vinous Varieties of So-called Mammary Carcinoid Tumors Solid and

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1 and vinous Varieties of Socalled Mammary Carcinoid Tumors EDWIN R. FISHER, M.D., ALKA S. PALEKAR, MD., AND NSABP COLLABORATORS Fisher, Edwin R., Palekar, Alka S., and NSABP collaborators: and mucinous varieties of socalled mammary carcinoid tumors. Am J Clin Pathol 7: ,1979. Three neoplasms that had histologic features reminiscent of carcinoid tumors of other sites were encountered in a review of,00 examples of invasive mammary cancer in women (.09%). One of these showed cytoplasmic argyrophilia. This, as well as the two putative carcinoids, lacked argentaffinity. Attention is directed to the occurrence of variable' numbers of argyrophilic cells in eight of 19 socalled mucinous cancers of the breast studied. Further, neurosecretorytype granules were observed in cells of all four mucinous cancers suitably prepared for electron microscopic examination. The possible reasons for the lack of universal argyrophilic reactions in these lesions is discussed. It is concluded that there may be two types of mammary carcinoid tumors, the solid and mucinous varieties. patient who had the latter type had experienced treatment failure after five years of observation. Various numbers of ductal epithelial cells in four of 4 examples of banalfibrocysticdisease showed cytoplasmic argyrophilia, and neurosecretorytype granules were found in two of eight examples suitably prepared for electron microscopic examination. Whether this demonstration establishes the existence of precursor elements for the development of the carcinoid tumors is at present uncertain. (Key words: Breast; Carcinoid; Carcinoma: Cystoplasmic argyrophilia; Fibrocystic disease; Mucinous carcinoma; Neurosecretory granules.) THERE IS ACCUMULATING EVIDENCE to indicate that a small number of carcinomas of the breast may actually represent carcinoid tumors of this organ. In a previous report relating to the pathologic characteristics of 1,000 mammary cancers, we described the occurrence of several mammary neoplasms with histologic features mimicking those of carcinoid tumors, but argentaffin and argyrophil reactions were absent. Because of this, they were noncommittally designated basaloid infiltrating ductal carcinomas. More recently, Cubilla and Woodruff have recounted the clinical and pathologic features of the cases of eight patients (one had two tumors in the same breast and another, bilateral lesions) whose tumors had histologic features con Received September, 1978; received revised manuscript and accepted for publication vember Supported by a Breast Cancer Task Force Contract (NIHNOI 78976) from the U. S. Public Health Service and a Grant (RC11) from the American Cancer Society. Address reprint requests to Dr. Fisher: Shadyside Hospital, Institute of Pathology, 0 Centre Avenue, Pittsburgh, Pennsylvania /79/100/0909 $00.90 A Department of Pathology, Shadyside Hospital and University of Pittsburgh, Pennsylvania sonant with those of carcinoid tumors. Argyrophilic granules were demonstrated by a modified Grimelius stain in all, and neurosecretory granules were identified in three of the examples in which the tissue was suitably prepared for electron microscopic examination. Argyrophilic granules were not observed in 40 cancers of other histologic types. Surprisingly, for these patients with tumors having carcinoid characteristics the prognosis was comparable to that for carcinoma of the breast in general. Kaneko and associates' have also recently recorded the occurrence of bilateral carcinoid tumors of the breast, in a 78yearold man, associated with the urinary excretion of a moderate amount of norepinephrine. He died of metatasis from a carcinoma of the urinary bladder. It is noteworthy that Feyrter and Hartmann, more than a decade previously, had described the characteristics of two tumors they encountered among 170 mammary cancers that they regarded as examples of carcinoids. Both showed an argyrophilic reaction, and hydroxytryptamine was extracted from the tumor of one patient; the urine of the other patient contained increased amounts of hydroxyindoleacetic acid. The clinical courses were apparently banal. The detection of mucin in these two tumors prompted the authors to speculate that there might be a spectrum represented by the solid carcinoid variety on one hand and a mucinous form on the other, hence the designation "carcinoma solidum gelatinosum." It is germane that Cubilla and Woodruff mentioned the presence mucicarminophilic material in cells of two of the tumors they designated carcinoids of the breast. Although Lillie and Glenner 16 discounted the presence of mucin in enterochromaffin tissue and its tumors, there have been numerous reports attesting to the presence of mucinsecreting adenocarcinomas with carcinoid foci in the gastrointestinal tract. 17 " 119 Indeed, Goldenberg and Fisher 8 found a carcinoid tumor of the large intestine to assume the morpholigic characteristics of a signetring mucinsecreting adenocarcinoma in the hamster cheek pouch after heterotransplantation. Further, we have ican Society of Clinical Pathologists Downloaded from by guest on 1 vember

2 Downloaded from by guest on 1 vember 018 FIG. I (upper, left). Basaloid appearance of mammary cancer designated pseudocarcinoid because of intracytoplasmic glycogen and lack of argyrophilia. Its appearance closely resembles that of the putative carcinoid depicted in Figure. Hematoxylin and eosin. xloo. FIG. (upper, right). An example of pseudocarcinoid that closely mimics the appearance of "solid" carcinoid tumors. However, glycogen was found in many cytoplasms. Hematoxylin and eosin. x0. FIG. (lower, left). Area of basaloid mammary cancer of Case, which did not contain glycogen or show argyrophilia, but because of its appearance was designated "putative" carcinoid. Hematoxylin and eosin. x 100. Inset demonstrates its extremely well differentiated cellular population. x0. FIG 4 (lower, right). Area of an example of a nonglycogencontaining solid tumor of breast with features highly reminiscent of carcinoid tumor (Case ). However, cellular argyrophilia was lacking. Hematoxylin and eosin. x0. 910

3 Vol MAMMARY CARCINOIDS 911 also found and depicted intracytoplasmic densecore granules of neurosecretory type in cells comprising a classic mucinous carcinoma of the breast, 4 although at the time these were designated simply secretory granules. This information prompted us to reexplore the possible "carcinoid" nature of some mucinous mammary cancers, as well as those tumors with a more solid, classic carcinoid appearance. Some of the tinctorial technics utilized for this purpose have also been applied to examples of fibrocystic disease in an attempt to discern possible precursors cells of such lesions. A previous ultrastructural study of mammary diseases disclosed the presence of neurosecretorytype cytoplasmic granules in one of the examples of benign ductal hyperplasia examined by this technic. 4 Materials and Methods There were eight mammary neoplasms from a repository of,00 examples of invasive cancers of the breast (National Surgical Adjuvant Breast Projects) which comprised uniform, welldifferentiated epithelial cells with absent or scant mitotic activity arranged in basaloid, acinar, or festooned patterns reminiscent of carcinoid tumors of other sites. However, after careful review and assessment of results obtained by use of special stains {vide infra), glycogen was found in the neoplastic cells of five (Figs. 1 and ). These neoplasms were designated "pseudocarcinoids," leaving three that were strongly suspect as representing carcinoids (Figs., 4, and ). Nineteen examples of mammary cancers that had the classic appearance of mucinous carcinoma characterized by solid clusters or tubules or acini of welldifferentiated neoplastic cells within varioussized pools of mucin were also retrieved. Ages of the patients and information about their tumors are presented in Tables 1 and. The average periods during which patients were available for study were five years for both the suspect carcinoids and mucinous neoplasms. Also available for examination were 147 specimens from areas of fibrocystic disease from 0 patients whose ages ranged from to 60 years. Sections were prepared from blocks of tissue that had been fixed in formalin in the conventional manner. These were stained by a modified Grimelius method 14 and the GomoriBurtner 1 methenamine silver technic for argyrophilic and argentaffin substances respectively; ferric ferricyanide 1 for reducing substances; periodic acidschiffalcian blue sequence with and without antecedent diastase digestion for glycogen, mucin and other polysaccharide substances; Sudan black B; peracetic acidschiff; and Nile blue sulfate 1 ; and examined unstained for autofluorescence for the demonstration of Iipofuscins. Control sections for the argyrophilic and argentaffin reactions consisted of similarly fixed and prepared sections of appendix, bronchial carcinoid, islet cell tumor of the pancreas, and carcinoid of the small intestine. These were simultaneously stained with the sections of the various mammary lesions. Electron micrographs and grids containing ultrathin sections of four mucinous cancers and eight examples of fibrocystic disease obtained from tissue previously prepared for electron microscopic examination 4 were available for review. Results Neoplastic cells of only one example of the three putative mammary carcinoids showed a positive Grimelius reaction (Fig. 6). The argyrophilic granules appeared most concentrated in perinuclear portions of the cytoplasms. The argentaffin and other reactions were negative except for an occasional cell containing ferric ferricyanidepositive material and a slight amount of mixed mucin in occasional cells from Case. Regional nodal metastases were found in one of the two patients whom axillary dissections were performed. One patient succumbed to her disease five years after treatment, whereas the other two remain free of recurrence. ne of the lesions designated "pseudocarcinoid" had a positive Grimelius or argentaffin reaction. In three lesions, mucin was detected in addition to glycogen. Grimeliuspositive, principally perinuclear cytoplasmic granules were variably observed in cells comprising eight of the 19 mucinous cancers (Figs. 7 and 8). The argentaffin reaction was negative in every case, as were the ferric ferricyanide and peracetic acidschiff reactions. Occasional cells in four cases had positive Sudan black, Nile blue sulfate, and glycogen reactions, as well as autofluorescence. These reactions, however, did not appear to coincide with the cytoplasmic argyrophilia, and were most often found in cells of tumors lacking the latter. In addition, there was no statistically significant correlation between the occurrence of Grimeliuspositive cells and the pattern of growth, i.e., solid, alveolar or tubular; type of mucin; or nuclear grade of the tumor (Fisher's exact t test). Neurosecretorytype cytoplasmic granules measuring 100 fxm were encountered in cells of all the mucinous cancers from which tissues were appropriately fixed and prepared for electron microscopic examination (Fig. 9). Positive argyrophilic reactions were seen in various numbers of the tumor cells in only two of these cases. ne of the patients with mucinous cancer has shown evidence of treatment failure. Argyrophilic granules were variably found in ductal epithelial cell cytoplasms in five of the 0 cases of fibro Downloaded from by guest on 1 vember 018

4 91 FISHER AND PALEKAR A.J.C.P. December 1979 Downloaded from by guest on 1 vember 018 FIG. (upper, left). Alveolated and cordlike pattern of the neoplasm of Case 1, highly reminiscent of carcinoid tumor. Hematoxylin and eosin. x 100. Inset reveals its well differentiated character. A few foci of necrosis are evident. x0. FIG. 6 (upper, right). Intense argyrophilic cytoplasmic reaction in cells of tumor in Case 1. (Grimelius stain. x40). FIG. 7 (lower, left). Area of classic mucinous cancer. Hematoxylin and eosin. xloo. FIG. 8 (lower, right). Cells of the mucinous cancer depicted in Figure 7 show an intense Grimelius reaction. x0.

5 vol MAMMARY CARCINOIDS Table I. Clinical and Other Characteristics of Examples of "" Carcinoids of the Breast 91 Patient's Age (Yr) Tumor Size (cm) des/ 0 Grimelius Mucin Recurrence Time Patient Available for Study Case I (48) Case (4108) Case (4104) /0 1/1 0/0t tt Yes 6 yr Syr* yr * Died of disease at one year, t Simple mastectomy. t Occasional cell. cystic disease examined (Fig. 10). The granules were disposed at various sites within the cells of sclerosing adenosis and ductal hyperplasia. Some intracellular deposits of Sudan black B, ferric ferricyanide, and peracetic acidschiff, and autofluorescent material, were also found, but their locations did not appear to coincide with that of the argyrophilic granules. The argentaffin reaction was consistently negative. Electron microscopic examination revealed various numbers of densecore granules of the neurosecretory type, measuring 100 ^.m in some cells comprising two of the eight examples of fibrocystic disease examined by this technic (Fig. 11). ne of the cells containing such granules possessed any features of myoepithelial elements. Discussion It is suspected that the recently demonstrated interest in mammary carcinoid tumors will result in a surfeit of recorded examples. Yet, as mentioned in Case 1 (494) Case (4818) Case (486) Case 4 (410) Case (41) Case 6 (41410) Case 7 (417) Case 8 (4999) Case 9 (460) Case 10 (479) Case 11 (48) Case 1(481) Case 1 (49) Case 14 (4948) Case 1(410) Case 16(41119) Case 17(4106) Case 18(41404) Case 19(41648) Table. Clinical and Other Characteristics of Examples of Mucinous Cancer of the Breast Patient's Age (Yr) * Simple mastectomy. t A alcian blue; P = PAS; M mixed. Tumor Size (cm) des/ 0 17/0 /0 11/0 /0 /1 0/1 17/ 1/0 /0 9/0 Grimelius t t t t Type of Mucint A P, M P P, M P P P P, M,, P Histologic Pattern Nuclear Grade our preliminary investigations, in this study some neoplasms resembling carcinoids, after careful inspection, proved to have intracytoplasmic glycogen within their neoplastic cells. Although we can find no direct information concerning the presence of such a moiety in cells of carcinoid tumors, our own experience indicates that cells of most, if not all, bona fide carcinoid tumors lack such material. 6 Although we do not regard necrosis as a common feature of carcinoid tumors, it is of interest that the one Grimeliuspositive example of carcinoid tumor did contain rare foci of this change. This information has prompted us to regard only three cases as putative carcinoids. A positive argyrophilic reaction was evident in only one of the latter. Thus, the incidence of mammary carcinoid tumors of the solid variety in our material was either 0.09% or 0.0%, depending upon the stringency of diagnostic criteria used for identification. The failure to detect cytoplasmic argyrophilic granules in all three examples of carcinoid tumors was disquieting but not totally surprising. The polemics re Recurrence X Electron microscopy demonstrates neurosecretory granules. Time Patient Available for Study (Yr) Downloaded from by guest on 1 vember 018

6 914 FISHER AND PALEKAR A.J.C.P. December 1979 garding the diagnostic criteria for carcinoid tumors is well known. It is of interest in this regard that Lillie and Glenner 16 have estimated that the diagnoses of approximately 80% of gastrointestinal carcinoids recorded in the medical literature were based solely upon histologic features. On the other hand, some have been unwilling to accept only cytoplasmic argyrophilia as sufficient evidence for enterochromaffin neoplasms, demanding not only an argentaffin reaction but also positive azo coupling and Gibbs reactions. 17 Yet, there is sufficient evidence to indicate that an argentaffin reaction most often may be negative in cells comprising carcinoids of foregut or hindgut origin. Our experience with oat cell carcinoma of the lung, a malignant variant of bronchial carcinoid, indicates that argyrophilic granules may be quite variable and often undetectable. 6 The ultrastructural identification of neurosecretory granules in such lesions appears to be more diagnostically sensitive. This was found in some of the examples of mucinous cancers investigated in this study. Gould and Chejfec9 have recently recorded a similar disparity between the histochemical and ultrastructural characteristics of a small group of colonic neoplasms they regard as representing neuroendocrine or carcinoid tumors. This information also tends to support the view relating the inconsistency of tinctorial reactions seen in carcinoids to their heterogeneous chemical composition. 18 Unfortunately, in our cases of solid carcinoid tumors, material appropriately prepared to allow for their ultrastructural evaluation was not available. The variability of tinctorial reactions, as well as the dichotomy of our results in this regard and those reported by Cubilla and Woodruff, might have been related to differences in technic. Although all of the material was formalinfixed and stained simultaneously with controls, it was obtained from various institutions, each of which, it is suspected, possesses its own minor variations of socalled "standard" technics. The capriciousness of the various silver methods employed for the demonstration of these lesions appears to be aptly exemplified by the various modifications that have been proposed. Despite these theoretical explanations, we have chosen to regard the one example showing a positive Grimelius reaction as a true carcinoid and the remaining two as putative examples of this tumor type. Downloaded from by guest on 1 vember 018 FIG. 9. Portions of cells from a mucinous mammary cancer containing occasional cytoplasmic granules of the neurosecretory type (arrows), x0,000.

7 Vol MAMMARY CARCINOIDS K... x_ N '' J v? v.,, 4 **, ^#r' \v. v v m FIG. II. Electron micrograph of ductal epithelial cells in a case offibrocysticdisease, demonstrating a variety of lysosomes as well as neurosecretory granules (arrows), x 17,0. *.**, Downloaded from by guest on 1 vember 018 FIG. 10. A focus of ductal hyperplasia in a breast with fibrocystic disease, revealing occasional argyrophilic granules in the epithelial cells. Grimelius stain x0. * The findings in this study also indicate that there may be another form of carcinoid tumor of the breast or, at least, a tumor type possessing carcinoid elements, the mucinous variant of cancer of the breast. Although this histologic type generally has not been regarded in the context of carcinoid tumors, the degree of differentiation and structural disposition of its cellular elements are not dissimilar to that encountered in the latter. Approximately half of these tumors in our material also contained argyrophilic granules. However, neurosecretory granules were observed in all cases in which tissue was suitably prepared for such study. In some cases the granules were few, again indicating a greater sensitivity to the ultrastructural method, since some of the positive electron microscopic cases were negative histochemically. We have no certain explanation for the lack of universal tinctorial reactions in this group of cases, although the explanations offered previously in regard to the solid variety seem appropriate. Further, we could detect no particular features common to those tumors with and 91

8 916 FISHER AND PALEKAR A.J.C.P. December 1979 without cytoplasmic argyrophilia. It is of interest that in one of the solid tumors occasional droplets of mucin were encountered, a feature also seen in several cases by Cubilla and Woodruff. This observation provides some support for the speculation by Feyrter and Hartmann' 1 regarding the occurrence of a spectrum of solid and mucinous breast tumors of paraendocrine nature. Such an occurrence appears analogous to that observed in occasional colonic neoplasms in which both mucinsecreting and carcinoid foci may be encountered. Last, the failture to detect recurrence or treatment failure in any of the patients with mucinsecreting cancers in this select group of patients is not only in accord with previous studies indicating the favorable clinical course of patients with this type of mammary cancer, but also is compatible with the behavior of the majority of carcinoid tumors. It appears warranted to conclude that the prognosis of patients with the mucinous form of carcinoid lesions appears more favorable than that of those who have the solid variety. Although there have been six recorded examples of carcinoid tumors metastatic to the breast, 10 none of our patients has shown any evidence for another primary site of origin for her carcinoid tumor during the fiveyear period of observation. Attempts to identify possible precursor cells of the mammary carcinoid tumors generally have been unsuccessful. Feyrter and Hartmann, as well as Vogler, 0 have suggested that the ductal basilar lucent cells of myoepithelial type may represent analogs of the paraendocrine system in the breast. Yet, Vogler 0 notes that such elements are rarely argyrophilic in the normal organ, and he found argyrophilic ductal cells in only one of 18 examples of fibrocystic disease examined. Cubilla and Woodruff could not find any argyrophilic cells in limited numbers of normal breasts, normal areas of cancerbearing breasts, or samples of a variety of alterations common to fibrocystic disease. Similarly, we have not been successful in identifying an argyrophilic reaction in histologically normalappearing areas of breasts harboring cancers. On the other hand, cells in ducts of approximately 10% of the examples of fibrocystic disease examined contained argyrophilic granules, and neurosecretory granules were found in two of eight specimens examined by electron microscopy. In neither were these cytoplasmic inclusions found in cells with myoepithelial characteristics. Whether such cells do indeed represent precursor elements of the carcinoid tumor of breast is not certain at this time. References 1. Bates HR Jr, Belter LF: Composite carcinoid tumor (argentaffinomaadenocarcinoma) of the colon: Report of cases. Dis Colon Rectum 10:467470, Cubilla AL, Woodruff JM: Primary carcinoid tumor of the breast. Am J Surg Pathol 1:89, Feyrter F, Hartmann G: Uber die carcinoide Wuchsform des Carcinoma mammae, insbesondere das Carcinoma solidum (gelatinosum) mammae. Frankf Z Pathol 7:4, Fisher ER: Ultrastructure of the human breast and its disorders. Am J Clin Pathol 66:917, Fisher ER, Gregorio RM, Fisher B: The pathology of invasive breast cancer. A syllabus derived from thefindingsof the National Surgical Adjuvant Breast Project (Protocol. 4). Cancer 6:18, Fisher ER, Palekar A, Paulson JD: Comparative histopathologic, electron microscopic and tissue culture studies of bronchial carcinoids and oat cell carcinomas of lung. Am J Clin Pathol 69:1617, Gibbs, NM: The histogenesis of carcinoid tumors of the rectum. J Clin Pathol 16:0614, Goldenberg DM, Fisher ER: Histogenetic relationship between carcinoids and mucinsecreting carcinomas of colon as revealed by heterotransplantation. Br J Cancer 4:610614, Gould VE, Chejfec G: Neuroendocrine carcinomas of the colon. Ultrastructural and biochemical evidence of their secretory function. Am J Surg Pathol :18, Harrist TJ, Kalisher L: Breast metastasis: An unusual manifestation of a malignant carcinoid tumor. Cancer 40:10 106, Hernandez FJ, Reid JD: Mixed carcinoid and mucinsecreting intestinal tumors. Arch Pathol 88:489496, Klein HZ: Mucinous carcinoid tumor of the vermiform appendix. Cancer :770777, Kaneko H, Hojo H, Ishikawa S, et al: repinephrineproducing tumors of bilateral breasts. A case report. Cancer 41:0007, Lack E, Mercer L: A modified Grimelius argyrophil technique for neurosecretory granules. Am J Surg Pathol 1:777, Lillie RD: Histopathologic Technic and Practical Histochemistry. New York, McGraw Hill, 196, pp Lillie R, Glenner GG: Histochemical reactions in carcinoid tumors of the human gastrointestinal tract. Am J Pathol 6: 661, Pearse AGE: Histochemistry, Theoretical and Applied. Boston, Little Brown, 19, pp Pearse AGE: The APUD cell concept and its implications in pathology, Pathology Annual. Edited by SC Sommers. New York, AppletonCentury Crofts, 1974, pp Toker C: Observations on the composition of certain colonic tumors. Cancer 4:660, Vogler E: Uber das basilare HelleZellenorgan der meschlichen Brustdrude. Klin Med :19168, 1947 Downloaded from by guest on 1 vember 018