a-FETOPROTEIN IN YOLK SAC TUMOR Yoshiaki Tsuchida, Yasuo Endo, Yoshinori Urano, and Masanobu Ishida Department of Pediatric Surgery, First Department of Internal Medicine, and Department of Pathology Faculty of Medicine, University of T o k y o T o k y o , Japan
Although the a-fetoprotein ( A F P ) concentration is most frequently elevated in the serum of patients with hepatocellular carcinoma, as many as 6 2 % of patients wth teratocarcinoma have been found to have abnormally high serum A F P concentrations.1 In 1967, Abelev and his co-workers 2 first reported that A F P was positive in 13 of 32 cases with testicular teratoblastoma and the rate of the positivity higher in those with the features of embryonal carcinomas.3 Masopust and his associates published a paper in 1968 stating that A F P was found in the sera of patients with ovarian, testicular, and presacral teratocarcinomas. Thereafter, more than 50 AFP-positive teratocarcinomas, originating in various sites including the ovary, testis, and the sacrococcygeal, mediastinal,: and intracranial ti regions, have been reported u p to now, and many of the authors”’ have observed that determination of serum A F P concentrations reflected not only the progress of the illness but also the effectiveness of therapy, as it does frequently in hepatocellular carcinomas. T h e occurrence of the A F P in hepatocellular carcinomas and in some hepatic disorders has been well explained in connection with the synthesis of this protein by the fetal liver. However, the phenomenon of A F P synthesis by teratocarcinomas has not been well explained until recently. Specifically, unsolved questions have been ( I ) whether all malignant teratomas are AFPpositive and all benign teratomas AFP-negative, ( 2 ) why A F P is positive in teratocarcinomas, and ( 3 ) which part of the tumor tissue of teratocarcinoma synthesizes AFP. Concerning the second question mentioned above, the authors reported in 1973 three cases of AFP-positive teratocarcinomas that were originally classified as embryonal carcinoma * I but reclassified as yolk sac tumor (endodermal sinus tumor) after reexamination.!’ They pointed out that the occurrence of A F P in teratocarcinomas is best explained by the concept of a yolk sac tumor, because a yolk sac tumor (endodermal sinus tumor) is of yolk sac origin by definition, l 3 and because it is known that large amounts of A F P are synthesized by the fetal yolk sac as well as by the fetal liver.ll-lk The same explanation has been given independently by Ballas in 1972,l!’ and Wilkinson et al. in 1973.”’ To confirm this explanation, it was necessary to review a much larger series of AFP-positive teratocarcinomas with the concept of yolk sac tumor in mind.!’ I t is especially important to know which part of the tumor tissue of teratocarcinoma produces AFP, but there have been only a few works 2 1 - 4 3 that demonstrated immunofluorescent localization of A F P in teratocarcinomas since the first report by Mawas and his colleagues in l969.?’ This paper consists of a survey of A F P incidence in benign and malignant
22 1
Annals New York Academy of Sciences
222
a-FETOPROTEIN
IN
TABLE1 TERATOMAS : A SURVEY
OF
33
INSTITUTIONS IN
JAPAN
AFP-Positive
AFP-Negative
Malignant Benign
25 9
8 46
Total
34
54
teratomas, a histologic reevaluation of 19 AFP-positive teratocarciomas and a report of immunofluorescent localization of AFP in one of our cases. Incidence of a-Fetoprotein in Teratomas
A questionnaire on AFP in teratomas was sent to leading pediatric surgeons all over Japan. Information was requested relative to positivity of AFP, the site and histologic type of the teratomas, the age and sex of the patients and the method and concentration of the AFP determinations. This survey was done by Hasegawa and Tsuchida and the results will be reported in detail elsewhere.*5 At the moment, 88 cases in which AFP was studied were collected from 33 institutions in Japan. Only those AFP levels over 200 ng/ml were counted as “positive” in the following statistics. Both mature and so-called “immature” 26 teratomas were classified as benign teratomas. Thirty-three cases were malignant (TABLE 1). They occurred in the mediastinum in one, in the retroperitoneum in one, in the sacrococcygeal region in 14, in the testicle in 11 and in the ovary in 6 cases (TABLE 2). Of the 33 malignant teratomas, 25 were AFP-positive and 8 AFP-negative. Fifty-five cases were benign. Nine of these were AFP-positive and 46 AFPnegative (TABLE 1 ) . The occurrence of AFP in benign teratomas was studied according to age in TABLE3. AFP-positive benign tumors were seen only at the age of one month or younger. There were no AFP-positive cases at all in patients older than two months of age.
TABLE2 €2-FETOPROTEIN I N MALIGNANTTERATOMAS : A IN JAPAN
SURVEY OF
AFP-Positive
33
INSTITUTIONS
AFP-Negative
~
Mediastinal Retroperitoneal Sacrococcygeal Testicular Ovarian Total
1
1 10 8 5
4 3 1
25
8
223
Tsuchida et al.: AFP in Yolk Sac Tumor OCCURRENCE
OF
TABLE 3 a-FETOPROTEIN IN BENIGNTERATOMAS ACCORDING TO AGE A SURVEY OF 33 INSTITUTIONS IN JAPAN
Age
AFP-Positive
< 1 mo.
AFP-Negative 5 2 5 2 2
6 3
1 mo. 2 mos. 3 mos. 4 mos. 5 mos. 6-1 1 mos. 1 yr. 2 yrs. 3 yrs. 4 yrs. 5 yrs. 6-10 yrs. 11-15 yrs.
3 11 5 1 2 1 4 3
Total
9
46
Histologic Reevaluation of A FP-Positive Teratmnas As already mentioned, the occurrence of A F P in teratocarcinomas will be best explained if every AFP-producing tumor is yolk sac tumor, because it was demonstrated by Gitlin l.l-lx that large amounts of A F P are synthesized by the yolk sac and by the fetal liver during early fetal life (FIGURE1 ) . The concept of yolk sac tumor is not universely accepted at the present moment. Some pathologists adopt this nomenclature into their classification (TABLE 4),L'li-ZH but others d o not. W e had a chance to study the microscopic sections of 19 of the 25 AFPpositive malignant teratomas, which had had various initial histologic diagnoses such as embryonal carcinoma, malignant teratoma, orchioblastoma, teratocarcinoma, teratoblastoma, etc. Of the 19 cases we studied, 4 were from the University of and the other 15 from 1 1 other institutions in Japan with generous permission of the investigators. One of the authors (Y.T.) reexamined all of the cases himself with the concept of a yolk sac tumor in
Lim 1 mammals HuMl
FIGUREI . Sites of AFP synthesis in fetuses. Data from Gitlin et al.ll-ls
Rm
0 0
2 birda
-
GlTrKt
0 0
-
0
0
-
3 fish shlrlr
Yolk Sac
Annals New York Academy of Sciences
224
TABLE4 HISTOLOGIC CLASSIFICATION OF TEMTOMAS * I. Benign (mature) teratomas 2. Teratomas with mature and immature (embryonic) tissues 3. Malignant teratomas
(a) (b) (c) (d)
Germinoma (seminoma. dysgerminoma) Embryonal carcinoma Yolk sac carcinoma Choriocarcinoma
* From Mahour et al.= mind. Only those tumors that fit the criteria by Teilum (TABLE 5 ) were diagnosed as yolk sac tumor. TABLE6 summarizes the results of our reexamination, with information relative to the serum A F P level and the original site of the teratocarcinoma. Sixteen were reclassified as definitely yolk sac tumors. In one, this diagnosis is possible but not definite. In two cases no diagnostic structure of yolk sac tumor was found in the available sections, but malignant neural elements were present. Yolk sac tumors have two sub-types; endodermal sinus tumor and polyvesicular vitelline tumors.’:’. 3o All of the 16 definite and the one possible yolk sac tumors in TABLE 6 were endodermal sinus tiimorsl2*l3under this subclassification. Many tumors showed papillary appearance with loose stroma, and the most commonly seen diagnostic structures in these were Schiller-Duval bodies (FIGURE 2) ,12.l:’ although some of them were atypically arranged. Less frequently, markedly vacuolated meshworks (FIGURE 3) and only in a few instance, festoon shaped configurations (FIGURE4 ) were found. TABLE5 HISTOLOGICAL APPEARANCE OF ENDODERMAL SINUSTUMORS (YOLKSAC TUMORS) * The endodermal sinus tumors may show the following characteristic patterns: I . A loose vacuolated network with wide meshes or cystic spaces, lined with flat mesothelioid cells and showing foci of active hemopoiesis in the underlying capillary spaces. The meshes often contain hyaline, acidophilic, and PAS-positive globules or mucoid precipitates. 2. A myxoid reticulum resembling the “magma reticulare” or extra-embryonic mesoderm of the exocoelom. 3. Endodermal sinus structures with mantling of vessels by yolk sac endoderm. 4. Cystic structures in regular arrangement of small cavities, lined by a layer of flat cells with protruding nuclei and continuous with the parietal lining of the endodermal sinuses. Individual cysts showing a clear, columnar, in part mucinous epithelium may occur in the loose myxoid or vacuolated stroma with mesoblastic mesothelial-like elements and early differentiation of yolk sac endoderm, forming characteristic festoon-shaped configurations. 5 . Compact aggregates of undifferentiated embryonal cells merging with the reticular network of the extra-embryonic mesoblast or the endodermal sinus structures. .-
’> See reference 13.
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Annals New York Academy of Sciences
FIGURE3. Loose vacuolated meshwork with honeycomb appearance (Case 5 in TABLE 6 ) . (H& E, x 150.)
FIGURE 4. Festoon shaped configuration (Case 13 in TABLE6 ) . (H& E, x 150.)
Tsuchida et al.: AFP in Yolk Sac Tumor
227
lrnmunopuorescent Localization of A FP Synthesis
Immunofluorescent localization of A F P synthesis was studied in a threeyear-old female child admitted to the University of Tokyo Hospital with the diagnosis of sacrococcygeal yolk sac Fluorescein isothiocyanatelabeled goat anti-serum against rabbit 7-globulin was applied t o the sections which were fixed by modified Hamashimak technique 32 and pretreated with rabbit anti-serum against human AFP.”‘ To establish the specificity of the staining reaction, control experiments were performed both with non-immune rabbit serum and without any rabbit anti-serum. Monospecificity of anti-human A F P serum was confirmed by immunoelectrophoresis (FIGURE 5). AFP-specific immunofluorescence was seen in the tumor cells arranged in 6 ) . Most of the tumor cells showed a strong bright papillary pattern (FIGURE staining in their respective cytoplasms, while some others showed a relatively weak immunofluorescence. There was a Schiller-Duval in the stained field. A strong positive reaction was shown in the surrounding endodermal cell layer, but 319
FIGURE 5. Monospecificity of anti-human AFP serum was confirmed by immunoelectrophoresis. Serum of a patient with hepatoma in center, anti-serum against human serum in top, and anti-serum against human AFP that was used for the immunofluorescent study, in bottom.
there was no staining in the vascular core (FIGURE 7).32 T h e relationship between PAS-positive granules and AFP-specific immunofluorescence was not always parallel.:42 Discussion
AFP-positive benign tumors were seen only at the age of one month or younger in this survey (TABLE 3 ) . Serum A F P levels of normal infants were reported to be extremely high at this age,33 although the exact normal values have not yet been given. It therefore seems that w e should not consider these abnormally high A F P levels as “positive,” but rather as “biological” in very young infants. With this exception, it would appear that determination of A F P concentrations is valuable in the differential diagnosis of benign and malignant teratomas.
228
Annals New York Academy of Sciences
FIGURE6. AFP specific immunofluorescencewas seen in the tumor cells (Case 8 in TABLE 6 ) . ( x 150.)
FIGURE 7. A strong immunofluorescent staining was shown in the surrounding endodermal cell layer of a Schiller-Duval body. (Case 8 in TABLE6). ( x 400.) From Urano et al." By permission of the publishers of La Nouvelle Presse Medicale.
Tsuchida et al.: AFP in
229
Yolk Sac Tumor
The concept of yolk sac tumor has been advocated by Teilum since 1959.12 Teilum pointed out that there is a group of tumors having characteristics resembling epithelium, stroma, and epithelium-stroma combination of a yolk sac in so-called “embryonal carcinomas” and proposed a new histologic entity of yolk sac tumor (endodermal sinus tumor) after his extensive comparative studies of human teratocarcinomas and the rat’s placenta.12 Originally, Teilum used the term, endodermal sinus tumor, which of course means that the tumor is of yolk sac origin. Later he added another less frequently seen histologic entity of yolk sac origin; that is polyvesicular vitelline tum0r.l:’ Therefore, both endodermal sinus tumor and polyvesicular vitelline tumor are yolk sac tumors. Huntington and his co-workers prefer to use the term, yolk sac tumor, even in the place of endodermal sinus tumors,31,:Iz and since their publications “yolk sac tumor” has been used as a synonym for “endodermal sinus tumor” by several other authors.19,21. 27. 2‘ At the moment, the term, endodermal sinus tumor or yolk sac tumor, is sometimes omitted from classifications by those TABLE7 AFP-POSITIVEYOLK SAC TUMORSREPORTEDI N
THE
LITERATURE
Authors
Age
Sex
Site
Balas (1972)’” Wilkinson et a / . ( 1973)20 Ohrnori et a / . (1974)”. Itoh e t a / . (1974)*’
20 yrs. 13 yrs. 8 yrs. 22mos. 1 yr. 40 yrs. 46yrs. 10 mos. 1 yr. 3 yrs. 24 yrs. 2yrs. 21 mos. 7 yrs.
F F
Ovarian Ovarian Ovarian Abdominal Wall Testicular Ovarian Ovarian Vaginal Ovarian Sacrococcygeal Ovarian Testicular Testicular Intra-cranial
Teilum et al. (1974)” Furihata (1974)O
* M.O. =micro-Ouchterlony
F M
M F F F F
F F M
M F
AFP (nglrnl) 25,000
206,000
positive positive positive positive positive positive positive positive positive positive
M.O.)* M.O.) M.O.) M.O.)
2,2 10 7,000
method.
who d o not support the concept by Teilum, but it would appear that pathologists who support this idea are gradually increasing in number.1!1-21* 21i-2’, :W Gitlin disclosed that synthesis of A F P occurs in the liver, G-I tract, and 1 ) .14-1‘ Synthesis of A F P yolk sac in the fetuses of various animals (FIGURE by the yolk sac was again demonstrated by Uriel using estrogen binding technique.37 According t o Gitlin, in the fetuses of mammals, A F P synthesis occurs mainly in the liver and the yolk sac, with only traces of synthesis in the gastrointestinal tract.’ I - l ‘ These findings explain the occurrence of A F P in hepatocellular carcinomas, in yolk sac tumors. and in a few instances of gastric carcinomas.!) It was shown in this study that at least 16 of the 19 AFP-positive teratocarcinomas were yolk sac tumors. The number of our observations may be still insufficient to warrant a final conclusion, but, in addition to ours, more than 10 AFP-positive cases have been reported in the literature under the nomenclature of yolk sac tumor (TABLE 7).1!1-22. These findings tend to
230
Annals New York Academy of Sciences
make us conclude that the production of AFP is due to the tumor cells of yolk sac origin in teratocarcinomas. A possible explanation of two exceptional cases in which malignant neural elements without yolk sac structures were found could be that yolk sac structures were perhaps present outside of the available sections. Alternatively, organs other than the yolk sac epithelium may produce very minute amounts of AFP. More direct evidence of AFP synthesis was provided by immunofluorescent techniques (FIGURES 6 and 7). Immunofluorescent localization of AFP synthesis in teratomas was first demonstrated by Mawas et al. in 1969,24 and thereafter by a couple of other authors.Z1-Z3 Among these investigators,Z1-24 Itoh et al.*l and Teilum et aL2? reported immunofluorescent localization of AFP under the specific nomenclature of yolk sac tumor (endodermal sinus tumor), as in the present paper. We demonstrated a bright immunofluorescence in the surrounding endodermal cell layer of a Schiller-Duval body (FIGURE 7). Teilum and his co-workers similarly noted a bright staining in the cells lining the characteristic endodermal sinuses.22 Itoh et al. and Teilum et al. also found a marked staining of the PAS-positive intra- and extra-cytoplasmic hyaline grobules.21q2 2 As Teilum states in his textbook,':' yolk sac tumor consists of various characteristic histologic features even inside of a single tumor. So, further histochemical studies of AFP are needed to determine in its minute detail which structures synthesize AFP and which do not. It is of interest to note that FIGURE 7 seems to show immunochemical evidence of yolk sac origin of that layer that has long been defined as of yolk sac origin morphologically. Gitlin and his associates demonstrated AFP-specific immunofluorescence in the liver and in the yolk sac in fetuses:'" Immunofluorescent localization of AFP was also shown in murine teratocarcinomas by Engelhardt and his colleagues in 1973.41 Present studies seem to offer some evidence for a conclusion that AFP is synthesized not by the whole components but by the yolk sac components of teratomas. Therefore, it seems to us quite possible that there is discordance of human chorionic gonadotropin and AFP in testicular teratocarcinoma as reported by Braunstein et a1.42 or dissociation of serum AFP and tumor growth as pointed out by Esterhay et al.,1 because it is not uncommon for a teratocarcinoma to consist of various components. Combined determinations of AFP and chorionic gonadotropin would have more value. It may be difficult to explain the occurrence of AFP in teratocarcinomas in so far as we continue to use the present classification (FIGURE 8 ) , but with the concept of yolk sac tumor in mind, it can be very easily explained (FIGURE 9). Conversely, the occurrence of AFP may lend an additional support to the morphologic contention that these tumors are of yolk sac origin.
TUMORS
FIGURE8. AFP in malignant teratoma.
Tsuchida et al.: AFP in Yolk Sac Tumor
23 1
FIGURE9. AFP in yolk sac tumor.
FETUS
Summary
The phenomenon of A F P synthesis by teratocarcinomas was studied. A survey of A F P occurrence in benign and malignant teratomas revealed that determination of A F P concentrations is valuable in the differential diagnosis between benign and malignant teratomas. unless the patient is at the age of one month or younger. High A F P levels in these very young infants should not be regarded as positive but rather as biological. Histologic sections of 19 AFP-positive teratocarcinomas, originating in the mediastinum, retroperitoneum, sacrococcygeal region, testicle and ovary, were reviewed with the concept of yolk sac tumor in mind. Sixteen of the 19 cases showed a typical yolk sac tumor (endodermal sinus tumor) as defined by Teilum and one other was compatible with this diagnosis. The occurrence of A F P in teratocarcinomas is best explained by the concept of yolk sac tumor, because it is known that large amounts of A F P are synthesized not only by the fetal liver but also by the yolk sac during early embryonic life, and because the diagnosis of yolk sac tumor implies that the tumor is of yolk sac origin morphologically. More direct evidence of A F P synthesis was demonstrated by imrnunofluorescent techniques in one of our cases. A bright immunofluorescence was seen only in that cell layer that has long been defined as of yolk sac origin morphologically. Acknowledgments
The authors are particularly grateful to Drs. J. Ukijima in Tokyo, H. Watanabe in Kanazawa, T. Todani in Okayama. H . Ogata in Chiba, T. Koide in Tokyo, I. Watanabe in Sendai, K. lkeda and H . Ohgami in Fukuoka, H. Yano in Kurume, A. Endo in Yonago, T. Sugito in Nagoya and T. Hiei in Kyoto for their generous permission to study their cases. They also wish to express their heartfelt thanks to Dr. H. Hasegawa, National Cancer Center, Tokyo and Dr. K. Ohmi, Department of Pediatrics, University of Tokyo, Tokyo for their help and encouragement throughout these studies. Their thanks are also due to Prof. Y. Aoyama, Institute of Medical Science, University of Tokyo, Tokyo and Prof. T. Shikata, Department of Pathology, University of Tokyo, Tokyo, who gave valuable advice in the completion of immunofluorescent studies.
232
Annals New York Academy of Sciences
I . ESTERHAY,R. J., JR., H. M. SHAPIRO,1. C. SUTHERLAND, K. R. MCINTIRE& P. H. WIERNIK. 1973. Serum alpha fetoprotein concentration and tumor growth dissociation in a patient with ovarian teratocarcinoma. Cancer 31: 835-839. N. A. KRAEVSKY, S. D. PEROVA & N. I. PEREVOD2. ABELEV,G . l., V. ASSECRITOVA, CHIKOVA. 1967. Embryonal serum alpha-globulin in cancer patients-diagnostic value. Int. J. Cancer 2: 551-558. 3. ABEDEV,G. 1. 1968. Production of embryonal serum alpha-globulin by hepatomas-review of experimental and clinical data. Cancer Res. 28: 1344-1350. & L. KOTAL. 1968. Occur4. MASOPUST,J., K. KITHIER,J. RADL,J. KOUTECKY rence of fetoprotein in patients with neoplasms and non-neoplastic diseases. Int. J. Cancer 3: 364-373. H. 1974. Personal communication. 5. WATANABE, 6. FURIHATA, S. 1974. Personal communication. D. BUFFE, 0. SCHWEISGUTH & P. BURTIN. 7. MAWAS,C., M. KOHEN,J. LEMERLE, 1969. Serum alpha-I-foetoprotein (fetuin) in children with malignant ovarian or testicular teratomas-preliminary results. Int. J. Cancer 4: 76-79. & M. D. POULIK.1972. Effect of therapy 8. KITHIER,K., J. LUSHER,J. BROUGHT on the serum level of alpha-I fetoprotein in embryonal cell carcinoma. J. Pediat. 81: 71-75. 9. TSUCHIDA,Y., S. SAITO,M. ISHIDA. K. OHMI,Y.URANO,Y. ENDO& T. ODA. 1973. Yolk sac tumor (endodermal sinus tumor) and alpha-fetoprotein-a report of three cases. Cancer 32: 917-921. 10. KAHAN,B. & L. LEVINE. 1971. The occurrence of a serum fetal alpha-1-protein in developing mice and murine hepatomas and teratomas. Cancer Res. 31: 930-93 6. Y., K. OHMI & Y. ENDO. 1973. Embryonal carcinoma and alpha1 1 . TSUCHIDA, fetoprotein. Gann Monogr. Carxer Res. 14: 14 1-147. 12. TEILUM,G . 1959. Endodermal sinus tumor of the ovary and testis-comparative morphogenesis of the so-called mesonephroma ovarii and of extra embryonic (yolk sac allantoic) structure of the rat’s placenta. Cancer 12: 1092-1105. 13. TEILUM,G. 197 1. Special Tumors of Ovary and Testis-Comparative Pathology and Histological Identification. Munksgaard. Copenhagen, Denmark. 14. GITLIN,D. & M. BOESMAN.1967. Sites of serum alpha-fetoprotein synthesis in the human and in the rat. J. Clin. Invest. 46: 1010-1016. 15. GITLIN,D. & J. KITZES. 1967. Synthesis of serum albumin, embryo-specific alpha-globulin and conalbumin by the chick yolk sac. Biochim. Biophys. Acta 147: 334-340. 16. GITLIN, D. & A. PERRICELLI.1970. Synthesis of serum albumin, prealbumin, alpha-foetoprotein, alpha-1-antitrypsin and transferrin by the human yolk sac. Nature 228: 995-997. 17. GITLIN,D. 1971. Sites of alpha-fetoprotein synthesis. N. Engl. J. Med. 285: 14361437. 18. GITLIN,D., A. PERRICELLI& G. M. GITLIN. 1972. Synthesis of alpha-fetoprotein by liver, yolk sac, and gastrointestinal tract of the human conceptus. Cancer Res. 32: 979-982. 19. BALLAS,M. 1972. Yolk sac carcinoma of the ovary with alpha fetoprotein in serum and ascitic fluid demonstrated by immunoosmophoresis. Am. J. Clin. Pathol. 57: 51 1-516. E. J., E. G. FRIEDRICH & T. A. HOSTY. 1973. Alpha-fetoprotein 20. WILKINSON, and endodermal sinus tumor of the ovary. Am. J. Obstet. Gynecol. 1 1 6 7117 14. 21. ITOH, T., T. SHIRAI,A. NAKA& S. MATSUMOTO.1974. Yolk sac tumor and alpha-fetoprotein-clinicopathological study of four cases. Gann 65: 21 5-226. & 9 . N~RGAARD-PEDERSEN. 1974. Immunofluo22. TEILUM,G., R. ALBRECHTSEN
Tsuchida et al.: AFP in Yolk Sac Tumor
23. 24.
25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.
233
rescent localization of alpha-fetoprotein synthesis in endodermal sinus tumor (yolk sac tumor). Acta Path. Microbiol. Scand. Sect. A. 82: 586-588. HARADA, S. 1974. Immunofluorescent studies of alpha-fetoprotein in malignant sacrococcygeal teratomas. Presentation before the 1 Ith annual meeting of the Japanese Society of Pediatric Surgeons. Tokyo, Japan. MAWAS,C., D. BUFFE, J . LEMERLE, 0. SCHWEISGUTH & P. BURTIN. 1969. Recherche immunologique de I’alpha-1-foeto-proteine ou fetuins dans les tumews primitives du foie et les teratomes malins de I’enfant. Arch. Fr. Pediatr. 2 6 779-790. HASECAWA, H. & Y. TSUCHIDA. Alpha-fetoprotein in teratomas-a survey in Japan. To be published. MAHOUR, G. H., M. M. WOOLLEY, S. N. TRIVEDI & B. H. LANDING.1974. Teratomas in infancy and childhooddxperience of 81 cases. Surgery 7 6 309318. CARNEY, J. A., D. P. THOMPSON, C. L. JOHNSON & H. B. LYNN. 1972. Teratoma in children-clinical and pathologic aspects. J. Pediatr. Surg. 7: 271282. Ross, M. M. & J. W. MORROW.1972. Yolk sac carcinoma of the testis. J. Urol. 10.3: 109-1 1 1 . TSUCHIDA, Y., Y. URANO,Y. EDNO,K. OHMI, K. HASHIZUME, S. SAITO,M. 1975. A study on alpha-fetoprotein and endodermal ISHIDA& H. HASEGAWA. sinus tumor. J. Pediatr. Surg. In press. HUNTINGTON, R. W., JR. & W. K. BULLOCK.1972. Endodermal sinus and other yolk sac tumours-a reappraisal. Acta Pathol. Microbiol. Scand. Sect. A. 8O(Suppl. 233): 26-31. HAMASHIMA, Y., J. G. HARTER& A. H. COONS. 1964. The localization of albumin and fibrinogen in human liver cells. J. Cell Biol. 20: 271-279. URANO, Y., Y. ENDO,Y. TSUCHIDA & K. OHMI. 1975. Localization tissulaire de I’alpha-foetoproteine d’un dysernbryome immature. Nouv. Presse Med. 4: 193. MASSEYEFF, R. 1975. Evolution of a-Fetoprotein serum levels throughout life in humans and rats, and during pregnancy in the rat. This volume. HUNTINGTON, R. W., JR. & W. K. BULLOCK.1970. Yolk sac tumors of extragonadal origin. Cancer 25: 1368-1376. HUNTINGTON, R. W., JR. & W. K. BULLOCK.1970. Yolk sac tumors of the ovary. Cancer 25: 1355-1365. SCHNAUFER, L. 1974. Personal communication. URIEL,J. 1975. Liver differentiation and the estrogen-binding properties of afetoprotein. This volume. OHMORI,M., T. HAYASHI, K. MATSUOKA & N. NANBU. 1973. AFP-positive embryonal carcinoma of the ovary. Geka 35: 1024-1026. OHMORI, M. 1974. Personal communication. GITLIN,D., J. KITZES& M. BOESMAN.1967. Cellular distribution of serum alpha-foetoprotein in organs of the foetal rat. Nature 215: 534. ENGELHARDT, N. V., V. S . POLTORANINA & A. K. YAZOVA.1973. Localization of alpha-fetoprotein in transplantable murine teratocarcinomas. Int. J. Cancer 11: 448-459. BRAUNSTEIN, G. D., K. R. MCINTIRE & T. A. WALDMANN. 1973. Discordance of human chorionic gonadotropin and alpha-fetoprotein in testicular teratocarcinomas. Cancer 31: 1065-1068. ABELEV,G. 1. 1971. Alpha-fetoprotein in ontogenesis and its association with malignant tumors. Advan. Cancer Res. 14: 295-358.
Although the a-fetoprotein ( A F P ) concentration is most frequently elevated in the serum of patients with hepatocellular carcinoma, as many as 6 2 % of patients wth teratocarcinoma have been found to have abnormally high serum A F P concentrations.1 In 1967, Abelev and his co-workers 2 first reported that A F P was positive in 13 of 32 cases with testicular teratoblastoma and the rate of the positivity higher in those with the features of embryonal carcinomas.3 Masopust and his associates published a paper in 1968 stating that A F P was found in the sera of patients with ovarian, testicular, and presacral teratocarcinomas. Thereafter, more than 50 AFP-positive teratocarcinomas, originating in various sites including the ovary, testis, and the sacrococcygeal, mediastinal,: and intracranial ti regions, have been reported u p to now, and many of the authors”’ have observed that determination of serum A F P concentrations reflected not only the progress of the illness but also the effectiveness of therapy, as it does frequently in hepatocellular carcinomas. T h e occurrence of the A F P in hepatocellular carcinomas and in some hepatic disorders has been well explained in connection with the synthesis of this protein by the fetal liver. However, the phenomenon of A F P synthesis by teratocarcinomas has not been well explained until recently. Specifically, unsolved questions have been ( I ) whether all malignant teratomas are AFPpositive and all benign teratomas AFP-negative, ( 2 ) why A F P is positive in teratocarcinomas, and ( 3 ) which part of the tumor tissue of teratocarcinoma synthesizes AFP. Concerning the second question mentioned above, the authors reported in 1973 three cases of AFP-positive teratocarcinomas that were originally classified as embryonal carcinoma * I but reclassified as yolk sac tumor (endodermal sinus tumor) after reexamination.!’ They pointed out that the occurrence of A F P in teratocarcinomas is best explained by the concept of a yolk sac tumor, because a yolk sac tumor (endodermal sinus tumor) is of yolk sac origin by definition, l 3 and because it is known that large amounts of A F P are synthesized by the fetal yolk sac as well as by the fetal liver.ll-lk The same explanation has been given independently by Ballas in 1972,l!’ and Wilkinson et al. in 1973.”’ To confirm this explanation, it was necessary to review a much larger series of AFP-positive teratocarcinomas with the concept of yolk sac tumor in mind.!’ I t is especially important to know which part of the tumor tissue of teratocarcinoma produces AFP, but there have been only a few works 2 1 - 4 3 that demonstrated immunofluorescent localization of A F P in teratocarcinomas since the first report by Mawas and his colleagues in l969.?’ This paper consists of a survey of A F P incidence in benign and malignant
22 1
Annals New York Academy of Sciences
222
a-FETOPROTEIN
IN
TABLE1 TERATOMAS : A SURVEY
OF
33
INSTITUTIONS IN
JAPAN
AFP-Positive
AFP-Negative
Malignant Benign
25 9
8 46
Total
34
54
teratomas, a histologic reevaluation of 19 AFP-positive teratocarciomas and a report of immunofluorescent localization of AFP in one of our cases. Incidence of a-Fetoprotein in Teratomas
A questionnaire on AFP in teratomas was sent to leading pediatric surgeons all over Japan. Information was requested relative to positivity of AFP, the site and histologic type of the teratomas, the age and sex of the patients and the method and concentration of the AFP determinations. This survey was done by Hasegawa and Tsuchida and the results will be reported in detail elsewhere.*5 At the moment, 88 cases in which AFP was studied were collected from 33 institutions in Japan. Only those AFP levels over 200 ng/ml were counted as “positive” in the following statistics. Both mature and so-called “immature” 26 teratomas were classified as benign teratomas. Thirty-three cases were malignant (TABLE 1). They occurred in the mediastinum in one, in the retroperitoneum in one, in the sacrococcygeal region in 14, in the testicle in 11 and in the ovary in 6 cases (TABLE 2). Of the 33 malignant teratomas, 25 were AFP-positive and 8 AFP-negative. Fifty-five cases were benign. Nine of these were AFP-positive and 46 AFPnegative (TABLE 1 ) . The occurrence of AFP in benign teratomas was studied according to age in TABLE3. AFP-positive benign tumors were seen only at the age of one month or younger. There were no AFP-positive cases at all in patients older than two months of age.
TABLE2 €2-FETOPROTEIN I N MALIGNANTTERATOMAS : A IN JAPAN
SURVEY OF
AFP-Positive
33
INSTITUTIONS
AFP-Negative
~
Mediastinal Retroperitoneal Sacrococcygeal Testicular Ovarian Total
1
1 10 8 5
4 3 1
25
8
223
Tsuchida et al.: AFP in Yolk Sac Tumor OCCURRENCE
OF
TABLE 3 a-FETOPROTEIN IN BENIGNTERATOMAS ACCORDING TO AGE A SURVEY OF 33 INSTITUTIONS IN JAPAN
Age
AFP-Positive
< 1 mo.
AFP-Negative 5 2 5 2 2
6 3
1 mo. 2 mos. 3 mos. 4 mos. 5 mos. 6-1 1 mos. 1 yr. 2 yrs. 3 yrs. 4 yrs. 5 yrs. 6-10 yrs. 11-15 yrs.
3 11 5 1 2 1 4 3
Total
9
46
Histologic Reevaluation of A FP-Positive Teratmnas As already mentioned, the occurrence of A F P in teratocarcinomas will be best explained if every AFP-producing tumor is yolk sac tumor, because it was demonstrated by Gitlin l.l-lx that large amounts of A F P are synthesized by the yolk sac and by the fetal liver during early fetal life (FIGURE1 ) . The concept of yolk sac tumor is not universely accepted at the present moment. Some pathologists adopt this nomenclature into their classification (TABLE 4),L'li-ZH but others d o not. W e had a chance to study the microscopic sections of 19 of the 25 AFPpositive malignant teratomas, which had had various initial histologic diagnoses such as embryonal carcinoma, malignant teratoma, orchioblastoma, teratocarcinoma, teratoblastoma, etc. Of the 19 cases we studied, 4 were from the University of and the other 15 from 1 1 other institutions in Japan with generous permission of the investigators. One of the authors (Y.T.) reexamined all of the cases himself with the concept of a yolk sac tumor in
Lim 1 mammals HuMl
FIGUREI . Sites of AFP synthesis in fetuses. Data from Gitlin et al.ll-ls
Rm
0 0
2 birda
-
GlTrKt
0 0
-
0
0
-
3 fish shlrlr
Yolk Sac
Annals New York Academy of Sciences
224
TABLE4 HISTOLOGIC CLASSIFICATION OF TEMTOMAS * I. Benign (mature) teratomas 2. Teratomas with mature and immature (embryonic) tissues 3. Malignant teratomas
(a) (b) (c) (d)
Germinoma (seminoma. dysgerminoma) Embryonal carcinoma Yolk sac carcinoma Choriocarcinoma
* From Mahour et al.= mind. Only those tumors that fit the criteria by Teilum (TABLE 5 ) were diagnosed as yolk sac tumor. TABLE6 summarizes the results of our reexamination, with information relative to the serum A F P level and the original site of the teratocarcinoma. Sixteen were reclassified as definitely yolk sac tumors. In one, this diagnosis is possible but not definite. In two cases no diagnostic structure of yolk sac tumor was found in the available sections, but malignant neural elements were present. Yolk sac tumors have two sub-types; endodermal sinus tumor and polyvesicular vitelline tumors.’:’. 3o All of the 16 definite and the one possible yolk sac tumors in TABLE 6 were endodermal sinus tiimorsl2*l3under this subclassification. Many tumors showed papillary appearance with loose stroma, and the most commonly seen diagnostic structures in these were Schiller-Duval bodies (FIGURE 2) ,12.l:’ although some of them were atypically arranged. Less frequently, markedly vacuolated meshworks (FIGURE 3) and only in a few instance, festoon shaped configurations (FIGURE4 ) were found. TABLE5 HISTOLOGICAL APPEARANCE OF ENDODERMAL SINUSTUMORS (YOLKSAC TUMORS) * The endodermal sinus tumors may show the following characteristic patterns: I . A loose vacuolated network with wide meshes or cystic spaces, lined with flat mesothelioid cells and showing foci of active hemopoiesis in the underlying capillary spaces. The meshes often contain hyaline, acidophilic, and PAS-positive globules or mucoid precipitates. 2. A myxoid reticulum resembling the “magma reticulare” or extra-embryonic mesoderm of the exocoelom. 3. Endodermal sinus structures with mantling of vessels by yolk sac endoderm. 4. Cystic structures in regular arrangement of small cavities, lined by a layer of flat cells with protruding nuclei and continuous with the parietal lining of the endodermal sinuses. Individual cysts showing a clear, columnar, in part mucinous epithelium may occur in the loose myxoid or vacuolated stroma with mesoblastic mesothelial-like elements and early differentiation of yolk sac endoderm, forming characteristic festoon-shaped configurations. 5 . Compact aggregates of undifferentiated embryonal cells merging with the reticular network of the extra-embryonic mesoblast or the endodermal sinus structures. .-
’> See reference 13.
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Annals New York Academy of Sciences
FIGURE3. Loose vacuolated meshwork with honeycomb appearance (Case 5 in TABLE 6 ) . (H& E, x 150.)
FIGURE 4. Festoon shaped configuration (Case 13 in TABLE6 ) . (H& E, x 150.)
Tsuchida et al.: AFP in Yolk Sac Tumor
227
lrnmunopuorescent Localization of A FP Synthesis
Immunofluorescent localization of A F P synthesis was studied in a threeyear-old female child admitted to the University of Tokyo Hospital with the diagnosis of sacrococcygeal yolk sac Fluorescein isothiocyanatelabeled goat anti-serum against rabbit 7-globulin was applied t o the sections which were fixed by modified Hamashimak technique 32 and pretreated with rabbit anti-serum against human AFP.”‘ To establish the specificity of the staining reaction, control experiments were performed both with non-immune rabbit serum and without any rabbit anti-serum. Monospecificity of anti-human A F P serum was confirmed by immunoelectrophoresis (FIGURE 5). AFP-specific immunofluorescence was seen in the tumor cells arranged in 6 ) . Most of the tumor cells showed a strong bright papillary pattern (FIGURE staining in their respective cytoplasms, while some others showed a relatively weak immunofluorescence. There was a Schiller-Duval in the stained field. A strong positive reaction was shown in the surrounding endodermal cell layer, but 319
FIGURE 5. Monospecificity of anti-human AFP serum was confirmed by immunoelectrophoresis. Serum of a patient with hepatoma in center, anti-serum against human serum in top, and anti-serum against human AFP that was used for the immunofluorescent study, in bottom.
there was no staining in the vascular core (FIGURE 7).32 T h e relationship between PAS-positive granules and AFP-specific immunofluorescence was not always parallel.:42 Discussion
AFP-positive benign tumors were seen only at the age of one month or younger in this survey (TABLE 3 ) . Serum A F P levels of normal infants were reported to be extremely high at this age,33 although the exact normal values have not yet been given. It therefore seems that w e should not consider these abnormally high A F P levels as “positive,” but rather as “biological” in very young infants. With this exception, it would appear that determination of A F P concentrations is valuable in the differential diagnosis of benign and malignant teratomas.
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Annals New York Academy of Sciences
FIGURE6. AFP specific immunofluorescencewas seen in the tumor cells (Case 8 in TABLE 6 ) . ( x 150.)
FIGURE 7. A strong immunofluorescent staining was shown in the surrounding endodermal cell layer of a Schiller-Duval body. (Case 8 in TABLE6). ( x 400.) From Urano et al." By permission of the publishers of La Nouvelle Presse Medicale.
Tsuchida et al.: AFP in
229
Yolk Sac Tumor
The concept of yolk sac tumor has been advocated by Teilum since 1959.12 Teilum pointed out that there is a group of tumors having characteristics resembling epithelium, stroma, and epithelium-stroma combination of a yolk sac in so-called “embryonal carcinomas” and proposed a new histologic entity of yolk sac tumor (endodermal sinus tumor) after his extensive comparative studies of human teratocarcinomas and the rat’s placenta.12 Originally, Teilum used the term, endodermal sinus tumor, which of course means that the tumor is of yolk sac origin. Later he added another less frequently seen histologic entity of yolk sac origin; that is polyvesicular vitelline tum0r.l:’ Therefore, both endodermal sinus tumor and polyvesicular vitelline tumor are yolk sac tumors. Huntington and his co-workers prefer to use the term, yolk sac tumor, even in the place of endodermal sinus tumors,31,:Iz and since their publications “yolk sac tumor” has been used as a synonym for “endodermal sinus tumor” by several other authors.19,21. 27. 2‘ At the moment, the term, endodermal sinus tumor or yolk sac tumor, is sometimes omitted from classifications by those TABLE7 AFP-POSITIVEYOLK SAC TUMORSREPORTEDI N
THE
LITERATURE
Authors
Age
Sex
Site
Balas (1972)’” Wilkinson et a / . ( 1973)20 Ohrnori et a / . (1974)”. Itoh e t a / . (1974)*’
20 yrs. 13 yrs. 8 yrs. 22mos. 1 yr. 40 yrs. 46yrs. 10 mos. 1 yr. 3 yrs. 24 yrs. 2yrs. 21 mos. 7 yrs.
F F
Ovarian Ovarian Ovarian Abdominal Wall Testicular Ovarian Ovarian Vaginal Ovarian Sacrococcygeal Ovarian Testicular Testicular Intra-cranial
Teilum et al. (1974)” Furihata (1974)O
* M.O. =micro-Ouchterlony
F M
M F F F F
F F M
M F
AFP (nglrnl) 25,000
206,000
positive positive positive positive positive positive positive positive positive positive
M.O.)* M.O.) M.O.) M.O.)
2,2 10 7,000
method.
who d o not support the concept by Teilum, but it would appear that pathologists who support this idea are gradually increasing in number.1!1-21* 21i-2’, :W Gitlin disclosed that synthesis of A F P occurs in the liver, G-I tract, and 1 ) .14-1‘ Synthesis of A F P yolk sac in the fetuses of various animals (FIGURE by the yolk sac was again demonstrated by Uriel using estrogen binding technique.37 According t o Gitlin, in the fetuses of mammals, A F P synthesis occurs mainly in the liver and the yolk sac, with only traces of synthesis in the gastrointestinal tract.’ I - l ‘ These findings explain the occurrence of A F P in hepatocellular carcinomas, in yolk sac tumors. and in a few instances of gastric carcinomas.!) It was shown in this study that at least 16 of the 19 AFP-positive teratocarcinomas were yolk sac tumors. The number of our observations may be still insufficient to warrant a final conclusion, but, in addition to ours, more than 10 AFP-positive cases have been reported in the literature under the nomenclature of yolk sac tumor (TABLE 7).1!1-22. These findings tend to
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Annals New York Academy of Sciences
make us conclude that the production of AFP is due to the tumor cells of yolk sac origin in teratocarcinomas. A possible explanation of two exceptional cases in which malignant neural elements without yolk sac structures were found could be that yolk sac structures were perhaps present outside of the available sections. Alternatively, organs other than the yolk sac epithelium may produce very minute amounts of AFP. More direct evidence of AFP synthesis was provided by immunofluorescent techniques (FIGURES 6 and 7). Immunofluorescent localization of AFP synthesis in teratomas was first demonstrated by Mawas et al. in 1969,24 and thereafter by a couple of other authors.Z1-Z3 Among these investigators,Z1-24 Itoh et al.*l and Teilum et aL2? reported immunofluorescent localization of AFP under the specific nomenclature of yolk sac tumor (endodermal sinus tumor), as in the present paper. We demonstrated a bright immunofluorescence in the surrounding endodermal cell layer of a Schiller-Duval body (FIGURE 7). Teilum and his co-workers similarly noted a bright staining in the cells lining the characteristic endodermal sinuses.22 Itoh et al. and Teilum et al. also found a marked staining of the PAS-positive intra- and extra-cytoplasmic hyaline grobules.21q2 2 As Teilum states in his textbook,':' yolk sac tumor consists of various characteristic histologic features even inside of a single tumor. So, further histochemical studies of AFP are needed to determine in its minute detail which structures synthesize AFP and which do not. It is of interest to note that FIGURE 7 seems to show immunochemical evidence of yolk sac origin of that layer that has long been defined as of yolk sac origin morphologically. Gitlin and his associates demonstrated AFP-specific immunofluorescence in the liver and in the yolk sac in fetuses:'" Immunofluorescent localization of AFP was also shown in murine teratocarcinomas by Engelhardt and his colleagues in 1973.41 Present studies seem to offer some evidence for a conclusion that AFP is synthesized not by the whole components but by the yolk sac components of teratomas. Therefore, it seems to us quite possible that there is discordance of human chorionic gonadotropin and AFP in testicular teratocarcinoma as reported by Braunstein et a1.42 or dissociation of serum AFP and tumor growth as pointed out by Esterhay et al.,1 because it is not uncommon for a teratocarcinoma to consist of various components. Combined determinations of AFP and chorionic gonadotropin would have more value. It may be difficult to explain the occurrence of AFP in teratocarcinomas in so far as we continue to use the present classification (FIGURE 8 ) , but with the concept of yolk sac tumor in mind, it can be very easily explained (FIGURE 9). Conversely, the occurrence of AFP may lend an additional support to the morphologic contention that these tumors are of yolk sac origin.
TUMORS
FIGURE8. AFP in malignant teratoma.
Tsuchida et al.: AFP in Yolk Sac Tumor
23 1
FIGURE9. AFP in yolk sac tumor.
FETUS
Summary
The phenomenon of A F P synthesis by teratocarcinomas was studied. A survey of A F P occurrence in benign and malignant teratomas revealed that determination of A F P concentrations is valuable in the differential diagnosis between benign and malignant teratomas. unless the patient is at the age of one month or younger. High A F P levels in these very young infants should not be regarded as positive but rather as biological. Histologic sections of 19 AFP-positive teratocarcinomas, originating in the mediastinum, retroperitoneum, sacrococcygeal region, testicle and ovary, were reviewed with the concept of yolk sac tumor in mind. Sixteen of the 19 cases showed a typical yolk sac tumor (endodermal sinus tumor) as defined by Teilum and one other was compatible with this diagnosis. The occurrence of A F P in teratocarcinomas is best explained by the concept of yolk sac tumor, because it is known that large amounts of A F P are synthesized not only by the fetal liver but also by the yolk sac during early embryonic life, and because the diagnosis of yolk sac tumor implies that the tumor is of yolk sac origin morphologically. More direct evidence of A F P synthesis was demonstrated by imrnunofluorescent techniques in one of our cases. A bright immunofluorescence was seen only in that cell layer that has long been defined as of yolk sac origin morphologically. Acknowledgments
The authors are particularly grateful to Drs. J. Ukijima in Tokyo, H. Watanabe in Kanazawa, T. Todani in Okayama. H . Ogata in Chiba, T. Koide in Tokyo, I. Watanabe in Sendai, K. lkeda and H . Ohgami in Fukuoka, H. Yano in Kurume, A. Endo in Yonago, T. Sugito in Nagoya and T. Hiei in Kyoto for their generous permission to study their cases. They also wish to express their heartfelt thanks to Dr. H. Hasegawa, National Cancer Center, Tokyo and Dr. K. Ohmi, Department of Pediatrics, University of Tokyo, Tokyo for their help and encouragement throughout these studies. Their thanks are also due to Prof. Y. Aoyama, Institute of Medical Science, University of Tokyo, Tokyo and Prof. T. Shikata, Department of Pathology, University of Tokyo, Tokyo, who gave valuable advice in the completion of immunofluorescent studies.
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Annals New York Academy of Sciences
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rescent localization of alpha-fetoprotein synthesis in endodermal sinus tumor (yolk sac tumor). Acta Path. Microbiol. Scand. Sect. A. 82: 586-588. HARADA, S. 1974. Immunofluorescent studies of alpha-fetoprotein in malignant sacrococcygeal teratomas. Presentation before the 1 Ith annual meeting of the Japanese Society of Pediatric Surgeons. Tokyo, Japan. MAWAS,C., D. BUFFE, J . LEMERLE, 0. SCHWEISGUTH & P. BURTIN. 1969. Recherche immunologique de I’alpha-1-foeto-proteine ou fetuins dans les tumews primitives du foie et les teratomes malins de I’enfant. Arch. Fr. Pediatr. 2 6 779-790. HASECAWA, H. & Y. TSUCHIDA. Alpha-fetoprotein in teratomas-a survey in Japan. To be published. MAHOUR, G. H., M. M. WOOLLEY, S. N. TRIVEDI & B. H. LANDING.1974. Teratomas in infancy and childhooddxperience of 81 cases. Surgery 7 6 309318. CARNEY, J. A., D. P. THOMPSON, C. L. JOHNSON & H. B. LYNN. 1972. Teratoma in children-clinical and pathologic aspects. J. Pediatr. Surg. 7: 271282. Ross, M. M. & J. W. MORROW.1972. Yolk sac carcinoma of the testis. J. Urol. 10.3: 109-1 1 1 . TSUCHIDA, Y., Y. URANO,Y. EDNO,K. OHMI, K. HASHIZUME, S. SAITO,M. 1975. A study on alpha-fetoprotein and endodermal ISHIDA& H. HASEGAWA. sinus tumor. J. Pediatr. Surg. In press. HUNTINGTON, R. W., JR. & W. K. BULLOCK.1972. Endodermal sinus and other yolk sac tumours-a reappraisal. Acta Pathol. Microbiol. Scand. Sect. A. 8O(Suppl. 233): 26-31. HAMASHIMA, Y., J. G. HARTER& A. H. COONS. 1964. The localization of albumin and fibrinogen in human liver cells. J. Cell Biol. 20: 271-279. URANO, Y., Y. ENDO,Y. TSUCHIDA & K. OHMI. 1975. Localization tissulaire de I’alpha-foetoproteine d’un dysernbryome immature. Nouv. Presse Med. 4: 193. MASSEYEFF, R. 1975. Evolution of a-Fetoprotein serum levels throughout life in humans and rats, and during pregnancy in the rat. This volume. HUNTINGTON, R. W., JR. & W. K. BULLOCK.1970. Yolk sac tumors of extragonadal origin. Cancer 25: 1368-1376. HUNTINGTON, R. W., JR. & W. K. BULLOCK.1970. Yolk sac tumors of the ovary. Cancer 25: 1355-1365. SCHNAUFER, L. 1974. Personal communication. URIEL,J. 1975. Liver differentiation and the estrogen-binding properties of afetoprotein. This volume. OHMORI,M., T. HAYASHI, K. MATSUOKA & N. NANBU. 1973. AFP-positive embryonal carcinoma of the ovary. Geka 35: 1024-1026. OHMORI, M. 1974. Personal communication. GITLIN,D., J. KITZES& M. BOESMAN.1967. Cellular distribution of serum alpha-foetoprotein in organs of the foetal rat. Nature 215: 534. ENGELHARDT, N. V., V. S . POLTORANINA & A. K. YAZOVA.1973. Localization of alpha-fetoprotein in transplantable murine teratocarcinomas. Int. J. Cancer 11: 448-459. BRAUNSTEIN, G. D., K. R. MCINTIRE & T. A. WALDMANN. 1973. Discordance of human chorionic gonadotropin and alpha-fetoprotein in testicular teratocarcinomas. Cancer 31: 1065-1068. ABELEV,G. 1. 1971. Alpha-fetoprotein in ontogenesis and its association with malignant tumors. Advan. Cancer Res. 14: 295-358.
