifiable cytoplasm was present throughout 12 tumors, yielding the appearance of intermediate small cell carcinoma (Figs. 1 and 2). Six tumors had an oat-cell appearance throughout; in contrast to intermediate small cell carcinoma, these lacked distinct cytoplasm and had fusiform nuclei (Fig. 3). Twenty tumors had admixtures
rch 1991
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Two of us (A.P.B., K.M.S.) independently classified the tumors into three categories based on hematoxylin and eosin and periodic acid-Schiff(PAS) -stained slides: small cell carcinoma, poorly differentiated adenocarcinoma, and undifferentiated carcinoma. For small cell carcinoma, we used criteria similar to those published for small cell carcinomas of the lung and colon. 113 Poorly differentiated adenocarcinomas were classified on the basis of intracytoplasmic mucin vacuoles present on PAS stain, in areas where glandular differentiation was not appreciated on hematoxylin and eosin stain. Undifferentiated carcinomas lacked both glands and intracytoplasmic mucin.1 Categorization was done without knowledge of clinical findings and immunohistochemical results. Mitotic counts were performed by one of us (A.P.B.) on 5-fim sections with the use of a X40 power objective (490-jim field diameter), without knowledge of clinical or other pathologic data. The highest count in ten consecutive fields in up to 50 viewed (depending on the size of tumor) was recorded; the count was expressed in mitoses/HPF.
Cytokeratin AE1/3 Synaptophysin
BURKE, SHEKITKA, AND SOBIN
317
Small Cell Colorectal Carcinomas TABLE 2. CLINICOPATHOLOGIC FEATURES: SMALL CELL, POORLY DIFFERENTIATED ADENOCARCINOMAS AND UNDIFFERENTIATED CARCINOMAS Poorly Differentiated Adenocarcinoma (15 tumors)
Undifferen tiated Carcinoma (6 tumors)
63 years 22:16 22R, 7L, 8Re 45% 26% 24%
69 years 7:8 12R, 3L 27% 33% 0
56 years 5:1 4R, 1L, IRe 33% 17% 17%
31% 37% 84% 18% 56% 16% 81% 89% 89% 71% 36%
0 0 20% 0 13% 0 31% 100% 73% 7% 7/9
40% 50% 83% 17% 67% 0 50% 100% 83% 50% 2/4
Re = rectum; R = right colon; L - left colon. • Well- or moderately differentiated adjacent areas.
t At the time of surgery. t Cases for which five-month follow-up was available.
of intermediate small cell and oat-cell carcinoma. Intracytoplasmic mucin was invariably absent, although intraluminal mucin was present in four cases that had acinarlike structures. Overlying adenomas were present in 17 cases (Fig. 4) and squamous differentiation in 9 (Fig. 5). Poorly Differentiated Adenocarcinomas (15 tumors). These tumors did not show gland formation on routine stains but demonstrated PAS-positive cytoplasmic mucin vacuoles (Fig. 6). In contrast to small cell carcinomas, there were prominent nucleoli and nuclear clearing. None of these tumors had squamous areas; in eight cases wellor moderately differentiated areas were present and four had overlying adenomas. Undifferentiated Carcinomas (six cases). These tumors had nuclear features similar to those of poorly differentiated adenocarcinomas but lacked intracytoplasmic mucin (Fig. 7). Two had overlying adenomas. Four cases resembled large cell carcinoma of the lung. Two cases lacked epithelial characteristics and resembled lymphoma (Fig. 8); a positive stain for cytokeratin was necessary to establish a diagnosis of carcinoma. One of these tumors had numerous argyrophil cells by the Churukian-Schenk method and was positive for chromogranin. Results of Tests for Endocrine
Differentiation
More than 80% of the small cell carcinomas were positive for NSE and dense-core granules (Table 2). Positivity for chromogranin, Leu-7, and argyrophilia was often focal or patchy. Synaptophysin positivity was usually more diffuse (Fig. 9). The squamous areas were negative for en-
docrine markers with the exception of occasional weak NSE positivity; overlying adenomas and adenocarcinomas were generally negative for all endocrine markers, with the exception of rare positive cells. The undifferentiated carcinomas showed endocrine features in three of six cases. Four of 15 poorly differentiated adenocarcinomas had dense-core granules by electron microscopic examination but were usually negative for other endocrine markers. Frequency of Liver Metastases:
Follow-up
The frequency of liver metastases was higher at the time of surgery for the patients with small cell carcinoma than for those with poorly differentiated adenocarcinoma. The patients with small cell carcinomas received a variety of treatment regimens, including radiation, Cytoxan® (cyclophosphamide; Bristol-Myers/Mead Johnson Oncology, Evansville, IN), vincristine, Adriamycin® (doxorubicin HCI; Adria Laboratories, Dublin, OH), dacarbazine, 5fluorouracil, lomustine, and streptozocin. The variety of treatment regimens and the small numbers of cases make statements regarding efficacy of treatment impossible, but most patients did not respond to therapy. A single patient received combination anti-oat cell therapy (Cytoxan, vincristine, Adriamycin, D T I Q a n d is alive at 12 months despite liver metastases. Two-thirds of patients with small cell carcinomas were dead at five months. Histology of Metastases Histologic material was available for 21 small cell tumors. All metastases had the appearance of small cell car-
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Mean age M:F Location Adenoma Adenocarcinoma* Squamous change Positivity for Churukian-Schenk Chromogranin Neuron-specific enolase Leu-7 Synaptophysin Serotonin Dense-core granules Transmural extension Lymph node metastases Liver metastasesf Alive at 5-month follow-up:):
Small Cell Carcinoma (38 tumors)
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FIG. 5 (upper, left). Small cell carcinoma. The small cell carcinoma merges with squamous cell elements. Hematoxylin and eosin (X250). FIG. 6 (upper, right). Poorly differentiated adenocarcinoma. Unlike small cell carcinoma, there is nuclear clearing and prominent nucleoli. Periodic acid-Schiff-positive mucin vacuole (arrow). Hematoxylin and eosin (X400). FIG. 7 (lower, left). Undifferentiated carcinoma. Large cells, prominent nucleoli (not features of small cell carcinoma); mucin stains were negative (not shown). Hematoxylin and eosin (X400). FIG. 8 (upper, right). Undifferentiated carcinoma. Tumor composed of sheets of cells with prominent nucleoli and a lack of nuclear molding, resembling lymphoma. Tumor stained diffusely positive for cytokeratin, chromogranin, synaptophysin, and Leu-7 (not shown). Hematoxylin and eosin (XI50).
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ANATOMIC PATHOLOGY Original Article
FIG. 9. Small cell carcinoma. Cells positive in a patchy distribution for synaptophysin. Peroxidase-antiperoxidase (X250).
Statistical Analysis The effect of individual variables on liver metastasis at the time of surgery was studied for 34 transmural small cell carcinomas, 15 transmural poorly differentiated adenocarcinomas, and 6 transmural undifferentiated carcinomas. Liver involvement versus no liver involvement was compared by the chi-squared statistic (Fisher's exact test) with each histologic, immunohistochemical, and ultrastructural variable (Table 3). Small cell histologic characteristics were more significant in predicting liver metastases than other variables, although chromogranin and NSE were also significant. To assess independence of variables, a step-wise mulTABLE 3. VARIABLES ASSOCIATED WITH LIVER METASTASIS: SMALL CELL, POORLY DIFFERENTIATED ADENOCARCINOMAS AND UNDIFFERENTIATED CARCINOMAS Variable
P Value
Small cell histologic characteristics Neuron-specific enolase* Chromograninf Squamous differentiationf Dense-core granules*
0.0001 0.002 0.005 0.006 0.04
* Not independently significant by step-wise regression. t Minimally independently significant by step-wise regression (see text).
tiple regression was performed. Small cell histologic characteristics correlated with liver metastasis (r = 0.70); combined with small cell histologic characteristics, chromogranin positivity (r = 0.74) and squamous change (r = 0.79) increased the correlation. Other immunohistochemical variables did not show an independent correlation with liver metastasis. DISCUSSION The clinical course of the patients with small cell carcinomas in our study was marked by a high frequency of liver metastasis and early death. These findings agree with other reports of small cell carcinoma of the colon.2"4 One of our patients with liver metastases did well for 12 months with oat-cell chemotherapy; responses of small cell colorectal carcinomas to oat-cell treatment have been reported anecdotally.16 In our series, a variety of chemotherapeutic regimens were used, possibly because the tumors, despite similar histologic characteristics, were given different diagnoses by contributing pathologists. Uniform diagnostic criteria should be applied to obtain meaningful assessments of treatment responses. All of the small cell tumors in our series demonstrated endocrine differentiation by at least one special technique. The endocrine nature of small cell carcinomas of the lung and colorectum is well documented with the use of ultrastructure and immunohistochemical stains for NSE, chromogranin, synaptophysin, and Leu-7.17"21 We have found that many markers lack sensitivity, and the most sensitive marker, NSE, is known to lack specificity.19 The
A.J.C.P. • March 1991
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cinoma, with the exception of one case: one liver metastasis from a mixed small cell carcinoma-adenocarcinoma showed only moderately differentiated adenocarcinoma.
BURKE, SHEKITKA, AND SOBIN
32]
Small Cell Colorectal Carcinomas lack of sensitivity necessitates the use of multiple markers in some cases to demonstrate endocrine differentiation. In addition, four of six undifferentiated carcinomas and one-third of the poorly differentiated adenocarcinomas also reacted for endocrine markers, indicating that endocrine differentiation is not specific for any one histologic type of carcinoma. Because of this lack of specificity, tests for endocrine differentiation are not always reliable in distinguishing small cell carcinoma and undifferentiated carcinoma. Not all of the small cell tumors had densecore granules by electron microscopic examination, but this may be a result of sampling and the small numbers of granules present.
REFERENCES I. JassJR, Sobin LH. Histological typing of intestinal tumors. WHO International Histological Classification of Tumors, 2nd ed. Berlin: Springer-Verlag, 1989;33.
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Many small cell carcinomas in our series expressed divergent differentiation, namely glandular and squamous. However, virtually all metastatic lesions from small cell carcinomas were of small cell type. Several tumors had an overlying adenoma with little or no transition to the small cell carcinoma, leading to the initial diagnosis of metastatic small cell carcinoma. The association between colonic adenomas and endocrine tumors has been documented previously.2,22'23 We could not meaningfully correlate pathologic parameters with survival data because of differences in treatment. Early liver metastasis is a relatively good indicator of tumor aggressiveness, however. Our data indicate that, although some tests of endocrine differentiation are highly associated with liver metastases, small cell histologic characteristics are the best single predictor of liver metastasis among colorectal carcinomas of low differentiation. Other variables were independently not significant, with the exception of chromogranin and squamous change, which were minimally so. For these reasons, we believe that the histologic appearance is the simplest and most reliable method for the classification of colorectal carcinomas. However, because all small cell tumors had evidence of endocrine differentiation, these markers may be helpful in diagnosis and chromogranin positivity may prove to be of prognostic significance in itself. In conclusion, small cell carcinomas of the large intestine have endocrine features that may require multiple techniques for demonstration. They often show coexistent squamous and glandular differentiation. Their behavior is aggressive, marked by early liver metastases. Additional studies to assess treatment response depend on uniform, reproducible classification of these lesions. Although endocrine markers may be helpful in diagnosis, they are not specific; the routine light microscopic appearance is sufficient for clinically relevant classification.
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