Utility of a paraffin section-reactive CD56 antibody (123C3) for characterization and diagnosis of lymphomas.

Although expression of CD56 (neural cell adhesion molecule, a natural killer cell marker) is uncommon among lymphomas, this feature has defined a distinctive and important category of lymphoma: the putative natural killer (NK) cell lymphoma, which shows a predilection for the upper aerodigestive tract, skin, skeletal muscle, and other extranodal sites and pursues an aggressive clinical course. Thus far, CD56 expression can be reliably analyzed only on fresh or frozen tissues. In this study, we evaluated the sensitivity and specificity of a CD56 antibody, 123C3, when applied on routine formalin-fixed, paraffin-embedded tissues for analysis of lymphomas, by comparing the staining results with those obtained on frozen tissues using the CD56 antibody NKH1. The 123C3 antibody worked on paraffin sections only with prior antigen retrieval using a pressure cooker or a microwave oven. Among 32 CD56+ T/NK cell lymphomas and one CD56+ B-lymphoblastic lymphoma, the neoplastic cells showed crisp membrane staining with 123C3 in all cases. None of the 24 CD56- T-cell lymphomas and 50 CD56-B-cell lymphomas stained with 123C3. In normal or reactive lymphoid tissues from a variety of sites, there were few small lymphocytes (< 0.1%) that showed cell membrane staining with 123C3, although occasional plasma cells might show cytoplasmic staining. We conclude that with suitable antigen retrieval procedures, 123C3 can be reliably applied on routine paraffin sections for detection of CD56 expression in lymphomas. Furthermore, this antibody can be used to support a diagnosis of lymphoma or to detect residual disease for cases of CD56+ T/NK cell lymphoma in which the neoplastic lymphoid cells are small and show minimal atypia, especially in small biopsies.

Discordant CD3 expression in lymphomas when studied on frozen and paraffin sections.

The results of CD3 staining in the T or putative natural killer (NK) cell lymphomas of nasal and extranasal sites as reported in the literature have been confusing, with some studies reporting a low rate of CD3 positivity and others a high frequency of CD3 staining. The former studies were performed on fresh or frozen tissues, whereas the latter were performed on paraffin sections using a polyclonal antiserum (poly-CD3). Although previous studies have suggested a high concordance rate of CD3 staining between fresh/frozen tissues and paraffin sections, many CD3- cases have not been studied, and the more reliable antigen retrieval techniques have not been applied. In this study, we addressed the question of discordant CD3 expression by comparing the results of CD3 staining in lymphomas as studied on frozen sections and as studied on paraffin sections (with antigen retrieval effected by pressure cooking). This series was biased toward inclusion of a high percentage of cases of putative NK cell lymphomas, which are prevalent among Asians and usually show a CD2+ CD3(Leu4)- CD56+ immunophenotype. Among 35 cases of CD3(Leu4)- T- and T/NK-cell lymphomas, 30 (86%) showed staining with poly-CD3 on paraffin sections. All 15 CD3(Leu4)+ T-cell lymphomas showed positive staining with poly-CD3 on paraffin sections. None of 60 B-cell lymphomas were stained by poly-CD3, confirming no loss of specificity of staining with this antiserum despite use of an effective antigen-retrieval technique. The discordance rate of CD3 staining in T- and T/NK-cell lymphomas in this series was 60%, and this phenomenon was most commonly observed in the CD56+ T/NK-cell lymphomas: CD3(Leu4)- in frozen sections but poly-CD3+ in paraffin sections. Therefore, to avoid confusion, we propose designating the results based on fresh/frozen tissues CD3(f) and those based on poly-CD3 application on paraffin sections CD3(p) in future reporting of CD3 immunophenotype.

The MIC2 antibody 013. Practical application for the study of thymic epithelial tumors.

The lymphocytes that accompany thymomas express an immature T-cell phenotype, as usually demonstrated by CD1 or TdT immunoreactivity. Even when thymomas metastasize or occur in ectopic sites, the infiltrating T lymphocytes show this unique immature phenotype, contrasting with thymic and nonthymic carcinomas, in which the infiltrating T lymphocytes typically show a mature phenotype (CD1 and TdT negative). Therefore, the presence of an immature T-cell population in an epithelial tumor strongly supports a diagnosis of thymoma. The availability of an antibody that consistently marks immature T-cells in routine paraffin sections would be of great help in the study of thymic tumors. In this report, we describe the use of MIC2 antibody (013), which has been widely used for the diagnosis of Ewing's sarcomas and peripheral primitive neuroectodermal tumors because it intensely stains thymocytes. Immunohistochemical staining was performed on paraffin sections of normal/hyperplastic thymus (18 cases), thymoma (62 cases), thymic carcinoma (nine cases), tumors showing borderline features between thymoma and thymic carcinoma (three cases), and ectopic hamartomatous thymoma (two cases). T-cell and B-cell antibodies were also applied to aid in the interpretation. In the normal thymus, almost all lymphocytes in the cortex stained with 013, whereas fewer than 5% of those in the medulla were 013 positive. In thymomas, including the three ectopic thymomas and the single case of metastatic thymoma, most lymphocytes were 013 positive, except the spindle-cell foci (medullary thymoma or medullary component of mixed thymoma), in which the percentage of 013-positive lymphocytes was lower (5-30%). Within the pale foci of "medullary differentiation" and the perivascular spaces of lymphocyte-rich thymomas, few lymphocytes showed 013 positivity, indicating that the T lymphocytes in these areas were more mature. None of the thymic carcinomas harbored 013-positive lymphocytes. Among the three cases of borderline thymoma/thymic carcinoma, only one harbored 013-positive lymphocytes. The 013-positive lymphocytes were not seen in the ectopic hamartomatous thymomas. In normal lymph nodes and nonthymic carcinomas studied as controls, there were no or at most small numbers of isolated 013-positive lymphocytes. We conclude that interpreted in the proper context, MIC2 antibody can serve as a useful marker of immature T-cells and thus help in the confirmation of a diagnosis of thymoma in small biopsy specimens, ectopic thymoma, or metastatic thymoma; in the distinction between invasive thymoma and thymic carcinoma; and in the classification of thymomas.

Lymphangiomyomatosis and angiomyolipoma: closely related entities characterized by hamartomatous proliferation of HMB-45-positive smooth muscle.

Angiomyolipoma is a hamartomatous condition which can occur as a component of the tuberous sclerosis complex. Lymphangiomyomatosis, another hamartomatous lesion occurring predominantly in the lungs, has long been suspected to be related to angiomyolipoma and tuberous sclerosis because of occasional clinical associations. We undertook this study to provide further support for the close relationship between these two entities. Five cases of lymphangiomyomatosis and 20 case of angiomyolipoma were retrieved for histological review and immunohistochemical studies. The antibodies used were anti-muscle specific actin (HHF-35), anti-desmin (D33) and anti-melanoma (HMB-45). Lesions featuring smooth muscle proliferation were used as controls. The proliferated smooth muscle cells in both lymphangiomyomatosis and angiomyolipoma were much plumper and paler or even clear, when compared with the deeply eosinophilic cytoplasm of the normal spindly smooth muscle cells and those of leiomyomas. Their nuclei were round to oval and pale rather than elongated and dark. Cells with bizarre nuclei were commoner in angiomyolipoma (18/20 cases) than lymphangiomyomatosis (1/5). In 12 cases of angiomyolipoma there were foci indistinguishable from lymphangiomyomatosis, i.e. plump spindle cells arranged in short fascicles around ramifying endothelium-lined spaces. All five cases of lymphangiomyomatosis stained for muscle-specific actin, desmin and HMB-45. For angiomyolipomas, the positivity rates for these markers were: 20/20, 17/20 and 18/20, respectively, including one case that was negative for both desmin and HMB-45. The various smooth muscle proliferations and tumours selected as controls were uniformly HMB-45 negative. The distinctive cytological features, morphological overlap and immunophenotypic profile all support a close relationship between lymphangiomyomatosis and angiomyolipoma, which probably represent different morphological manifestations of hamartomatous proliferation of a peculiar form of HMB-45-positive smooth muscle.

Multiclefted nuclei. A helpful feature for identification of intermediate trophoblastic cells in uterine curetting specimens.

Intermediate trophoblast is a distinct form of trophoblast, the presence of which in uterine curettings is considered a reliable indicator of intrauterine pregnancy even in the absence of chorionic villi. However, the appearance of intermediate trophoblastic cells have not been described in sufficiently specific terms to permit their reliable identification, and distinction from decidual cells can be difficult. We have noticed for some time that the intermediate trophoblastic cells often show multiple deep clefts in the nuclei, and the present study was performed to address the issue of whether this nuclear feature is reliable for their identification. We reviewed 242 uterine curettings of intrauterine pregnancy, documented by the presence of chorionic villi, and were able to find a distinct population of cells with large, hyperchromatic, multiclefted nuclei scattered in the decidua in 88% of the cases. In most instances, these cells produced a characteristic variegated pattern that was readily recognizable at low magnification. Positive immunostaining for cytokeratin (CAM 5.2) in these isolated cells within the decidua confirmed their trophoblastic nature. In contrast, multiclefted nuclei were absent in the 51 negative control cases, which included decidualized endocervical polyps (40 cases), uterine curettings from patients with tubal pregnancy (10 cases), and endometriosis with decidual change (one case). We conclude that intermediate trophoblastic cells can usually be reliably identified in curettings of intrauterine pregnancy by their characteristic nuclear multiclefting.

Characterization of central actions of neuropeptide Y on food and water intake in rabbits.

The effects of a 36-amino acid peptide, neuropeptide Y (NPY), on feeding and drinking behaviors were studied in young and adult rabbits. Intraventricular injection of NPY to adult rabbits induced feeding and drinking in a dose-related manner. While the lowest doses tested (0.2 micrograms) was without effect, other doses (0.5 and 1 microgram) elicited feeding and drinking almost instantaneously. When 1, 5 and 10 microgram doses were injected into young rabbits, immediate increases in feeding and drinking were evident, but differences in the magnitude of responses among these dosages were significant only in water consumption. Unlike studies in rats, in these rabbits NPY elicited a more pronounced response in drinking than in feeding. The drinking response after NPY administration was not a consequence of food intake because it occurred in the absence of food. With ad lib feeding, the majority of enhanced food consumption was confined to the first 30-min after NPY injection; however, an increased motivation to eat was retained for at least 2 hr after NPY when food was withheld and then returned. These observations are consistent with specific stimulatory effects of NPY on food and water intake.

Dietary arginine deprivation and delayed puberty in the female rat.

Dietary arginine deprivation was found to delay puberty in the female rat. Physiological pinealectomy by exposing to constant light suggests this gland is not involved in this delay. Compensatory ovarian hypertrophy (COH) was used to test the hypothalamic sensitivity to negative steroid feedback. COH occurred in hemiovariectomized immature rats ad libitum fed the control or arginine-deficient diet but failed to occur in hemiovariectomized, underfed, growth-matched control rats, which suggests that feed restriction and arginine deficiency do not exert identical effects on the hypothalamic-pituitary-gonadal axis. Puberty, as defined by vaginal opening, first ovulation and the initiation of estrous cycles, was advanced by a week in the immature female rat fed a control diet after treatment with estradiol benzoate, 0.05 microgram/(100 g body weight X day) starting at 26 days of age. The time of first estrus and the first ovulation was not advanced in arginine-deficient rats by the same dosage of estrogen when administration began at 26 days of age. Treatment at an older age (40 or 54 days) or with a higher dosage [0.25 microgram/(100 g body weight X day)] at 26 days of age did advance puberty. The failure of estrogen to induce a vaginal cyclicity suggests an insufficient amount of endogenous estrogen to trigger a gonadotropin surge to cause the onset of puberty in the rat fed an arginine-deficient diet.

Dietary arginine and sexual maturation of the female rat.

The effects of dietary arginine deficiency on sexual maturation were examined in the female rat. Consumption of a diet devoid of arginine since weaning delayed the onset of puberty. Weight at vaginal opening and weight at first estrus were significantly greater in the arginine-deficient rat than in the control rat. Arginine deficiency appeared to have a specific effect on the development of reproductive function since puberty in arginine-deficient animals was delayed more than could be accounted for by impairment of growth. The specific effect of arginine deficiency was also indicated by reduced weights of the reproductive tissues after 15 weeks of experimental feeding when compared with the growth-matched controls. Sexual maturation was also examined in rats fed diets containing graded levels of arginine (1.12, 0.84, 0.56, 0.28 or 0%). Vaginal opening and first estrus were delayed by feeding diets containing 0.28 or 0% arginine. Uncoupling of these two events also occurred in the majority of the rats fed these two diets. In spite of normal timing of puberty, maturing rats fed a diet with 0.56 of 0.84% arginine had reduced ovarian weight and first ovulation rate when compared with rats fed the control diet (1.12% arginine). It is concluded that more than 0.84% dietary arginine was required for growing rats to support normal sexual maturation.

Effect of arginine deficiency on mammary gland development in the rat.

The effect of dietary arginine deficiency on rat mammary gland development was investigated in pregnant rats and ovariectomized nonpregnant rats. Consumption of an arginine-deficient diet from day 0 of pregnancy significantly reduced the total DNA and RNA content of the mammary gland by day 20. Pair-feeding studies revealed that reduced food intake associated with consumption of the arginine-deficient diet was not responsible for the altered nucleic acid content of the gland. Restriction of food intake by pair-feeding did reduce the wet weight of the mammary gland as well as its fat content when compared with ad libitum fed controls, but the degree of reduction did not fully account for the depressing effect on the wet weight and fat content observed when the arginine-deficient diet was fed. The ratio of RNA to DNA in the mammary tissues of rats fed the arginine-deficient diet indicated normal protein synthetic activity per cell. The extent of reduction in mammary nucleic acid content by arginine deficiency was similar for pregnant rats and ovariectomized nonpregnant rats treated with estrogen and progesterone. These studies show that dietary arginine is required for optimal mammary growth. The possible mechanism(s) by which arginine can modify mammary growth are discussed.

Arginine deficiency during gestation and lactation in the rat.

The effects of dietary arginine deprivation during gestation and lactation were studied in the rat. Placental development, as indicated by the total protein and RNA content, of rats fed a diet devoid of arginine was inferior to that of pair-fed controls. Fetal cerebral protein plus body weights were reduced by maternal arginine deprivation. Urinary excretion of orotic acid and citric acid was significantly increased in arginine-deficient rats throughout the gestational period. Pups nursed by arginine-deficient dams during lactational periods had smaller body and organ weights than those nursed by pair-fed control dams regardless of their prenatal nutritional states. Nursing performance of the dam was most affected by feeding arginine-free diet throughout gestation and lactation. Consumption of an arginine-free diet by the dam during gestation resulted in lower birth weight and higher perinatal mortality of the pups nursed. Deletion of arginine from the diet after parturition significantly reduced nursing performance after 18 days of lactation. The reduction in nursing performance caused by arginine deficiency during gestation could be corrected by replenishing arginine after parturition. Mammary gland development, as indicated by nucleic acid content, was reduced by either reducing dietary arginine or food intake during lactation. All these results provided evidence that dietary arginine is required during gestation and lactation for optimal reproductive response and nursing performance in rats.