Immunocytochemical analysis of p63 and 34ßE12 in fine needle aspiration cytology specimens for breast lesions: a potentially useful discriminatory marker between intraductal papilloma and ductal carcinoma in situ.

OBJECTIVE: We applied immunocytochemistry to fine needle aspiration (FNA) breast lesion slides in an attempt to enhance their objectivity and specificity. METHODS: We analysed 56 FNA specimens from patients with histologically confirmed breast lesions, using 34ßE12 and p63 antibodies. Immunostained slides were examined in terms of both solitary positive cells (within clusters and non-clusters) and positive clusters within a slide. RESULTS: Positive scores (≥2) for p63(+) cells and percentages of p63(+) clusters differed significantly (P < 0.001) between malignant (3 of 34; 9%) and benign (11 of 22; 50%) cases and varied between benign and malignant groups: intraductal papilloma (IDP) (2 of 8), other benign lesions (9 of 14), ductal carcinoma in situ (DCIS) (1 of 11) and invasive carcinoma (IC) (2 of 23). Similarly, 34ßE12 scores were higher in benign cases (IDP, 8 of 8; other benign, 9 of 14) than in malignant cases (DCIS, 1 of 11; IC, 3 of 23). As well as a significant difference between benign and malignant cases (17 of 22, 77% versus 4 of 34, 12%; P < 0.001), the percentage of 34ßE12(+) clusters was significantly higher in IDP compared with DCIS (P = 0.001). CONCLUSIONS: The immunostaining of FNA breast specimens for p63 and 34ßE12 may help in difficult diagnoses.

Increased expression of proapoptotic BMCC1, a novel gene with the BNIP2 and Cdc42GAP homology (BCH) domain, is associated with favorable prognosis in human neuroblastomas.

Differential screening of the genes obtained from cDNA libraries of primary neuroblastomas (NBLs) between the favorable and unfavorable subsets has identified a novel gene BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1). Its 350 kDa protein product possessed a Bcl2-/adenovirus E1B nineteen kDa-interacting protein 2 (BNIP2) and Cdc42GAP homology domain in the COOH-terminus in addition to P-loop and a coiled-coil region near the NH2-terminus. High levels of BMCC1 expression were detected in the human nervous system as well as spinal cord, brain and dorsal root ganglion in mouse embryo. The immunohistochemical study revealed that BMCC1 was positively stained in the cytoplasm of favorable NBL cells but not in unfavorable ones with MYCN amplification. The quantitative real-time reverse transcription-PCR using 98 primary NBLs showed that high expression of BMCC1 was a significant indicator of favorable NBL. In primary culture of newborn mice superior cervical ganglion (SCG) neurons, mBMCC1 expression was downregulated after nerve growth factor (NGF)-induced differentiation, and upregulated during the NGF-depletion-induced apoptosis. Furthermore, the proapoptotic function of BMCC1 was also suggested by increased expression in CHP134 NBL cells undergoing apoptosis after treatment with retinoic acid, and by an enhanced apoptosis after depletion of NGF in the SCG neurons obtained from newborn mice transgenic with BMCC1 in primary culture. Thus, BMCC1 is a new member of prognostic factors for NBL and may play an important role in regulating differentiation, survival and aggressiveness of the tumor cells.

Assessment of hemorrhage in pituitary macroadenoma by T2*-weighted gradient-echo MR imaging.

BACKGROUND AND PURPOSE: Intratumoral hemorrhage occurs frequently in pituitary macroadenoma and manifests as pituitary apoplexy and recent or old silent hemorrhage. T2*-weighted gradient-echo (GE) MR imaging is the most sensitive sequence for the detection of acute and old intracranial hemorrhage. T2*-weighted GE MR imaging was used to investigate intratumoral hemorrhage in pituitary macroadenomas. MATERIALS AND METHODS: Twenty-five consecutive patients who underwent total or subtotal resection of pituitary macroadenoma with heights from 17 to 53 mm, including 1 patient with classic pituitary apoplexy, underwent MR imaging before surgery, including T2*-weighted GE MR imaging. For histologic assessment of the hemorrhage in whole surgical specimens, we used hematoxylin-eosin staining. RESULTS: T2*-weighted GE MR imaging detected various types of dark lesions, such as "rim," "mass," "spot," and "diffuse" and combinations, indicating clinical and subclinical intratumoral hemorrhage in 12 of the 25 patients. The presence of intratumoral dark lesions on T2*-weighted GE MR imaging correlated significantly with the hemorrhagic findings on T1- and T2-weighted MR imaging (P < .02 and <.01, respectively), and the surgical and histologic hemorrhagic findings (P < .001 and <.001, respectively). CONCLUSION: T2*-weighted GE MR imaging could detect intratumoral hemorrhage in pituitary adenomas as various dark appearances. Therefore, this technique might be useful for the assessment of recent and old intratumoral hemorrhagic events in patients with pituitary macroadenomas.

Requirement of thyrotropin-releasing hormone for the postnatal functions of pituitary thyrotrophs: ontogeny study of congenital tertiary hypothyroidism in mice.

We recently reported that TRH-deficient mice showed characteristic tertiary hypothyroidism. In the present study, we investigated how this tertiary hypothyroidism occurred particularly in pre- and postnatal stages. Immunohistochemical analysis revealed a number of TSH-immunopositive cells in the TRH-/- pituitary on embryonic day 17.5 and at birth. The mutant pituitary at birth in pups born from TRH-deficient dams also showed no apparent morphological changes, indicating no requirement of either maternal or embryonic TRH for the development of pituitary thyrotrophs. In contrast, apparent decreases in number and level of staining of TSH-immunopositive cells were observed after postnatal day 10 in mutant pituitary. Similar decreases were observed in the 8-week-old mutant pituitary, while no apparent changes were observed in other pituitary hormone-producing cells, and prolonged TRH administration completely reversed this effect. Consistent with these morphological results, TRH-/- mice showed normal thyroid hormone levels at birth, but the subsequent postnatal increase was depressed, resulting in hypothyroidism. As expected, TSH content in the TRH-/- pituitary showed a marked reduction to only 40% of that in the wild type. Despite hypothyroidism in the mutant mice, both the pituitary TSHbeta and alpha mRNA levels were lower than those of the wild-type pituitary. These phenotypic changes were specific to the pituitary thyrotrophs. These findings indicated that 1) TRH is essential only for the postnatal maintenance of the normal function of pituitary thyrotrophs, including the normal feedback regulation of the TSH gene by thyroid hormone; 2) neither maternal nor embryonic TRH is required for normal development of the fetal pituitary thyrotroph; and 3) TRH-deficient mice do not exhibit hypothyroidism at birth. Moreover, reflecting its name, TRH has more critical effects on the pituitary thyrotrophs than on other pituitary hormone-producing cells.

Dysplastic ganglioneurocytoma with increased glucose metabolism: a heterotopia with unique histopathology.

A 1 year and 7 month old boy was incidentally found to have an intracranial mass lesion at the frontal base. The mass was 45 x 54 x 47 mm in size, contained a calcification, a few small cysts, and extended downward to the sphenoid sinus and upper pharynx. The signal intensity of the lesion on magnetic resonance imaging was iso-high on T1-weighted images, and slightly high on T2-weighted images, and it did not enhance with gadolinium injection. Although there was no obvious mass effect, 18F-fluorode-oxyglucose positron-emission tomography demonstrated increased uptake, and a surgical resection was performed suspecting a neoplastic lesion. Histologically, the lesion consisted of small to large anomalous neurons and glial cells but lacked normal cortical architecture. Cellularity was high in some portion with MIB-1 labeling index of 2%, but there was no cellular atypia suggestive of neoplasm. Therefore, this lesion was considered to be a dysplasia that does not fit into the previously described entity. We suggest this lesion would be better described as dysplastic ganglioneurocytoma.

A monoclonal antibody that specifically recognizes a novel mitochondrial protein of human astrocytes.

We established a monoclonal antibody to human astrocytes using a human glial cell-rich fraction as the immunogen. The antibody, named PRAS-4, specifically labeled populations of astrocytes in a fine granular manner immunohistochemically. In formalin-fixed, paraffin-embedded tissue sections, PRAS-4-positive astrocytes were extensively distributed in the gray matter of the central nervous system, namely the cerebral cortex, basal ganglia, diencephalon, midbrain, various nuclei of the brain stem, cerebellar cortex and nuclei, and spinal cord. In the white matter, a few positive astrocytes were located mostly in the perivascular area. The reaction was lost after protease digestion and it resisted periodic acid, suggesting that the epitope is of a protein. The molecular weight of the antigen was estimated as 62 kDa. Ultrastructurally, the immunoreaction was localized on the outer and inner membranes of astrocytic mitochondria, and unlabeled mitochondria coexisted in the same cells. Extra-mitochondrial regions were not stained. PRAS-4 preferentially labeled astrocytes of the protoplasmic type, and may be applicable to studies on the development, specific functions and neoplasms of astrocytes.

Oligodendroglial microtubular tangles in olivopontocerebellar atrophy.

Oligodendroglial microtubular tangles (OMT), a distinctive oligodendroglial change, was observed in seven of eight cases of olivopontocerebellar atrophy (OPCA). This change was a well-defined glassy cytoplasmic structure showing intense argyrophilia. OMT were distributed in the brain stem, cerebellum, and basal ganglia where severe neurodegenerative changes were consistently observed. In 45 control cases, no OMT were found regardless of the presence or absence of neurological disorders. The OMT were immunostained by anti-tubulin antibodies, but no other antibodies reacted with them. Each OMT consisted of a meshwork of randomly oriented fibrils studded with granular and fuzzy material. The fibrillary elements were between 20 and 30 nm in diameter. It is suggested that OMT are primarily composed of altered microtubules, and are related to the neurodegenerative process of OPCA.

Expression of interleukin-1beta mRNA and protein in human gliomas assessed by RT-PCR and immunohistochemistry.

To characterize the expression and localization of interleukin (IL)-1beta in human gliomas, both reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were used on surgically excised human gliomas, human malignant glioma xenografts, and human glioblastoma cell lines. The RT-PCR products for IL-1beta mRNA were quantified by computerized image analysis. IL-1beta mRNA was detectable in 30 out of 35 (86%) surgically resected gliomas. An abundant expression of IL-1beta mRNA was often found in the glioblastomas, anaplastic astrocytomas, and pilocytic astrocytomas, but not in other types of gliomas. Quantitatively, in both the grade 2 astrocytomas and the oligodendrogliomas, the IL-1beta mRNA levels were significantly (p < 0.05) lower than those of the grade 3/4 astrocytomas. Immunohistochemically, IL-1beta was localized in the pleomorphic tumor cells of the astrocytic tumors and in macrophages. In contrast to the astrocytic tumors, low and high grade oligodendrogliomas showed no or little expression of IL-1beta antigen. IL-1beta was present less frequently than IL-1alpha and IL-1 receptor type 1 in 4 malignant gliomas transplanted into nude mice by RT-PCR. All 2 cell lines showed IL-1beta expression at both the mRNA and protein levels. It is concluded that in human gliomas, both high-grade astrocytomas and pilocytic astrocytomas often express high IL-1beta production, and that IL-1beta is mainly localized in astrocytic tumor cells and macrophages.

Swollen cortical neurons in Creutzfeldt-Jakob disease contain a phosphorylated neurofilament epitope.

The distribution of swollen neurons and the presence of a phosphorylated neurofilament protein (NFP) epitope in these cells were studied in six cases of Creutzfeldt-Jakob disease (CJD). Swollen neurons are widely distributed in the cerebral cortex and are most abundant in the cingulate and parahippocampal gyri. They are more numerous in the panencephalopathic type of CJD than in the subacute spongiform encephalopathic type. A phosphorylated epitope of NFP was detected in the perikarya of swollen neurons by an immunocytochemical method using a series of monoclonal antibodies that distinguish phosphorylated and nonphosphorylated epitopes of NFP. This abnormal distribution of phosphorylated NFP epitopes indicates that the process of NFP phosphorylation is altered in neurons affected by CJD. This investigation, in accordance with previous studies, suggests that the abnormal post-translational modification of the neurofilament may play an important role in the pathogenesis of several neurodegenerative disorders.

Infantile neuroaxonal dystrophy: perinatal onset with symptoms of diencephalic syndrome.

In a neonatal case of infantile neuroaxonal dystrophy, there was emaciation, nystagmus, and endocrinologic disorder suggesting the diencephalic syndrome. At autopsy, spheroid bodies were widely disseminated, particularly in the hypothalamus, infundibulum, and neurohypophysis. The pathologic process may have started in utero.

Massive apoptosis detected by in situ DNA nick end labeling in neuroblastoma.

To seek evidence that tumor regression in neuroblastoma might result from massive apoptosis, we investigated tumor cell death in 39 neuroblastomas. Characteristic histologic features of apoptosis, condensed nuclear fragments and eosinophilic cytoplasm, were observed in all specimens. A ladder of DNA fragments induced by apoptosis was demonstrated by means of DNA agarose gel electrophoresis in 18 of the 19 tumors examined. In situ DNA nick end labeling (TUNEL) stained the nuclei with DNA fragmentation in 16 of 39 neuroblastomas. The TUNEL -positive cells were distributed in a scattered fashion in 10 tumors. In the remaining six tumors, they were densely located around nonviable areas of calcifications, where karyorrhectic or pyknotic cells were frequently observed. Five of six patients with such tumors were under 12 months of age, but there was no significant difference between the two groups in the patient age, origin of the primary lesion, or tumor stage. Biological features, including histology. DNA ploidy, and N-myc amplification, were not significantly different . Double fluorescent staining for bcl-2 oncoprotein and TUNEL showed that bcl-2 oncoprotein was expressed in the cytoplasm of tumor cells that were negative for TUNEL staining. This accumulated evidence suggests that massive apoptosis of tumor cells occurs in some neuroblastomas and may be related to tumor regression, whereas inhibition of apoptosis by bcl-2 oncoprotein expression might be associated with the tumorigenesis of neuroblastomas, as reported in our previous study.

A case of cimetidine-induced acute tubulointerstitial nephritis associated with antineutrophil cytoplasmic antibody.

We present a case of acute tubulointerstitial nephritis (ATIN) that developed in a 63-year-old man who had been taking cimetidine for treatment of a gastric ulcer. The constellation of clinical, laboratory, and histopathologic findings suggested drug-induced ATIN. Of interest, the patient had antineutrophil cytoplasmic antibody (ANCA) in his sera, reactive with myeloperoxidase, elastase, and lactoferrin. Prominent renal histological features included marked plasmacyte infiltration into the renal interstitium. Withdrawal of cimetidine resulted in complete resolution of renal findings, and the titers of ANCA concomitantly declined. Thus, cimetidine may have played a causative role in the development of ANCA-associated ATIN.

A neuronal fibrillary inclusion shares the epitope of p24 of human immunodeficiency virus.

A monoclonal antibody to p24 of human immunodeficiency virus (HIV) was demonstrated to react with degenerated neurons immunohistochemically. In a case of Pick's disease, the neurons of the dentate gyrus were labeled with the antibody, while normal-looking ones and glial components were negative. The positive structures showed argyrophilia by the Bielschowsky and Bodian methods. Antibodies to ubiquitin, paired helical filament and tau did not react with the inclusions. Ultrastructurally, they consisted of skeins of fuzzy-surfaced fibrils with a diameter of 15 nm. In cases of Alzheimer type dementia, thread-like positive profiles were observed in some neurofibrillary tangle-bearing neurons. These studies revealed the cross-reactivity of the anti-HIV p24 antibody to unknown types of neuronal inclusions and provide a new aspect for research into neurodegenerative disorders.

Solitary fibrous tumor of the orbit with extraorbital extension: case report.

OBJECTIVE AND IMPORTANCE: Solitary fibrous tumors (SFTs) are rare tumors of mesenchymal origin that typically arise in the pleura. Only 24 cases of SFTs in the orbit have been reported, all located within the orbit and generally with a benign course. We report the first case of an orbital SFT with extraorbital extension and short-term regrowth. CLINICAL PRESENTATION: A 54-year-old man presented with proptosis and double vision that had persisted for 7 months. The tumor extended from the right extraconal inferolateral orbit to the extradural middle cranial fossa and cavernous sinus, via the superior orbital fissure, on magnetic resonance imaging scans. Positron emission tomography with [(18)F]fluorodeoxyglucose demonstrated faint uptake in the orbital portion. INTERVENTION: Resection of the tumor was performed twice, because of short-term regrowth of the residual tumor in the orbit. The histological diagnosis was a SFT. The MIB-1 labeling index was 7% and the mitotic count was 5 mitotic figures/10 high-power fields at the time of the second operation. These findings indicate the malignant nature of the tumor. CONCLUSION: The natural history of SFTs of the orbit remains unclear, and the importance of careful and continued follow-up monitoring of the tumor should be emphasized.

Recurrent anaplastic ganglioglioma: pathological characterization of tumor cells. Case report.

A total resection of a left frontal lobe tumor in a 26-year-old man revealed differentiated ganglioglioma with small foci of atypical glial cells exhibiting mild atypia. Six and one-half years later, a large, well-demarcated tumor recurred; at that time, histological analysis revealed both typical ganglioglioma and highly cellular anaplastic areas, the latter predominating. Although the patient subsequently underwent total and subtotal resections, radiation therapy, and chemotherapy, tumors continued to recur at progressively shorter intervals and he died at the age of 35 years. Biopsies of tissue obtained at the last three resections and the autopsy revealed only anaplastic tumor cells. Routine histological examinations indicated that these tumors were uniformly composed of undifferentiated cells. However, pathological studies using immunohistochemical analysis, electron microscopy, and immunoblot analysis demonstrated that a small number of recurrent anaplastic cells had astrocytic features. Results of Ki-67/MIB-1 labeling and silver nucleolar organizer region counts for those cells were high for glial tumors. A retrospective study of the initial tumor showed slightly high MIB-1 labeling for atypical glial cells. This case is characterized by pathological findings of recurrent tumors that correspond to an unusual form of malignant glioma exhibiting slight astrocytic differentiation. The present case suggests that a longer follow-up period ( > 5 years) is necessary in cases of ganglioglioma with mild atypia and that careful examinations, including proliferating potential analysis of initial tumor cells, could be important for the diagnosis and treatment of ganglioglioma.

Intranuclear inclusions of meningioma associated with abnormal cytoskeletal protein expression.

We describe a case of meningothelial meningioma with a large number of intranuclear inclusions. Morphologically, these are divided into cytoplasmic inclusions and nuclear vacuoles. The cytoplasmic inclusion has a limiting membrane with cell organelles and filaments. Inclusions of this type are generally eosinophilic, like the cytoplasm. However, there are many inclusions that are more eosinophilic than the cytoplasm or that have a ground-glass appearance. Some of them may contain fine or coarse granules. On the other hand, the nuclear vacuole lacks a limiting membrane and appears empty. In most of the inclusions of this type, there is a faintly basophilic substance in the margin. Generally, the cytoplasmic inclusions are as immunopositive as cytoplasm with vimentin, but some of these cytoplasmic inclusions are more reactive. Under the electron microscope, abnormal aggregation of intermediate filaments is recognized in the cytoplasmic inclusions. It is considered that a strong reaction of cytoplasmic inclusions with vimentin immunostaining is due to abnormal aggregation of intermediate filaments. The present study distinctly demonstrates abnormal localization of intermediate filaments in the cytoplasmic inclusions, and it is suggested that the cytoskeleton participates in the evolution of the cytoplasmic inclusions.

Effects of recombinant human endostatin on a human neuroblastoma xenograft.

New antitumor agents must be added to the current neuroblastoma treatment regimens to improve the clinical results. We investigated whether recombinant human endostatin (rhEndostatin), an antiangiogenic agent, is effective against human neuroblastoma in the human neuroblastoma xenograft model designated TNB9. When tumors on the back of nude mice grew to a weight of 90-95 mg, rhEndostatin 10 mg/kg/day was administered subcutaneously every day for 10 consecutive days. Mean relative tumor weight in mice administered rhEndostatin (n=5) was significantly less than that in controls (n=12) on days 2, 4, and 6 after the start of administration (p<0.01 on day 2, p<0.05 on days 4 and 6), and regression of tumor growth (TRW<1.0) was marked on day 2. The maximum inhibition rate (MIR) by rhEndostatin was 46.4%, indicating inefficacy, but it may not be appropriate to apply Battelle Columbus Laboratories criteria to this experimental model because rhEndostatin is a protein. After day 8, tumors in the experimental group increased in weight and were not statistically significantly different from those in controls. Recombinant human endostatin was used in tumors in the arterial system of the mouse in this experiment because eventually rhEndostatin, not recombinant mouse endostatin, may be used to treat advanced neuroblastoma in the clinical setting. The results show that there is little cross-reactivity of rhEndostatin with the human and mouse models and indicate that rhEndostatin could become an effective agent for the treatment of human neuroblastoma.

Expression of non-glial intermediate filament proteins in gliomas.

Non-glial intermediate filament (IMF) proteins, as well as glial fibrillary acidic protein (GFAP) and vimentin, were studied by immunohistochemistry in 24 gliomas including low grade astrocytoma, pleomorphic xanthoastrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, ependymoma and ependymoblastoma, which were fixed in ethanol and embedded in paraffin. Cytoskeletal elements isolated from two glioblastomas were examined with immunoblot analysis. All tumors had GFAP-positive neoplastic cells and vimentin was also found in all the tumors except one oligodendroglioma. Twenty-three gliomas were immunostained with anti-desmin polyclonal antibody (DM-P), but anti-desmin monoclonal antibody reacted to only one glioblastoma. DM-P might crossreact with GFAP and vimentin. Cytokeratin expression was investigated with six antibodies. Twenty gliomas (83%) were positive for the antibody against epidermal keratin (CK-SE), however positive immunoreactivity varied from 58 to 8% with other cytokeratin antibodies. With the Western blot method, CK-SE had protein bands at 53 and 60-66 kDa. Neurofilament was expressed in one pleomorphic xanthoastrocytoma, one anaplastic astrocytoma, one glioblastoma and one ependymoblastoma. Expression of nonglial IMF proteins were observed in 21 tumors (88%), and coexpression of 4 or 5 classes of IMF proteins in 3 tumors (13%). We conclude that, in addition to GFAP and vimentin, gliomas express several types of non-glial IMF proteins.

Intracranial malignant fibrous histiocytoma: characterization of GFAP-positive cells in the tumor.

A case of malignant fibrous histiocytoma (MFH) arising in the cerebellopontine angle of a 57-year-old woman was reported. The immunohistochemical and electron microscopic findings indicated distinct fibrohistiocytic nature and myofibroblastic differentiation of this peculiar tumor. Immunohistochemistry disclosed that the tumor cells expressed monocyte/macrophage markers including CD-68, MAC 387, and alpha-1-antichymotrypsin. In addition, the neoplasm contained scattered glial fibrillary acidic protein (GFAP)-positive cells which made small nests in some areas. These GFAP-positive cells had bundles of densely packed intermediate filaments and scanty organellae in the cytoplasm, as demonstrated by an immunostained-semithin and serial-ultrathin section method. By immunostaining of MIB-1 and GFAP, and silver nucleolar organizer region (AgNOR) impregnation on serial sections, the GFAP-positive cells were not labeled by MIB-1 and their AgNOR counts averaged 1.13/nucleus. Thus, these GFAP-positive cells seem to have lower proliferating activity than neoplastic astrocytes. It is concluded that they may be nonneoplastic astrocytic cells involved by MFH.