[GANN, 57, ; October, 1966] UDC : HISTOCHEMICAL STUDY ON ALKALINE PHOSPHATASE ACTIVITY IN HUMAN NEOPLASMS

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1 [GANN, 57, ; October, 1966] UDC : HISTOCHEMICAL STUDY ON ALKALINE PHOSPHATASE ACTIVITY IN HUMAN NEOPLASMS (Plates LXIV-LXVIII) Iwao MURATA, Hiroshi KAWASHIMA, and Masahiko MORI (Department of Oral Surgery, Osaka University Dental School*) Synopsis Alkaline phosphatase activity was studied in 409 malignant and 225 benign human neoplasms employing a simultaneous azo-dye coupling technique. The enzymatic activity was not demonstrated in about 80% of malignant epithelial tumors. The neoplastic epithelium of oral squamous-cell carcinoma was completely devoid of the activity. In uterine cancers, endometrial adenocarcinoma showed an intense activity, and only about 4% of cervical cancers was positive. In gastric cancers, the enzymatic activity was negative in malignant epithelia in 78% of the cases, but there was no activity in other cancers of the digestive tract. In breast tumors, smalllobular type of fibroadenoma showed higher activity in periacinar aspects, whereas malignant epithelia in breast cancers hardly showed the activity. It seemed to be impossible for this study to establish a correlation between enzymatic activity and histopathologic pattern of tumors except for a few kinds of neoplasms. However, it is accepted that the occurrence of alkaline phosphatase in stromas of carcinomas was associated with inflammation, but the stromal activity appeared under a condition of little inflammation or none in some cancers; oral epidermoid carcinoma and adenocarcinoma in the stomach, the small and large intestine, the rectum, and the breast. INTRODUCTION Since the satisfactory technique for detecting alkaline phosphatase was described.17) a great deal of histochemical studies on enzymatic distribution in normal and pathological specimens has been reported.9,11,13,16) In recent year, the azo-dye coupling method using a new substituted naphthol complex as a substrate has been employed, the application of which has made the enzymatic localization more ideal in tissue sections than the previous technique using simple naphthyl phosphate,7) and many investigations using this new technique have been reported.6,8,20,23) However, there are almost no detailed descriptions about enzymatic activity patterns in many specimens. particularly the existence of the enzyme in neoplastic epithelia and stromas. In the present study, about 600 specimens of human neoplasms were dealt with for the histochemical demonstration, and this work was performed in an attempt to clarify a possible correlationship between the enzymatic behavior and histopathological pattern of tumors. MATERIALS AND METHODS Blocks were obtained from specimens removed surgically, which consisted of 225 benign and 409 malignant tumors. The blocks were frozen immediately in Dry Ice, and

2 frozen sections were fixed in 10% neutral formalin for minutes, and employed for a histochemical demonstration of alkaline phosphatase. When sections were intensely made into paraffin-embedded sections. In some cases, serial sections were stained by the periodic acid-schiff reaction for the demonstration of polysaccharides. Fixed frozen and paraffin sections were stained for alkaline phosphatase with a simultaneous azo-dye buffer of ph 9.2, and 20mg of Diazo Blue B. Another new azo-dye method7) was performed, in which a substrate solution consisted of 5mg of Naphthol AS-MX phosphate dissolved in 1ml of N,N-dimethylformamide, 30ml of Clark and Lub's buffer of ph were stained with Hematoxylin-Eosin for histopathological examination. RESULTS Alkaline phosphatase activity was generally confined to stromal elements of tumors except for some neoplasms, which showed an increased activity associated with inflammatory condition. Oral Cancer Fifty-six cases of epidermoid carcinoma, and one case each of adenocarcinoma with squamous-cell metaplasia, mucoepidermoid carcinoma in the oral cavity, and adenocarcinoma in parotid gland were examined (Table I). The neoplastic epi- Table I. Distribution of Alkaline Phosphatase Activity in Malignant Epithelial Tumor Cells +3 strong, +4 more intense. 514

3 Table II. Alkaline Phosphatase Activity in Stroma of Squamous-cell Carcinoma thelium of epidermoid carcinoma in the oral region was completely devoid of the alkaline phosphatase activity, and so was the normal oral epithelium. A few cases with necrosis or necrobiosis in the neoplastic epithelium were markedly stained for this enzyme. The enzymatic activity in stromal elements was observed in 73% with varied intensity (Table II), but in a few cases, the stromal activity for alkaline phosphatase was abundantly present irrespective of no or little inflammatory responses (Photo 1), and the enzymatic activity was almost absent in the stromal elements, though the stroma showed a marked inflammatory cell infiltration. Neoplastic epithelia of adenocarcinoma and mucoepidermoid carcinoma in the oral cavity showed no more enzymatic reaction than those in epidermoid carcinoma. In a case of adenocarcinoma in the parotid gland, the enzymatic activity was confined to the luminal area of malignant epithelium (Photo 4). Pleomorphic Adenoma of Salivary Glands Thirteen cases of pleomorphic adenoma, mixed tumor of the salivary gland, were studied (Table III). The adenomatous cells of pleomorphic adenoma exhibited almost no enzymatic reaction, though the Table III. Distribution of Alkaline Phosphatase Activity in Benign Tumor Cells +3 strong, +4 more intense. 515

4 enzymatic activity in normal salivary glands was demonstrable in the basal aspect of acini (Photo 2). The enzymatic reaction was found moderately in two of all cases. Ameloblastoma Seventeen cases of ameloblastoma in the lower jaw were examined. The enzymatic reaction in the neoplastic epithelium of the ameloblastoma was absent, which was very different from that of well diffferentiated ameloblasts in the developing tooth stained for alkaline phosphatase. The stromal elements were usually positive for alkaline phosphatase as in the case of epidermoid cancer. Other Tumors in the Oral Region Three cases of epulis fibromatosa, 2 cases each of papilloma and fibroma, and one case each of lipoma, cementoblastoma, nondentigerous cyst, follicular cyst, ossifying fibroma, and fibro-osteoma in the jaw were studied. The enzymatic reaction varied in epulis fibromatosa; the one reacted a trace, and the others moderately to intensely. In a case of cementoblastoma, calcified cementurn tissue had no enzymatic activity, the peripheral zone of which showed an intense reaction, and fibrous stromal elements reacted moderately (Photo 3). In a case of non-dentigerous cyst, enzymatic activity was absent in the cystic epithelium, but the odontogenic epithelium of follicular cyst was very strongly stained. Bony tissues and fibrous components of fibro-osteoma revealed no reaction, and peripheral zones of calcified bone of ossifying fibroma were stained more intensely than its central zones, and fibroblasts in stroma showed a marked activity. In cases of papilloma, fibroma, and lipoma, the enzymatic activity was not demonstrated. Gastric Tumors Eighty-six cases of well-differentiated adenocarcinoma, 18 cases of pylorocardiac glandular-cell adenocarcinoma, 17 cases of undifferentiated carcinoma, 10 cases each of scirrhous carcinoma and metastasis of adenocarcinoma, and 14 cases of polyp in the stomach were examined. The neoplastic epithelium in 78% of the present cases of gastric adenocarcinoma was completely devoid of the enzymatic activity without affording microscopic details (Table IV, Photo 7). With the exception of intensity and confinement of the enzymatic activity in a part of neoplastic epithelia, the presence of Table IV. Alkaline Phosphatase Activity of Neoplastic Epithelia in Various Types of Gastric Cancer +3 strong, +4 more intense. 516

5 517

6 alkaline phosphatase was observed in the rest of cases, which consisted of 13 cases of well-differentiated adenocarcinoma (Photo 9), one case each of adenocarcinoma with cystic formation, papillary formation, and squamous elements (adenoacanthoma), 7 cases of pylorocardiac glandular-cell adenocarcinoma (Photo 8), and 2 cases of metastasized adenocarcinoma. In addition, a high alkaline phosphatase activity was confined to the intestinal metaplastic epithelium. The neoplastic elements in which necrosis or necrobiosis took place revealed a marked enzymatic activity. Those positive areas occasionally contained a PAS-positive substance. In metastasis to the regional lymph nodes, the appearance of alkaline phosphatase was observed in a similar way as in the original tumors of the stomach. Undifferentiated and scirrhous carcinoma cells exhibited no or trace amount of the enzymatic activity in 31 out of 37 cases, and the remaining six cases were weakly or moderately reactive (Table IV). In 25% of the gastric cancers examined there was not only the enzymatic activity in small vessels in the stroma, but also in other stromal elements. A few cases showed a moderate to intense activity. Despite the presence of a marked cell infiltration, the stroma revealed no enzymatic activity, and vice versa. Various types of gastric cancers were divided into three main groups; well-differentiated adenocarcinoma, pylorocardiac glandular-cell adenocarcinoma, and undifferentiated carcinoma. In well-differentiated adenocarcinoma group, adenocarcinoma with cystic and papillary formation, and squamous elements were included, and undifferentiated carcinoma group contained scirrhous carcinoma. In each group, the relationship between intensity of the enzymatic activity and inflammatory response was as shown in Fig. 1. It was characteristically apparent that intensity of the enzymatic reaction in the tumor stroma was not always in proportion to the grade of inflammatory infiltration. In well-differentiated adenocarcinoma group, the percentage of the development of alkaline phosphatase activity in the stroma was higher than in the other groups. In undifferentiated carcinoma group, dense fibrous elements in the stroma was reactive in two cases. It was found that the ratio of cases of gastric adenocarcinoma which exhibited the stromal reaction was rather low, compared to cases of adenocarcinoma in other organs, i.e., small and large intestine, rectum, and breast (Fig. 1). The neoplastic epithelium, irrespective of no alkaline phosphatase activity in such tumor epithelium, was usually stained for PASreaction, but stromal elements with an abundant alkaline phosphatase were almost nonstainable to PAS-reaction. The polyp epithelium was absent for the enzymatic activity as well as adenocarcinoma in the stomach, but in one case, the highest activity was observed. Tumors of Small and Large Intestines, and Rectum Twenty-nine cases of adenocarcinoma in the rectum, 4 cases of adenocarcinoma in the colon, 2 cases of adenocarcinoma in the small intestine, and one casee of rectal polyp were examined (Tables I and III). No enzymatic activity was observed in neoplastic epithelia of adenocarcinoma in the intestines. The neoplastic cells in rectal adenocarcinoma showed no enzymatic reaction, and the stromal elements reacted weakly or moderately in 64% of the total cases, which were negative for PAS-stain. The epithelial elements in rectal polyp did not react (Photo 10). Necrotic areas in the tumor mass showed a positive enzymatic activity. 518

7 Pancreatic and Hepatic Cancers Two cases of adenocarcinoma in the pancreas and one case of hepatoma were studied (Table I). The malignant epithelium in pancreatic cancer exhibited no alkaline phosphatase activity. Alkaline phosphatase activity was the highest in hepatoma cells (Photo 5), though normal liver cells showed no enzymatic activity. Breast Tumors Thirty-five cases of adenocarcinoma and 69 cases of adenoma or fibroadenoma were examined (Table III). In the normal gland, alkaline phosphatase activity was positive in the glandular epithelium, and the highest in the myoepithlium and capillary vessels. In small lobular type adenoma the enzymatic activity was rather high in the acinar aspects (Photo 11). Sometimes, it was found more intensely in the luminal border in the lobule of adenoma. Duct cells with cystic dilatation showed varying stainability as acinar cells of the lobule and in duct papillomatosis, the outer layer of the ductal epithelium reacted intensely, and the rest showed a low reaction (Photo 13). The loose periductal and periacinous stroma contained a low stainability for alkaline phosphatase with the exception of an intense reaction in capillary elements. PASpositive materials were found in the periacinar region which showed the enzymatic activity. In the fibromatous component of fibroadenoma, the enzymatic activity was absent in hyalinized connective tissues, while alkaline phosphatase was present in proliferating connective tissue fibers (Photo 12). Furthermore, in 22 of 35 cases of breast cancers, malignant epithelia showed no enzymatic activity (Photos 14 and 15), and in the remaining cases, a moderate activity was demonstrated in 4 cases, slight in cases, and a trace 5 in 3 cases (Table I); the enzymatic activity in stroma was found in 69% of cases. The neoplastic cells which were negative or trace for this enzyme did not contain polysaccharides. Tumors of the Thyroid Gland Sixteen cases of adenocarcinoma, 43 cases of goiter, 3 cases of thyroid adenoma, one case each of Hashimoto's disease and Riedel's disease were examined (Tables I and III). Alkaline phosphatase activity in thyroid adenocarcinoma was absent in enoplastic cells. Stromal reaction was markedly observed in 5 of 16 cases, in addition to intense reaction in blood vessels which supported neoplastic epithelia. Epithelia of a large or small follicular type in struma were negative for alkaline phosphatase (Photo 16). The neoplastic epithelium in adenoma as well as in struma was devoid of the activity (Photo 17). Follicular colloids under the normal conditions and in the struma contained a large quantity of polysaccharides. In both Hashimoto's and Riedel's diseases, dense connective tissue fibers showed an intense activity (Photos 19 and 20). Tumors of Female Genital Organs Carvical Cancer Forty-five cases of squamous-cell carcinoma and 2 cases of adenocarcinoma were studied (Table 1). Almost all cases of squamous-cell carcinoma of uterine cervix were negative to alkaline phosphatase, but the malignant epithelia of 2 cases showed a positive enzymatic activity (Photo 22), though the normal squamous epithelia of the cervix were devoid of the enzymatic reaction. Alkaline phosphatase activity in stromal elements was developed with varying intensity in 34% of cases (Table II). The activity was rather high in the neighborhood of the neoplastic elements. The stromal reaction for alkaline phosphatase was rather lower in uterine squamous-cell carcinoma 519

8 than in oral epidermoid carcinoma. Alkaline phosphatase was observed in the least degree in neoplastic cells of cervical adenocarcinoma. Alkaline phosphatase-positive stromal elements did not show a strong PAS-staining. Endometrial Cancer Thirteen cases of adenocarcinoma were available for the present study (Table I). In neoplastic epithelia of adenocarcinoma originating from the endometrium, alkaline phosphatase appeared in variable degrees. Alkaline phosphatase activity in the periluminal aspect of malignant papillary structures and cystic architectures was apparently more intense (Photo 23). Most of the neoplastic epithelia proved to be PAS-negative. Ovarian Cancer Five cases of cystoadenocarcinoma and 3 cases of Krukenberg's tumor in the ovary were studied (Table I). The enzymatic activity was hardly observed in neoplastic cells of cystoadenocarcinoma, and it was low in Krukenberg's tumor (Photo 18). Others Three cases of chorioepithelioma, 2 cases each of dysgerminoma and dermoid cyst in the ovary, and one case each of cystoadenoma and malignant ovarian teratoma were studied (Tables I and III). Alkaline phosphatase in the chorioepithelioma was confined to chorio-placental epithelium, and that in cystoadenoma, malignant ovarian teratoma, and ovarian dermoid cyst was absent. A strong to moderate enzymatic reaction was found in cases of dysgerminoma of the ovary. Prostatic Tumors Two cases of adenocarcinoma and adenoma each in the prostate were examined (Tables I and III). Neoplastic epithelium of the prostatic adenocarcinoma was completely devoid of the enzymatic activity, and stromal elements showed an intense activity. Adenomatous cells in one of cases of prostatic adenoma reacted moderately, and in another, was negative. Bladder Cancer Eight cases of transitional-cell carcinoma of the urinary bladder were studied (Table I). Alkaline phosphatase was not present in the transitional-cell carcinoma, and it appeared very intensely in stroma. Seminoma One case of seminoma in the testis was examined (Table III). A strong to moderate enzymatic reaction was found in the tumorous epithelium (Photo 21). Adrenal Tumors Five cases of adrenocortical adenoma were studied (Table III). Alkaline phosphatase activity in adrenocortical adenoma was moderate to intense in 3 cases and trace in one case out of 5 cases. The enzymatic activity was confined to the sinusoid and capillary vessels in the normal status and to adenomatous cells. Tumors of the Nervous System Thirteen cases of meningioma, 8 cases of astrocytoma, 4 cases of neurinoma and craniopharyngioma, 3 cases each of pinealoma and oligodendroglioma, and one case each of ganglioneuroma, ganglioma, neurofibroma, and medulloblastoma were studied (Table V). Alkaline phosphatase was clearly found in meningioma and pinealoma. In menigothelial and fibrous type of a meningioma group, the enzymatic activity was found slightly to strongly in arachnoideal cells (Photos 24 and 25). A mosaic of islands of large round cells in pinealoma revealed an enzymatic staining (Photo 26). The enzymatic activity of neurinoma was negative, while that of neurofibroma was intensive, in which scattered cells with more intense reaction were observed. Astrocytic tumor cells in pilocytic astrocytoma, gemistocytic astrocytoma, spongioblastoma, and glioblastoma multiforme did not stain. Neoplastic epithelia of craniopharyngioma were devoid of the enzymatic activity, but one case showed a positive 520

9 Table V. Distribution of Alkaline Phosphatase Activity in Tumors of Nervous System activity. In ganglioneuroma, ganglioma, oligodendroglioma, and medulloblastoma, no, enzymatic activity was observed. Malignant Tumors of Mesodermal Origin Three cases of osteogenic sarcoma, 2 cases each of fibrosarcoma, lymphosarcoma, and reticulosarcoma, and one case each of leiomyosarcoma, small round cell sarcoma, liposarcoma, and fibroliposarcoma were studied (Table VI). Osteogenic sarcoma showed a moderate to intense activity in fibrous, chondroidal and spindle cells (Photo 6), and fibroliposarcoma a trace activity. Tumor cells of six mesodermal tumors showed no enzymatic staining. Table VI. Distibution of Alkaline Phosphatase Activity in Malignant Mesodermal Tumor Cells Others Two cases each of lympho-epithelioma and melanoma in the skin, 3 cases each of myxoma and plasmocytoma, and one case each of cylindroma and basalioma were examined (Table III). The enzymatic activity was moderately demonstrable in one case of myxoma and in one out of three cases of plasmocytoma. DISCUSSION The neoplastic epithelium of squamous-cell cancer and the homologous epithelium in human and animals generally lack alkaline phosphatase activity.16,10,21) In the 521

10 present investigation, the enzymatic activity was not observed in about 80% of malignant epithelial tumors in human, and in squamous cancer of cervix a positive enzymatic activity was demonstrated in 2 out of 45 cases. Furthermore, in gastrointestinal adenocarcinoma, almost all cases showed no alkaline phosphatase activity in the neoplastiec epithelium, but the activity was variably found in stromal elements. The surface epithelium of the normal gastric mucosa was generally negative for alkaline phosphatase, but small intestinal epithelia showed a large amount of the enzyme, as have been reported.15) Intestinal metaplastic epithelium of the stomach showed a strong activity for alkaline phosphatase, and occasionally coincided with the localization of aminopeptidase.25) It is suggested that the occurrence of alkaline phosphatase activity in the gastric epithelium usually exists in the intestinal metaplasia and does not always imply a malignant tumors. The appearance of alkaline phosphatase in the neoplasms, which originated from steroid hormone-linked organs, has been discussed by earlier workers.5,19) The enzymatic activity was moderately developed in the endometrium of humans,3,5,15) and cyclic variations of the enzyme were indicated.4,18) It has been reported that estradiol benzoate had a phosphatase-mobilizing effect elevating a low enzymatic reactivity in the uterus of castrated animals.2) In the present study, there was a positive staining in the neoplastic epithelium of endometrial adenocarcinoma, as has already been described.19) Experimentally induced adenocarcinoma in mice occasionally showed the presence of alkaline phosphatase activity.24) Moreover, it has been reported that alkaline phosphatase activity was elevated by estrogenic agents in the carcinogenic process of breast cancer in mice,22) and that the activity in normal mammary glands developed under influences of estrogen.10) In human breast diseases, alkaline phosphatase was markedly present in the lobules in adenoma with or without fibrosis, while the malignant epithelium of breast carcinoma was almost devoid of the enzymatic activity. In the present results, it was assumed that the appearance of the enzymatic activity in cervical squamous-cell cancer and endometrial adenocarcinoma might be partially accompanied with hormonal consequences, and that alkaline phosphatase was considered to be not essential to growth in such regions. However, the alkaline phosphatase activity in benign adenoma of the breast seems to afford a diagnostic approach to this kind of tumor. It has been accepted that alkaline phosphatase was confined to the stroma, essentially in reference to inflammatory conditions6,14,15,16,20) and proliferation of the connective tissue in tumor invasion.21) The development of alkaline phosphatase in tumor stroma was suggestive of its possible connection with the presence of proliferating mesenchymal cells, though inflammatory cells and regenerating collagen bundles showed a high alkaline phosphatase activity.15) Many cases of epidermoid carcinoma of the oral region in whose stromal tissue inflammatory processes were present, revealed a varying stromal reaction for alkaline phosphatase. This finding seems to be occasionally influenced by anatomical and physiological factors as well as being related to infectional factors. Gastric carcinomas showed almost no alkaline phosphatase in stromas, irrespective of the presence of ulcer and intense infiltration of inflammatory cells. It is understood that there is no parallel relationship between the distribution of alkaline phosphatase and the inflammatory response in tumor stromas arising in the gastrointestinal tract. Furthermore, in most of adenomas with fibrosis of the breast, alkaline phosphatase activity in periacinous 522

11 connective tissue fibers was low or trace, though they exhibited a predominant proliferation histopathologically. In fibroadenoma of the breast, the enzymatic reaction was low irrespective of fibrous proliferation in perilobular and intercanalicular regions. In spite of the presence of no or few inflammatory changes in the stroma, particularly in the case of squamous-cell carcinoma in the uterine cervix, alkaline phosphatase activity was present in non-inflammatory stroma. It has also been reported that the connective tissue without inflammatory infiltration, adjacent to invading neoplastic epithelia in experimental epidermoid carcinoma, showed an intense activity.21) The stromal substance in the prostatic cancer was markedly stained for alkaline phosphatase, in spite of no inflammatory conditions, and it has been indicated that the stromal component in the prostate is influenced by androgenic and estrogenic hormones.1) These facts seem to suggest that alkaline phosphatase in stromas of certain tumors is invariably dependent on hormonal influence. In other words, it could be concluded that the development of alkaline phosphatase in tumor stromas does not always run parallel with inflammatory responses and fibrous proliferation. Malignant mesodermal tumors in the present series were negative in alkaline phosphatase activity except for a positive reaction in osteosarcoma. It has been accepted that no enzymatic activity is generally observed in neoplastic cells of mesodermal tumors,20) and that a low enzymatic activity is present in the fibrillar components.20) The occurrence of alkaline phosphatase in cases of sarcoma with osteogenic character has already been described;12,13) there was a strong enzymatic reaction of tumor cells in osteogenic sarcoma as well as in normal osteogenic cells. This finding may be significant in the differential diagnosis of osteogenic tumor from others. It seems to be very significant that neoplastic elements did not show alkaline phosphatase activity in almost all of epithelia; all of oral epidermoid carcinoma and most of cervical squamous-cell carcinoma showed a negative reaction, and carcinoma of the gastrointestinal tract was devoid of the activity with the exception of a few cases. Absence of the activity in the neoplastic epithelium indicates that this enzyme is not essential to the tumor growth. The diminution of the enzymatic activity was not observed in tumor which originated from steroid hormone-related organs; endometrial adenocarcinoma showed an intense activity, and in breast fibroadenoma, the enzymatic activity was higher in periacinar aspects and sometimes intense in the luminal border of lobules. The present results seemed to indicate the occurrence of alkaline phosphatase in the tumor stroma depended on the inflammatory response, the proliferation of connective tissue fibers adjacent to invading neoplasms, and the mesenchymolysis due to the tumor invasion as the local factors, and hormonal influences as the general factor. 523

12 REFERENCES 1) Arcardi, J.A., Texas Repts. Biol. Med., 13, 592 (1955). 2) Atkinson, W.B., Elftman, N., Proc. Soc. Exptl. Biol. Med., 62, 148 (1946). 3) Atkinson, W.B., Gusberg, S.B., Cancer, 1, 248 (1948). 4) Atkinson, W.B., in "Menstruation and its Disorders," ed. E.T. Engle, 3 (1950). Charles C. Thomas, Springfield, Ill. 5) Idem, Texas Repts. Biol. Med., 13, 603 (1955). 6) Burstone, M.S., J. Natl. Cancer Inst., 21, 523 (1958). 7) Idem, ibid., 20, 601 (1958). 8) Idem, J. Histochem. Cytochem., 6, 322 (1958). 9) Cold, N.T., Gould, B.S., Arch. Biochem., 33, 155 (1951). 10) Fanger, H., Barker, B.E., Arch. Pathol., 67, 293 (1959). 11) Fell, H.B., Daniellie, J.E., Brit. J. Exptl. Pathol., 24, 196 (1943). 12) Gomori, G., Am. J. Clin. Pathol., 16, 347 (1946). 13) Idem, Texas Repts. Biol. Med., 13, 636 (1955). 14) Gould, B.S., Cold, N.T., Arch. Pathol., 52, 413 (1951). 15) Kabat, E.A., Furth, J., Am. J. Pathol., 17, 303 (1941). 16) Kawakatsu, K., Mori, M., Cancer Res., 23, 539 (1963). 17) Manheimer, L.H., Seligman, A.M., J. Natl. Cancer Inst., 9, 181 ( ). 18) McKay, D.G., Hertig, A.T., Bardawil, W.A., Velardo, J.T., Obst. Gynec., 8, 22 (1956). 19) Idem, ibid., 8, 140 (1956). 20) Monis, B., Rutenberg, A.M., Cancer, 13, 538 (1960). 21) Oka, R., Okamoto, Y., Go, J., Nagasuna, H., THIS JOURNAL, 52, 305 (1961). 22) Richardson, F.L., Pearson, B., J. Natl. Cancer Inst., 14, 1123 (1954). 23) Rutenberg, A.M., Barrnett, R.J., Tsou, K.C., Monis, B., Teague, R., J. Histochem. Cytochem., 6, 90 (1958). 24) Taki, I., Iijima, H., Doi, T., Uetsuki, Y., Mori, M., Am. J. Obstet. Gynecol., 94, 86 (1966). 25) Wattenberg, L.W., Arch. Pathol., 67, 281 (1959). EXPLANATION OF PLATES LXIV-LXVIII Photo 1. Epidermoid carcinoma. The enzymatic activity is confined to the stromal tissue, Photo 2. Pleomorphic adenoma. The adenomatous cells lack the activity, and stromal elements show almost no activity with exception for a marked reaction in blood vessels. (No. 942) Photo 3. Cementoblastoma in the lower jaw. Fibrous components show a moderate activity, and the peripheral region of calcified cementium indicates an intense reaction. (No. 1113) Photo 4. Adenocarcinoma in the parotid gland. The enzymatic stainability is demonstrated in the periluminal layer of malignant epithelia and blood vessels of the stromal tissues. (No. 954) Photo 5. Hepatoma. The enzymatic activity is abundant in malignant cells. (No. 863) Photo 6. Osteosarcoma in the lower jaw. The intense activity is present in osteogenic round Photo 7. Gastric adenocarcinoma. The neoplastic epithelium is completely devoid of the enzymatic activity and the stroma reveals a moderate activity despite the presence of a slight Photo 8. Pylorocardiac glandular-cell adenocarcinoma. Alkaline phosphatase activity is

13 Photo 9. Gastric papillary adenocarcinoma. The tumorous epithelium is rich in alkaline phosphatase, and the internal layer of neoplastic epithelia shows an intense reaction. Stromal tissue scarcely reacts irrespective of the presence of a moderate inflammatory response. (No. Photo 10. Rectal polyp. Epithelial elements are devoid of the stainability, and capillaries Photo 11. Breast adenoma (small lobular type). The glandular epithelium reveals the highest activity, and the loose periacinous stroma is almost negative with exception for an intense Photo 12. Breast fibroadenoma. The adenomatous epithelium reacts as strong as the one in the breast adenoma, the luminal border of which is rather high, and proliferating connective Photo 13. Breast adenoma (duct papillornatosis). The outer laver of the ductal element shows Photo 14. Breast adenocarcinoma. Malignant epithelia are completely devoid of the enzymatic staining, and stromal tissue with a moderate inflammatory infiltration is moderately stained. Photo 15. Comedocarcinoma of the breast. The malignant epithelium exhibits no enzymatic activity, and the basement membrane of comedocarcinoma displays a positive reaction. (No. Photo 16. Struma nodosa. The enzymatic activity in epithelia of a large or small follicle is Photo 17. Thyroid adenoma. The neoplastic epithelium is devoid of the activity, and only capillary elements supporting the tumorous epithelium are stained. (No. 918) (AS-MX) Photo 18. Krukenberg's tumor. The enzymatic activity is low in signet-ring cells, and the Photo 19. Hashimoto's disease. The epithelium of small follicles is absent of the activity, and Photo 20. Riedel's disease. Dense connective tissue fibers show an intense activity, and remain- Photo 21. Seminoma. Round or polygonal large tumor cells have a great deal of the activity, and the stromal tissue is strongly stained. The loose fibrous connective tissue adjacent to Photo 22. Squamous-cell carcinoma of the uterine cervix. The neoplastic epithelium reveals a moderate to intense activity, and stromal tissue is negative except for a high reaction in Photo 23. Endometrial adenocarcinoma. The stainability is demonstrated more intensely in the periluminal aspect of malignant cystic and papillary structures than in the other parts, and Photo 24. Meningothelial meningioma. Arachnoideal cells exhibit an intense reaction, and Photo 25. Fibroblastic meningioma. The enzymatic activity is found in arachnoideal cells, Photo 26. Pinealoma. A mosaic of islands of round cells reveals a marked reaction. (No. 996) 525

14 GANN, Vol. 57 PLATE LXIV

15 GANN, Vol. 57 PLATE LXV

16 GANN, Vol. 57 PLATE LXVI

17 GANN, Vol. 57 PLATE LXVII

18 GANN, Vol. 57 PLATE LXVIII