Transient elevation of messenger RNA encoding gastrin-releasing peptide, a putative pulmonary growth factor in human fetal lung.

Gastrin-releasing peptide (GRP), the mammalian homologue of the amphibian peptide bombesin, is present in pulmonary neuroendocrine cells and appears to be a growth factor for both normal and neoplastic pulmonary cells. Previously we have reported the cloning of the messenger RNAs (mRNAs) and gene that encode human GRP. We now report that GRP mRNAs are markedly elevated in human fetal lung during the canalicular phase of pulmonary development (from approximately 16 to 30 wk gestation). By RNA blot and in situ hybridization analyses, GRP mRNAs were first detectable in fetal lung at 9-10 wk, plateaued at levels 25-fold higher than in adult lungs from 16 to approximately 30 wk and then declined to near adult levels by 34 wk gestation. By contrast, GRP peptide levels remain elevated until several months after birth. Consistent with this, in situ hybridization and immunohistochemical studies showed that GRP mRNA and peptide consistently colocalized in early gestation lung but that in neonatal lung, many cells that contained GRP peptide no longer contained GRP mRNA. The transient expression of high levels of GRP mRNAs during an approximately 12-wk phase of fetal lung development suggests that the secretion of GRP or its COOH-terminal peptides from pulmonary neuroendocrine cells may play a role in normal lung development.

Coexpression of platelet-derived growth factor (PDGF) and PDGF-receptor genes by primary human astrocytomas may contribute to their development and maintenance.

The present studies investigated the expression of the two PDGF genes (c-sis/PDGF-2 and PDGF-1) and the PDGF-receptor b gene (PDGF-R) in 34 primary human astrocytomas. Northern blot analysis demonstrated the coexpression of the c-sis/PDGF-2 protooncogene and the PDGF-R gene in all astrocytomas examined. The majority of the tumors also expressed the PDGF-1 gene. There was no correlation between the expression of the two PDGF genes. Nonmalignant human brain tissue expressed the PDGF-R and PDGF-1 genes but not the c-sis/PDGF-2 protooncogene. In situ hybridization of astrocytoma tissue localized the expression of the c-sis and PDGF-R mRNA's in tumor cells. Capillary endothelial cells also expressed c-sis mRNA. In contrast, nonmalignant human brain tissue expressed only PDGF-R mRNA but not c-sis/PDGF-2 mRNA. The coexpression of a potent mitogenic growth factor protooncogene (c-sis) and its receptor gene in astrocytoma tumor cells suggests the presence of an autocrine mechanism that may contribute to the development and maintenance of astrocytomas. The expression of c-sis mRNA in tumor cells but not in nonmalignant brain cells may serve as an additional diagnostic criterion for the detection of astrocytomas in small tissue specimen using in situ hybridization for the detection of c-sis mRNA and/or immunostaining for the recognition of its protein product.

Expression of angiogenic growth factor genes in primary human astrocytomas may contribute to their growth and progression.

Astrocytomas are highly malignant brain tumors and are among the most neovascularized solid tumors. We have investigated the expression of the angiogenic growth factors acidic fibroblast growth factor and transforming growth factor-alpha, together with its receptor epidermal growth factor receptor, in 30 primary astrocytomas. Both acidic fibroblast growth factor and transforming growth factor-alpha, together with epidermal growth factor receptor, are found to be greatly overexpressed in these tumors when compared with normal brain. This overexpression of angiogenic growth factors may underlie the intense neovascularization characteristic of astrocytomas.

Prognostic significance of peritumoral lymphatic and blood vessel invasion in node-negative carcinoma of the breast.

The prognostic significance of intramammary lymphatic and blood vessel invasion was evaluated in a retrospective series of 221 patients with node-negative carcinoma of the breast treated with modified radical mastectomy. To facilitate identification of lymphatic and blood vessel invasion, the tumors were studied with an immunohistochemical technique using antibodies to endothelial markers. Peritumoral lymphatic and blood vessel invasion (PLBI) (encompassing both lymphatic and blood vessel invasion) was an adverse prognostic indicator independent of menopausal status, tumor size, and other histologic variables. Recurrence of disease and death resulting from carcinoma were significantly higher for patients with PLBI-present (+) tumors compared with patients with PLBI-absent (-) tumors (P less than .0001). The risk of recurrence for patients with PLBI+ tumors was 4.7 times that for their PLBI- counterparts. The presence of intratumoral lymphatic and blood vessel invasion (ILBI) is less important because few examples were found without concomitant PLBI. When PLBI was separated into lymphatic invasion and blood vessel invasion individually, the prognostic significance was retained in both groups. The immunohistochemical approach reduced both false-negative and false-positive observations and identified about 40% of PLBI that would have been missed by routine histologic examination alone. The presence of PLBI appears to be a potentially useful discriminant in predicting the outcome of patients with node-negative carcinoma of the breast.

Gastrin-releasing peptide gene expression in developing, hyperplastic, and neoplastic human thyroid C-cells.

Gastrin-releasing peptide (GRP), the mammalian homolog of bombesin, is often studied as a prototypic neuroregulatory hormone and growth factor, but its own regulation and physiological roles remain to be fully defined. We now demonstrate that the GRP gene is expressed in human thyroidal calcitonin (CT)-containing neuroendocrine cells (C-cells) in an ontogenic pattern similar to its expression in pulmonary neuroendocrine cells and is also expressed at high levels in C-cell hyperplasias and neoplasias (medullary carcinomas of the thyroid). Mean GRP-like immunoreactivity is 20 times higher in 3-week-old to 5-month-old infants than in normal adults, with six of seven infants having GRP levels 6- to 67-fold higher than those in normal adults, the highest levels occurring at 2-2.5 months. CT levels are about 100 times greater than GRP levels at all time intervals, with levels of GRP and CT being linearly correlated (r = 0.98). By RNA blot analysis, GRP mRNAs are increased in neonatal thyroids compared to adult thyroids. In situ hybridization and immunoperoxidase analyses localize GRP mRNAs and peptide to a majority of C-cells in fetuses and neonates, but to only 5-18% of C-cells in normal adults. The majority of developing C-cells have a dendritic morphology, suggesting a paracrine role, although this morphology is not observed in adult C-cells. In addition, for unknown reasons, an increased percentage of C-cells positive for GRP occurs in normal thyroid adjacent to GRP-negative follicular adenomas and papillary carcinomas, an association that we term perineoplastic. We hypothesize that GRP gene expression may play a role in both normal and neoplastic growth processes.

Localization of thyrotropin-releasing hormone prohormone messenger ribonucleic acid in rat brain in situ hybridization.

We studied the distribution of pro- TRH mRNA in rat brain by in situ hybridization histochemistry using radiolabeled single stranded cRNA probes to confirm the hypothesis that the TRH precursor is distributed beyond regions that contain immunoreactive TRH. All regions of the central nervous system previously recognized to contain TRH showed hybridization. Hypophysiotropic neurons in the medial parvocellular division of the paraventricular nucleus showed more intense hybridization than anterior parvocellular division cells, suggesting regional differences in expression. In addition, regions not previously recognized to contain TRH in neuronal perikarya by immunocytochemistry showed specific hybridization for pro-TRH mRNA. These include cells in the olfactory bulbs, dorsal motor nucleus of the vagus, ventrolateral periaqueductal gray, reticular nucleus of the thalamus, and anterior commissural nucleus. Only a single hybridizing band was observed on Northern blots of RNA extracts of the periaqueductal gray and reticular nucleus, identical to that seen in extracts of the paraventricular nucleus. The appearance of pro-TRH mRNA in neurons not previously recognized to contain TRH but which contain the prohormone suggests that non-TRH peptides within the TRH precursor may be preferentially expressed in certain regions of the brain.

Distribution of calcitonin-containing cells in the normal neonatal human thyroid gland: a correlation of morphology with peptide content.

C-cells have been mapped in the thyroid glands of 6 human neonates by means of immunoperoxidase localization of calcitonin and tissue calcitonin content as measured by radioimmunoassay. The C-cells were concentrated in a zone in the upper two-thirds of the lateral lobes bilaterally, where they were identified individually and in small groups in both an intrafollicular and parafollicular distribution. In contrast to findings in the adult, C-cells were predominantly intrafollicular in the neonate. The relative numbers of C-cells counted per unit area of thyroid tissue correlated strongly with the calcitonin content of immediately adjacent tissue sections. In areas rich in C-cells, as many as 75 immunoperoxidase-stained cells per low-power field were counted, and the concentration of calcitonin was as high as 540 to 2100 mU/g fresh weight, values that were as great as 10 times those observed in the normal adult thyroid gland. The prominence of the C-cell population and increased tissue calcitonin content in the human neonatal thyroid gland may reflect an as yet undefined physiologic role for calcitonin in the newborn.

Germline RET 634 mutation positive MEN 2A-related C-cell hyperplasias have genetic features consistent with intraepithelial neoplasia.

C-cell hyperplasias are normally multifocal in multiple endocrine neoplasia type 2A. We compared clonality, microsatellite pattern of tumor suppressor genes, and cellular kinetics of C-cell hyperplasia foci in each thyroid lobe. We selected 11 females from multiple endocrine neoplasia type 2A kindred treated with thyroidectomy due to hypercalcitoninemia. C-cell hyperplasia foci were microdissected for DNA extraction to analyze the methylation pattern of androgen receptor alleles and microsatellite regions (TP53, RB1, WT1, and NF1). Consecutive sections were selected for MIB-1, pRB1, p53, Mdm-2, and p21WAF1 immunostaining, DNA content analysis, and in situ end labeling. Appropriate tissue controls were run. Only two patients had medullary thyroid carcinoma foci. Nine informative C-cell hyperplasia patients showed germline point mutation in RET, eight of them with the same androgen receptor allele preferentially methylated in both lobes. C-cell hyperplasia foci showed heterogeneous DNA deletions revealed by loss of heterozygosity of TP53 (12 of 20), RB1 (6 of 14), and WT1 (4 of 20) and hypodiploid G0/G1 cells (14 of 20), low cellular turnover (MIB-1 index 4.5%, in situ end labeling index 0.03%), and significantly high nuclear area to DNA index ratio. MEN 2A (germline point mutation in RET codon 634) C-cell hyperplasias are monoclonal and genetically heterogeneous and show down-regulated apoptosis, findings consistent with an intraepithelial neoplasia. Concordant X-chromosome inactivation and interstitial gene deletions suggest clone expansions of precursors occurring at a point in embryonic development before divergence of each thyroid lobe and may represent a paradigm for other germline mutations.

Single strand conformation polymorphism analysis of androgen receptor gene mutations in patients with androgen insensitivity syndromes: application for diagnosis, genetic counseling, and therapy.

Recent studies indicate that mutations in the androgen receptor gene are associated with androgen insensitivity syndromes, a heterogeneous group of related disorders involving defective sexual differentiation in karyotypic males. In this report, we address the possibility of rapid mutational analysis of the androgen receptor gene for initial diagnosis, genetic counseling, and molecular subclassification of affected patients and their families. DNA from peripheral blood leukocytes of six patients from five families with various degrees of androgen insensitivity was studied. Exons 2 to 8 of the androgen receptor gene were analyzed using a combination of single strand conformation polymorphism analysis and direct DNA sequencing. Female family members were also studied to identify heterozygote carriers. Point mutations in the AR gene were identified in all six patients, and all mutations caused amino acid substitutions. One patient with incomplete androgen insensitivity was a mosaic for the mutation. Four of the five mothers, as well as a young sister of one patient, were carriers of the mutation present in the affected child. Our data show that new mutations may occur in the androgen receptor gene leading to sporadic androgen insensitivity syndrome. Molecular genetic characterization of the variant allele can serve as a primary tool for diagnosis and subsequent therapy, and can provide a basis for distinguishing heterozygous carriers in familial androgen resistance. The identification of carriers is of substantial clinical importance for genetic counseling.

Medullary thyroid carcinoma identified by cell-free translation of tumor messenger ribonucleic acid in a patient with a neck mass and the syndrome of ectopic adrenocorticotropin.

We report a patient who presented with a lateral neck mass and Cushing's syndrome secondary to the ectopic production of ACTH. The initial diagnosis was a nonchromaffin paraganglioma based on conventional light and electron microscopic studies. However, during this patient's hospital course, we determined that mRNA extracted from the tumor directed the translation of a calcitonin precursor in a cell-free system. This finding led to a consideration of the diagnosis of medullary thyroid carcinoma, which was then confirmed by further endocrine and histological evaluations. This is a report of the identification of a tumor type using techniques of molecular biology.

Hyperplastic lesions of the gastrointestinal endocrine cells.

A substantial body of knowledge is presently available on the morphologic, histochemical, ultrastructural, and functional characteristics of both the normal endocrine cell population of the gut and their related endocrine tumors. In contrast to this, we have only recently begun to recognize the existence of hyperplastic proliferations of various endocrine cell types, and information is therefore steadily accumulating on the morphologic criteria for their recognition, their clinicopathologic correlates and the clinical relevance of this morphologic finding. Hyperplastic proliferations of various endocrine cell types most often develop as a secondary phenomenon in a variety of clinical situations, and may modify the clinical course of the associated condition in a manner that underscores the functional interrelationships these endocrine cells have not only with each other but with other cell types as well. However, similar proliferations may also occur as a primary event (e.g. primary antral G-cell hyperplasia) and give rise to clinical and biochemical features attributable to the overproduction of their specific hormonal product (e.g. Zollinger-Ellison Syndrome, type I). This communication provides a broad overview of the current state of our knowledge of hyperplastic lesions of a variety of gut endocrine cell types in humans, their pathophysiologic significance, their relationship (if any) to the subsequent development of endocrine tumors (i.e. the hyperplasia-neoplasia sequence), and the utility of certain experimental models for the study of such proliferations in a variety of animal species.

Intramammary lymphatic invasion in breast carcinomas. Evaluation using ABH isoantigens as endothelial markers.

Recent studies indicate that intramammary lymphatic invasion represents an important prognostic parameter in breast carcinomas. However, the identification of intramammary lymphatic invasion in tissue sections is a subjective procedure, frequently hampered by factors such as fixation artefacts and interobserver variations. In this study, monoclonal antibodies to ABH isoantigens were applied on formalin-fixed, paraffin-embedded breast carcinoma tissue by using the avidin-biotin-peroxidase complex technique. In addition, the H antigen was localized using the Ulex europeus agglutinin I lectin binding technique. Isoantigen localization provided excellent delineation of lymphatics and blood vessels, in general unhampered by the retention of isoantigen expression in some breast carcinomas. In comparison, Factor VIII-related antigen localization required prior trypsin enhancement and was less sensitive and less consistent. The staining for isoantigens was more intense in vascular than in lymphatic endothelium. ABH isoantigen localization of lymphatic channels identified lymphatic tumor emboli peripheral to and within the carcinomas, and distinguished bona fide intramammary lymphatic invasion from tissue shrinkage artefacts. The applicability to routinely processed tissue permits retrospective studies and renders the identification of intramammary lymphatic invasion a more objective procedure. Further studies are needed to assess the role of this technique in evaluating the prognostic value of intramammary lymphatic invasion; the technique may be extended also to the study of other neoplasms.

Immunocytochemical diagnosis of alpha-1-antitrypsin deficiency.

Eight cases of liver disease associated with alpha-1-antitrypsin deficiency are described. Six of the cases, including the only childhood case, showed no evidence of lung disease. An occult but variable clinical course is defined in this disorder. A spectrum in the severity of tissue change was noted, and in some instances, extensive liver disease was correlated with relatively minor derangement in liver function. While this form of liver disease is uncommon, it should be included in the differential diagnosis of adult liver disease. Screening for alpha-1-antitrypsin globules in periodic acid-Schiff stained liver tissue sections should be considered in certain cases of cryptogenic liver disease in adults, particularly when advanced disease presents suddenly, where micronodular (portal) cirrhosis is unrelated to excessive alcohol use, or where tissue changes exceed those anticipated from serum biochemical abnormalities. In most of these cases, tissue findings from liver biopsy or autopsy, rather than clinical suspicion, led to the diagnosis. The availability of a simple and reliable immunoperoxidase technique, applicable to routinely processed tissue samples, allowed for rapid and specific diagnosis in all cases. This immunocytochemical method has proven its usefulness in the prospective and retrospective tissue diagnosis of alpha-1-antitrypsin deficiency and associated liver disease.

Leu-enkephalin-like immunoreactivity in proliferative lesions of the human adrenal medulla and extra-adrenal paraganglia.

Leu-enkephalin, a potent, endogenous, opiate-like regulatory peptide, is present in a subpopulation of normal adrenal medullary cells and in a spectrum of proliferative lesions of adrenal and extra-adrenal chromaffin cell origin. The presence, extent, and intensity of leu-enkephalin-like immunoreactivity is variable in normal and pathological states. While areas of diffuse medullary hyperplasia consistently exhibited leu-enkephalin-like immunoreactivity, approximately 50% of hyperplastic medullary nodules, pheochromocytomas, and paragangliomas were positively stained. Tumors of neuroblastic origin, on the other hand, did not contain leu-enkephalin-like immunoreactivity. Variations in leu-enkephalin-like immunoreactivity may be related to aberrations of feedback mechanisms, multicentric origins of lesions from chromaffin cells with or without the capacity for leu-enkephalin synthesis, or to a variety of other mechanisms, including defective innervation of hyperplastic and neoplastic chromaffin cells. The results of these studies indicate that leu-enkephalin-like immunoreactivity is a useful tissue marker for the demonstration of chromaffin cell hyperplasia and neoplasia and may also prove to be an important clinical marker for the assessment of chromaffin cell hyperfunction.

Duodenal carcinoids in patients with and without neurofibromatosis. A comparative study.

Nine duodenal carcinoids from patients with von Recklinghausen's neurofibromatosis (VRNF) were investigated for their morphologic, immunocytochemical, and ultrastructural characteristics, and were compared with seven similar tumors from patients without VRNF. Strong similarities were found between tumors in each group. Irrespective of their association with VRNF, duodenal carcinoids arose in adults and usually produced jaundice, upper intestinal bleeding, or obstruction. Tumors larger than 2.0 cm had already metastasized when first detected. All tumors showed a mixed architectural pattern; five tumors associated with VRNF were of the psammomatous type, as opposed to two of those without VRNF. While no tumors showed argentaffinity, stray argyrophil cells were present only in the three tumors not associated with VRNF. All of the tumors showed immunocytochemical evidence of somatostatinomas, and only one VRNF-associated tumor showed immunoreactivity for an additional regulatory substance, as opposed to three of those not associated with VRNF. Thus, while VRNF-associated duodenal carcinoids are not otherwise distinctive, they tend to be pure somatostatinomas (eight of nine cases), whereas similar tumors unassociated with VRNF are frequently multihormonal (three of seven cases). While many more duodenal carcinoids need to be investigated systematically for their immunocytochemical profile, detection of a pure somatostatinoma in the duodenum should alert one to the possibility of coexistent VRNF.

Polysialic acid of the neural cell adhesion molecule in the human thyroid: a marker for medullary thyroid carcinoma and primary C-cell hyperplasia. An immunohistochemical study on 79 thyroid lesions.

We recently reported that the gold-labeled monoclonal antibody MAb 735, reactive with a long-chain form of alpha-2,8-linked polysialic acid (polySia) found on the neural cell adhesion molecule (NCAM), is useful to immunohistochemically distinguish small-cell lung carcinomas from neuroendocrine carcinomas with higher grade of differentiation (carcinoids) and other types of lung carcinomas (Am J Pathol 1991;139:297). In this study, we tested the occurrence of polySia in various types of malignant thyroid tumors and C-cell hyperplasia to determine whether polySia is a useful adjunct for the differential diagnosis of medullary thyroid carcinoma (MTC) and other thyroid neoplasms and to distinguish primary from secondary (reactive) C-cell hyperplasia (CCH). We examined formaldehyde-fixed and paraffin-embedded sections of 79 thyroid lesions, consisting of 33 MTC (14 familial and 19 sporadic tumors), 13 follicular, 11 papillary, 16 anaplastic carcinomas, and four glands with primary and two with secondary CCH. We applied a direct and an indirect immunogold-silver technique for polySia, CT, and CEA detection, respectively. All 33 MTC showed a strong cell-surface-associated immunoreactivity for polySia, which was sensitive to endoneuraminidase digestion. The polySia immunoreactivity of nerves served as an internal control in all specimens. Immunoreactivity for CT and CEA was present in all MTC with the exception of one recurrent tumor with features of an anaplastic MTC type. All other thyroid neoplasms were nonreactive for polySia, CT, and CEA. Primary CCH associated with MTC showed a strong polySia immunostaining, which was less intense in primary CCH not combined with MTC. In normal-appearing C cells and in secondary CCH, staining for polySia was absent in the majority of cases. We conclude that polySia of NCAM is a valuable marker to distinguish medullary carcinomas from other types of thyroid carcinomas. Furthermore, it allows for the discrimination of primary from secondary C-cell hyperplasia and may be helpful to better define the normal range of C cells in unaffected members of a family with a history of multiple endocrine neoplasia (MEN)-II.

Alpha-lactalbumin as an immunohistochemical marker for metastatic breast carcinomas.

Alpha-lactalbumin (ALA), a milk protein, was demonstrated by immunohistochemistry with polyclonal antisera from three different sources in primary and metastatic breast carcinomas, and mammary Paget's disease. ALA localization was observed in 67% of mammary carcinomas, and in 62% of their metastases to sites which included lymph nodes, lung, bone, liver, pericardium, skin, and subcutaneous tissue. There was close correlation between primary and metastatic breast carcinomas in ALA positivity, but no correlation between ALA positivity and histologic differentiation. A variety of nonmammary neoplasms were examined for ALA immunoreactivity. In contrast to ALA immunoreactivity of breast tissue, which was removed by preabsorption of antiserum with ALA antigen, a number of skin appendage tumors, salivary gland tumors, and mesotheliomas demonstrated positive staining which was not abolished by preabsorption and was most likely due to the presence of cross-reacting antibodies. One commercial ALA antiserum also reacted with pancreatic islet cells in a distribution similar to glucagon. Our results demonstrate the presence of ALA in breast carcinomas and its potential value to the surgical pathologist in the workup of metastatic carcinomas of unknown primary sites. However, the staining encountered in some nonmammary tumors necessitates caution in its interpretation.

Rectal carcinoids as tumors of the hindgut endocrine cells: a morphological and immunohistochemical analysis.

The rectal mucosa is richly endowed with a constellation of amine and polypeptide hormone-producing endocrine cell types which may be identified by silver staining and immunohistochemical methods. In order to study the relationships of rectal carcinoid tumors to the normal hindgut endocrine cells, rectal carcinoids and normal rectal mucosa were compared for the presence of argentaffinity and argyrophilia and for the distribution of a battery of polypeptide hormones. Normal rectal mucosa contained frequent cells which stained for bovine pancreatic polypeptide (PP), human PP, and glucagon-like immunoreactivity (GLI. Somatostatin (SRIF) was present in a smaller proportion of rectal endocrine cells. Both argentaffin and argyrophil cells were encountered frequently in normal rectal mucosa. In the series of 13 rectal carcinoids examined, two cases were focally argentaffin-positive, while eight tumors revealed varying degrees of argyrophilia. Eight tumors contained immunoreactive bovine PP, and four of these tumors which were tested for human PP were also positively stained. SRIF was present in five cases, while GLI was identified in two tumors. Four of the tumors were multihormonal. Rectal carcinoids have a rich polypeptide hormone content which parallels that of the normal rectal mucosa. The distinctive hormonal profile and silver staining properties may prove to be of value as specific markers for carcinoid tumors of rectal or hindgut origin.

Psammomatous somatostatinomas of the duodenum.

The presence of psammoma bodies in carcinoid tumors of the gastrointestinal tract is a rare occurrence; it has also been reported to be associated with features of somatostatin production by the tumor cells. The morphologic features of three such tumors arising in the duodenum were studied by a combination of histochemical, immunocytochemical, and ultrastructural techniques in an effort to delineate their secretory profile and further subclassify them. All tumors showed a mixed architectural pattern with prominent areas of glandular differentiation. The psammoma bodies were almost exclusively located within the glandular lumina. In each instance, the majority of tumor cells showed histochemical and immunocytochemical features of somatostatin-containing cells, and one tumor studied ultrastructurally showed numerous large- and small-sized intracytoplasmic secretory granules, both of which contained somatostatin. In contrast to other endocrine tumors of the duodenum that frequently have a multihormonal secretory profile, psammomatous duodenal carcinoids are associated with the exclusive presence of somatostatin within tumor cells. While many more of such examples of this uncommon tumor need to be systematically investigated for their immunocytochemical and ultrastructural characteristics, duodenal somatostatinomas need to be included in the differential diagnosis of psammomatous tumors.

Immunocytochemical localization of oncodevelopmental proteins in human germ cell and hepatic tumors.

In a combined tissue and serum study alpha-1-antitrypsin (AAT) and alpha-fetoprotein are demonstrated in parallel within tumor tissue inclusions in both endodermal sinus (yolk sac) tumors and malignant hepatomas, and AAT is demonstrated as a marker in both neoplastic and preneoplastic liver lesions occurring in oral contraceptive users, all in association with normal serum AAT phenotype. The tumor inclusions in the first two instances differ immunocytochemically from AAT liver cell globules found in inherited AAT deficiency, which are unreactive for alpha-fetoprotein. It is concluded that unlike the molecular basis of storage associated with AAT phenotypic variation, the tumor inclusions reflect a separate, nongenetic mechanism of AAT storage, which may be epigenetic in nature. AAT and alpha-fetoprotein both are synthesized normally in yolk sac and fetal liver, a parallelism which disappears soon after birth. The reexpression of both proteins in two distinct tumor types arising from endodermal origins (yolk sac and liver), suggests that these markers may represent reemerging fetal gene products, a phenomenon previously proposed only for alpha-fetoprotein, a prototypic "oncofetal antigen."