Adrenal cortical tumors clinically mimicking pheochromocytoma.

The authors report five patients with adrenal cortical tumors in whom the preoperative diagnosis of pheochromocytoma was made. All patients had biochemical evidence of elevated catecholamine secretion in serum or urine. Clinically, two patients presented with symptoms suggestive of pheochromocytoma, and one patient had systemic hypertension that resolved following surgical excision of the tumor. Histologically, the findings were typical of adrenal cortical tumors: two adrenal cortical carcinomas and three adrenal cortical adenomas. Nevertheless, the immunohistochemical studies showed evidence of neuroendocrine differentiation in four tumors. Three tumors were positive for neuron-specific enolase and synaptophysin and a fourth tumor was positive for synaptophysin only. All neoplasms were negative for chromogranin. Electron microscopic studies in three tumors showed abundant endoplasmic reticulum and tubulovesicular cristae consistent with adrenal cortical cell origin. Neurosecretory granules, 150-300 mu in diameter, were found in one tumor. This current series of patients provides evidence that adrenal cortical neoplasms may be associated with clinical findings that simulate pheochromocytoma (so-called pseudo-pheochromocytoma). These clinical findings may be mediated by the presence of neuroendocrine features in these tumors.

Human relaxins in normal, benign and neoplastic breast tissue.

Immunoreactive relaxin is present in human breast cyst fluid and postpartum milk without concurrent detectable serum levels, suggesting that the breast is a site of relaxin synthesis. Monoclonal and polyclonal antibodies to human relaxin H2 have been used to immunolocalize relaxins in normal, benign and neoplastic breast tissues with the avidin-biotin immunostaining technique. In view of the similarities in amino acid sequence between H1 and H2 relaxins, these antibodies to H2 relaxin are likely to detect either or both relaxins present in tissue sections. Staining patterns with these antibodies were identical and showed positive diffuse cytoplasmic staining in normal, lobular and ductal epithelium and in myoepithelial cells in breast tissues from normal prepubertal, cyclic, gestational, lactational and postmenopausal females. Relaxin staining was also present in epithelial and myoepithelial cells of ducts and lobules in benign breast disease as well as in metaplastic epithelium of apocrine microcysts. All breast carcinomas (infiltrating ductal, tubular, medullary, intraductal and infiltrating lobular carcinomas) had strong uniform cytoplasmic staining within the neoplastic epithelial cells. All staining was abolished in normal and neoplastic tissues when the polyclonal antibody was preabsorbed with relaxin. It was necessary to distinguish between the possibilities of relaxins being sequestered by breast tissue and local synthesis. Therefore, the expression of the H1, H2 or both human relaxin genes in normal and neoplastic breast tissues was studied by the isolation of RNA, synthesis of first strand cDNA and amplification by PCR using primer sets which amplified either both H1 and H2, or specifically only H1 or H2 relaxin. The coamplification of both relaxin genes was verified by Southern analysis, diagnostic restriction enzyme digestion and sequencing. The primer set for H1 relaxin detected H1 gene expression in 1 out of 8 normal and 9 out of 12 neoplastic breast RNA samples. The H2 relaxin gene was found to be expressed in 3 out of 8 of the normal samples but in all 12 of the neoplastic samples, suggesting that this gene is expressed at higher copy number in the neoplastic tissues. This is the first demonstration of the cellular immunolocalization of relaxin and relaxin gene expression in normal and neoplastic breast. This should allow further exploration of relaxin's role(s) in normal breast physiology and in its tumorigenesis.

Stimulation of vasoactive intestinal peptide and neurotensin secretion by pentagastrin in a patient with VIPoma syndrome.

BACKGROUND: Pancreatic endocrine tumors (PETs) may secrete a variety of peptide hormones, either alone or in combination, and intravenously administered provocative agents have been used to stimulate hormone release to aid in the diagnosis and localization in suspected cases. These features of PETs led us to perform detailed biochemical investigations and provocative testing in a 26-year-old man with a 5 cm vasoactive intestinal peptide (VIP)-secreting tumor of the head of the pancreas. METHODS: Plasma hormone radioimmunoassays and immunohistochemical studies were performed for a panel of peptide hormones, including VIP, neurotensin, and pancreatic polypeptide (PP). Acid alcohol extracts of tumor specimens were analyzed for these peptide hormones as well. Before operation, four provocative test regimens were administered intravenously after an overnight fast: pentagastrin (0.5 microgram/kg/5 sec); rapid calcium infusion (2 mg/kg/min); a combination of calcium (2 mg/kg/min) followed by pentagastrin (0.5 microgram/kg/min); and secretin (2 clinical units/kg bolus). Blood samples were collected before each test and 1, 2, 3, 5, and 10 minutes after the infusions. RESULTS: Measurement of plasma hormone levels and tumor immunohistochemistry and hormonal extraction studies indicated secretion of VIP, neurotensin, and PP by the tumor. Coexpression of VIP and neurotensin was seen immunohistochemically within some individual tumor cells. Provocative testing resulted in maximal stimulation of VIP and neurotensin secretion with pentagastrin administration, which produced increases in plasma levels of VIP and neurotensin over basal levels of 81% and 87%, respectively. After operation, plasma levels of VIP, neurotensin, and PP were undetectable before and after administration of pentagastrin. CONCLUSIONS: The results emphasize the importance of comprehensive biochemical evaluation in patients with VIPoma syndrome to detect the production of a range of peptide hormones. Administration of intravenous pentagastrin appears to stimulate release of VIP and NT and should be evaluated further as a provocative agent for the diagnosis and follow-up of patients with these tumors.

Immunohistochemistry as a predictor of clinical outcome in patients given postoperative radiation for subtotally resected pituitary adenomas.

There is general agreement that postoperative radiation therapy is beneficial for patients with subtotally resected pituitary adenomas. We have identified 41 such patients treated during a 20-year period who received postoperative irradiation for a pituitary adenoma. The usual dose was 5040 cGy in 28 fractions. The mean follow-up time was 10.3 years. On routine hematoxylin and eosin (H&E) staining, there were thirty-three chromophobe, seven eosinophilic, and one basophilic adenoma. Tissue blocks were stained for growth hormone (GH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), prolactin (PRL), and/or adrenocorticotropin (ACTH) using the peroxidase-antiperoxidase immunohistochemistry (IHC) method. Routine H&E staining was a poor predictor of the IHC stain. While most patients with a known clinical endocrine syndrome stained positive on IHC for the suspected offending hormone, many patients without a clinical syndrome also stained positive indicating the presence of hormonally occult adenomas in this locally invasive group. The IHC stain results were compared to clinical outcome. The presence of positive GH IHC staining decreased the 15-year progression-free survival (PFS) from 100% to 64% compared to GH negative adenomas (p = 0.06). There was a trend toward decreased 15-year PFS in patients who did not stain for LH. Positive staining for prolactin, ACTH, or TSH had no influence on the progression-free survival. We conclude that additional prognostic information can be obtained in this subset of patients (by performing IHC analysis) that is not known by the clinical presentation or appearance on H&E stain.

Estrogen-receptor protein in thyroid neoplasms. An immunohistochemical analysis of papillary carcinoma, follicular carcinoma, and follicular adenoma.

Epidemiologic features of well-differentiated thyroid tumors and experimental evidence suggest that female sex hormones may exert effects on this gland and its neoplasms. This possibility was addressed by investigating the expression of estrogen-receptor protein in 80 thyroid neoplasms. Patients with papillary carcinomas, follicular carcinomas, and follicular adenomas were selected from each of the following groups: (1) postpubertal-premenopausal women (who are associated with the most favorable prognosis and greatest incidence of these neoplasms); (2) postmenopausal women; and (3) men of various ages. Sections from formalin-fixed paraffin-embedded tumors were stained with antiestrophilin antibody (clone H222) and the avidin-biotin-peroxidase complex method. In addition, other markers were included to distinguish thyroidal from other estrogen-receptor protein-reactive neoplasms; an anticytokeratin mixture, antithyroglobulin, and anti-gross cystic disease fluid protein-15 were applied in all cases. The expression of estrogen-receptor protein was detected in eight of 10, six of 10, and nine of 10 papillary carcinomas; four of eight, two of seven, and one of five follicular carcinomas; and none of 10, none of 10, and two of 10 follicular adenomas, in groups 1, 2, and 3, respectively. Nuclear staining was regional or multifocal in distribution. Cytokeratin and thyroglobulin were detected in all tumors. In contrast, none displayed anti-gross cystic disease fluid protein-15 immunoreactivity. These results indicate that the estrogen receptor may be detected immunohistochemically in thyroid neoplasms. However, no differences that could account for possible estrogen-related epidemiologic and prognostic variation in such tumors could be ascertained. Other discriminating immunostains, primarily including anti-gross cystic disease fluid protein-15 and thyroglobulin, are effective in distinguishing between thyroidal and extrathyroidal tumors that may express estrogen-receptor protein.

Calcium oxalate microcalcifications in the breast.

Calcium oxalate calcifications can be difficult to detect with routine histologic procedures. In the reported case, microcalcifications that were evident with radiography of the specimen and of the paraffin blocks could not be detected with light microscopy. Polarized light microscopy, however, revealed the calcifications to be calcium oxalate crystals. Use of polarized light microscopy may resolve radiologic-pathologic discrepancies in such cases.

Immunoreactivity for estrogen receptor protein in sweat gland tumors.

The histologic and immunophenotypic similarities between sweat gland carcinoma and breast cancer are well known. Indeed, these likenesses often preclude the diagnostic separation of primary cutaneous glandular neoplasms from metastatic mammary carcinomas, based on light microscopic and immunohistochemical features alone. To assess whether the presence of estrogen receptor protein (ERP) in breast carcinoma might serve as a diagnostic marker in this context, we analyzed 33 eccrine carcinomas, 24 sebaceous carcinomas, 15 intraepidermal apocrine carcinomas (extramammary Paget's disease), and 42 benign sweat gland tumors for ERP content. The monoclonal anti-ERP H222 was used with a modified avidin-biotin-peroxidase complex (ABC) method and paraffin sections. For comparison, eight cutaneous metastases of mammary carcinomas were similarly studied. ERP was identified in six of eight secondary neoplasms. However, this steroid-binding protein also was detected in 10 of 33 eccrine carcinomas. In three of 10 eccrine hidradenomas, each of two examples of hidradenoma papilliferum, and two of three chondroid syringomas, ERP-reactivity was noted as well. The remaining eccrine, apocrine, and sebaceous neoplasms were nonreactive. Among immunoreactive eccrine neoplasms, eight of 10 carcinomas occurred in males, whereas most ERP-positive benign eccrine tumors arose in females. The potential expression of ERP by sudoriferous malignancies reinforces the biologic similarities between mammary and cutaneous adnexal neoplasms. Moreover, ERP reactivity in the latter lesions underscores the inability of immunohistochemistry to distinguish primary and secondary glandular tumors of the skin with certainty.

Immunohistochemistry of GCDFP-24 and zinc alpha2 glycoprotein in benign sweat gland tumors.

The immunohistochemical localization of two other proteins that are present in breast gross cystic disease fluid, GCDFP-24 and zinc alpha 2 glycoprotein (Zn2GP), were studied in normal skin and in 41 benign sweat gland tumors. GCDFP-24 was localized to apocrine glands. There was no staining of eccrine glands or ducts. There was positive staining in the following sweat gland tumors: apocrine hidrocystoma (four of five), hidradenoma papilliferum (two of four), syringocystadenoma papilliferum (six of seven), mixed tumor (one of one), and glandular elements of cylindroma (one of four). No staining for GCDFP-24 occurred among the following SGT: eccrine hidrocystoma (two cases), eccrine poroma (three cases), syringoma (eight cases), eccrine spiradenoma (two cases), or clear cell hidradenoma (five cases). Zn2GP was localized to both apocrine glands and eccrine glands. Positive staining was seen in the following SGT: apocrine hidrocystoma (five of five), hidradenoma papilliferum (two of four), syringocystadenoma papilliferum (four of seven), mixed tumor (one of one), cylindroma (one of four), eccrine spiradenoma (two of two), and clear cell hidradenoma (one of five). No staining for Zn2GP was seen in the following SGT: eccrine hidrocystoma (two cases), eccrine poroma (three cases), or syringoma (eight cases). GCDFP-24 appears to be a discriminant of apocrine differentiation and function. Zn2GP was expressed predominantly in tumors of apocrine differentiation. However, it was also expressed in some tumors of eccrine differentiation.

Decreased expression of integrin adhesive protein receptors in adenocarcinoma of the breast.

The integrin superfamily represents a major class of receptors mediating cell-substrate adhesion. Our recent study of the tissue distribution of the alpha 2 beta 1 integrin, a cell-surface collagen receptor, revealed that high levels of receptor expression were associated with orderly, regulated epithelial cell proliferation. Those observations prompted the present investigation of alpha 2 beta 1 and other integrins in adenocarcinoma of the breast. The alpha 2 beta 1 integrin was highly expressed on the epithelium of the ducts and ductules of normal breast tissue. Normal or nearly normal levels of the receptor were expressed in fibroadenomas. In contrast, markedly decreased or undetectable alpha 2 beta 1 expression was typical of poorly differentiated adenocarcinomas. Well-differentiated lesions exhibited intermediate levels of expression. Similar, but less extensive, decreases in expression were observed for the alpha 5 beta 1 (fibronectin receptor) and alpha v beta 3 (vitronectin receptor). Significant expression of the beta 1 subunit on even poorly differentiated tumors suggests that the expression of other undefined members of the beta 1 family is not reduced to the same low level as alpha 2 beta 1 and alpha 5 beta 1. Expression of the alpha 2 beta 1 integrin was highly correlated with estrogen-receptor expression. Decreased expression of alpha 2 beta 1 and other integrin adhesive protein receptors probably contributes to the altered adhesive properties of tumor cells characteristic of the malignant phenotype.

Use of injectable cultured human fibroblasts for percutaneous tissue implantation. An experimental study.

Large quantities of cultured human facial dermal fibroblasts were propagated from randomly selected patients to determine their relative suitability as percutaneously injectable living implants. Volumetric and histologic comparisons were made between the following implants that were injected subcutaneously into athymic nude mice: (1) cultured human fibroblasts (HFb); (2) cultured human fibroblasts dispersed in Zyderm II collagen (HFb + Zyd); (3) Zyderm II collagen (Zyd); and (4) Zyplast collagen (Zyp). Both the HFb and HFb + Zyd implants were accepted as primary takes but regressed volumetrically at significantly greater rates than either the Zyd or Zyp implants. Correlative immunohistochemical staining revealed that, by 10 days, 90% of the cells within the HFb implants and 80% within the HFb + Zyd implants were of human origin; however, by 9 weeks, approximately 25% of the cells were of human origin in both types of implants. These results indicated that cultured human fibroblasts can be successfully injected as living grafts; however, the subsequent gradual attrition in the numbers of implanted cells, as noted in this model system, limits the long-term retention of the implants.

Expression of GCDFP-15 in breast carcinomas. Relationship to pathologic and clinical factors.

A retrospective immunoperoxidase staining study for a glycoprotein isolated from human breast gross cystic disease fluid (GCDFP-15) in 562 primary breast carcinomas in 539 patients was conducted to correlate its immunohistochemistry with pathologic and clinical factors. Overall, 55% of the carcinomas studied stained positively for GCDFP-15. In certain histologic subtypes, the percentage of carcinomas that stained positively was greater: those subtypes with apocrine histologic features (75%), intraductal carcinoma (70%), and infiltrating lobular carcinoma with signet-ring cell differentiation (90%). In contrast, only 5% of medullary carcinomas exhibited positive staining. Only 23% of breast carcinomas without apocrine features stained positively for GCDFP-15. Carcinomas that stained positively were more likely to have involved axillary lymph nodes (P less than 0.054). The staining was independent of nuclear grade, mitotic index, tumor size, and estrogen receptor status. Positive staining was related to a history of gross cystic disease but not to age, parity, menopausal status, or age at first birth. A positive stain was not related to risk of recurrence or survival.

Enhanced MHC class II expression in renal proximal tubules precedes loss of renal function in MRL/lpr mice with lupus nephritis.

Enhanced MHC class II (Ia) antigen expression is a common feature of autoimmunity. The authors investigated the occurrence of renal Ia expression in MRL/MpJ-lpr/lpr (MRL/lpr) mice with lupus nephritis. By immunoperoxidase staining, normal C3H/FeJ and the congenic strain MRL/MpJ-+/+ express Ia in mononuclear cells in the interstitium only, whereas MRL/lpr with nephritis have abundant Ia expression in proximal tubules (PT), mainly towards the basolateral membrane, and in the characteristic perivascular infiltrates. To a lesser extent, enhanced Ia expression is also observed in the interstitium and in the glomerular mesangium. By Northern blot analysis, the increase in Ia surface determinants correlates with an increased steady-state level of class II mRNA for both I-A and I-E. Ia expression on PT starts focally at around 2-months of age, often in proximity to vascular infiltrates, and precedes overt glomerulo-nephritis and proteinuria. Enhanced class II expression is not restricted to the kidney. MRL/lpr have also increased interstitial class II antigen expression in liver, lung, and spleen compared with normal C3H/FeJ mice. Thus, MRL/lpr mice have enhanced systemic Ia expression, but Ia antigen expression is particularly prominent in PT and may play a key role in the initiation and progression of lupus nephritis.

Small-cell undifferentiated carcinoma of the cervix. A clinicopathologic, ultrastructural, and immunocytochemical study of 15 cases.

The clinicopathologic, immunocytochemical, and ultrastructural features of 15 small-cell undifferentiated carcinomas (SCUC) of the uterine cervix are reported. Patients ranged in age from 25 to 87 (median, 42 years) and presented as stages IB (nine patients), IIA (one patient), IIB (two patients), IIIB (two patients), and IV (one patient). A variety of treatment regimens were employed. Ten patients died of disease (3-71 months; median, 11 months), one patient has a suspicious lung nodule 10 months after diagnosis, one patient is comatose with brain metastases 4 months after diagnosis, and three patients are alive and well 5, 11, and 78 months after diagnosis. Histologically and cytologically, the tumors were identical to pulmonary small-cell undifferentiated carcinoma. Six tumors were associated with other forms of carcinoma, in situ or invasive or both, including epidermoid carcinoma in situ (two cases), adenocarcinoma in situ and epidermoid carcinoma in situ (one case), adenocarcinoma (three cases), and epidermoid carcinoma (three cases). All 13 tumors expressed one or more epithelial markers and at least one neuroendocrine marker. Electron microscopy demonstrated dense-core granules in six of seven tumors, dendrite-like processes in seven tumors, filament bundles in four tumors, and intracytoplasmic lumina in one tumor. Small-cell undifferentiated carcinoma of the cervix is an aggressive tumor with a propensity for rapid metastasis and high mortality. These tumors may demonstrate multidirectional differentiation, including the frequent expression of neuroendocrine features.

Immunoperoxidase localization of GCDFP-15 with mouse monoclonal antibodies versus rabbit antiserum.

Five monoclonal antibodies (Mabs) raised against separate determinants on a breast gross cystic disease fluid protein of 15 KD (GCDFP-15) were compared to one another and to a rabbit antiserum (Rb) against GCDFP-15 by radioimmunoassay (RIA) and by immunoperoxidase localization in paraffin-embedded tissues. All five Mabs and the Rb were equivalent in recognition of GCDFP-15 in solution, as determined by RIA. However, two of the Mabs (A5, B15) showed only minimal binding to GCDFP-15 in paraffin-embedded tissues, whereas the other three Mabs (B1, B4, D6) were equivalent to the Rb in staining intensity. These latter three Mabs and the Rb were evaluated by the immunoperoxidase technique on a variety of benign and malignant neoplasms as well as normal tissues (150 specimens) for staining specificity. Immunoperoxidase staining by the three Mabs vs the Rb was equivalent in apocrine glands, metaplastic apocrine epithelium of breast, and breast carcinomas with apocrine features. No staining of the Mabs or Rb was seen in the other tissue specimens.

Immunohistochemistry of gross cystic disease fluid protein (GCDFP-15) in 65 benign sweat gland tumors of the skin.

Sixty-five cases of benign sweat gland tumors of the skin were studied for the expression and localization of gross cystic disease fluid protein-15 (GCDFP-15) by immunoperoxidase methods. There was positive staining of tumors of probable apocrine differentiation in 10 of 11 cases of apocrine hidrocystoma and five of five cases of hidradenoma papilliferum. There was no immunoreactivity for GCDFP-15 for tumors of probable eccrine differentiation, including five cases of eccrine hidrocystoma, five cases of eccrine poroma, five cases of eccrine spiradenoma, 10 cases of clear cell hidradenoma, and nine cases of syringoma. There was variable positive staining of tumors of more uncertain histogenesis, including eight of eight cases of syringocystadenoma papilliferum, one of four cases of cylindroma, and two of two cases of chondroid syringoma (mixed tumor). The above data support a functional differentiation of the expression of GCDFP-15 by eccrine compared to apocrine glandular epithelium with benign tumor development.