Cytological diagnostic clues in poorly differentiated squamous cell carcinomas of the breast: Streaming arrangement, necrotic background, nucleolar enlargement and cannibalism of cancer cells.

OBJECTIVE: Squamous cell carcinoma (SCC) is a rare histological type of breast cancer. The cytological diagnosis of non-keratinising, poorly differentiated SCC is often difficult, and distinguishing it from invasive ductal carcinoma or apocrine carcinoma (AC) is especially challenging. We aimed to define the diagnostic cytological features of poorly differentiated SCC of the breast. METHODS: We studied the cytological findings of poorly differentiated SCC (n=10) and compared them to those of IDC (n=15) and AC (n=14). The following six cytological features were evaluated: streaming arrangement, nucleolar enlargement, dense nuclei, cannibalism, atypical keratinocytes and necrotic background. RESULTS: SCC exhibited significantly higher frequencies of streaming arrangement (70% vs 6.7%, P=.002), nucleolar enlargement (80% vs 27%, P=.02), and necrotic background (80% vs 36%, P=.002) than invasive ductal carcinoma. The detection of two or three of these features yielded a higher sensitivity (80%) and specificity (93%) for the diagnosis of SCC. Streaming arrangement (70% vs 0%, P<.001), cannibalism (60% vs 0%, P=.002), and a necrotic background (80% vs 36%, P=.047) were all significantly more frequent in SCC than in AC. When distinguishing SCC from AC, the presence of two or three of these features yielded a high sensitivity (80%) and specificity (100%). CONCLUSIONS: Cytological features such as a streaming arrangement, a necrotic background, nucleolar enlargement and cannibalism are useful indicators for the diagnosis of SCC of the breast. As such, greater attention should be paid to these morphological features in daily clinical practice.

Expression of oestrogen receptor-beta in apocrine carcinomas of the breast.

AIMS: Apocrine carcinoma of the breast seldom expresses oestrogen receptors (ER) or progesterone receptors (PR), but frequently expresses androgen receptors (AR). Because of this unusual hormone receptor status, it has been suggested that oestrogens have a less important role in the pathogenesis of apocrine carcinoma. The ER status of apocrine carcinoma has been studied for one kind of ER, the classic receptor now named ER-alpha; however, the status of ER-beta, a secondary oestrogen receptor, has not been examined systematically in apocrine carcinoma. The aim was to study ER-beta status in apocrine carcinoma. METHODS AND RESULTS: The expression of ER-beta was examined immunohistochemically in 48 apocrine carcinomas and compared with clinicopathological factors and ER-alpha, PR and AR status. ER-beta positivity was observed in 35 cases (73%), regardless of any clinicopathological factors or the status of other receptors. The results of ER-beta mRNA analysis supported the immunohistochemical results. CONCLUSIONS: The significance of oestrogens in apocrine carcinoma should not be dismissed at present when the role of ER-beta remains to be determined. Studying the action of oestrogen or antioestrogen in apocrine carcinoma may reveal a role for ER-beta independent of ER-alpha and raise the potential of hormonal therapy for these tumours.

Expression of GCDFP-15 and AR decreases in larger or node-positive apocrine carcinomas of the breast.

AIMS: Apocrine carcinoma of the breast is typically, though not always, positive for gross cystic disease fluid protein-15 (GCDFP-15). In order to clarify the clinical significance of GCDFP-15 in apocrine carcinomas, GCDFP-15 expression was examined in apocrine carcinomas of different stages and compared with clinicopathological factors. Apocrine lesions reportedly exhibit an unusual immunohistochemical status, expressing androgen receptors (AR) instead of oestrogen receptors (ER), progesterone receptors (PR), or bcl-2. Their expression was also examined. METHODS AND RESULTS: Fifty-two apocrine carcinomas were examined immunohistochemically. Thirty-nine (75%) and 29 (56%) were positive for GCDFP-15 and AR, respectively. GCDFP-15 positivity was significantly lower in infiltrating carcinomas than intraductal carcinomas (P = 0.0111). In infiltrating carcinomas, GCDFP-15 positivity was significantly low in tumours > or = 15 mm (P = 0.0005) and node-positive tumours (P = 0.0004). Similar phenomena were observed for AR. Rare cases were positive for ER (3.8%), PR (5.8%), and bcl-2 (1.9%). CONCLUSIONS: GCDFP-15 positivity is transient and should not be considered a definitive marker of apocrine carcinomas. Cases which have apocrine features but lack GCDFP-15 expression should rather be considered as advanced apocrine carcinomas. ER/PR/bcl-2 negativity will sometimes be helpful to confirm the diagnosis of apocrine carcinoma, because it is more consistent than GCDFP-15/AR positivity.

Breast carcinoma in women over the age of 85: distinct histological pattern and androgen, oestrogen, and progesterone receptor status.

AIMS: The pathogenesis of breast carcinoma in very elderly women is of interest, because oestrogen levels are likely to be extremely low during the development of the disease. In an effort to understand the pathogenesis of breast carcinoma in these women, this study was undertaken to compare the histological patterns and hormone receptor status of breast carcinomas arising in very elderly and younger women. METHODS AND RESULTS: Thirty-seven breast carcinomas from women over the age of 85 years at the time of their operation were examined histologically and compared with those from a large group of premenopausal women. The proportions of mucinous carcinoma and apocrine carcinoma were significantly greater in older women. The expression of steroid hormone receptors was studied immunohistochemically. Androgen receptor-positive carcinomas were significantly more frequent among older women, whereas progesterone receptor-positive carcinomas were significantly less frequent. There was no statistically significant difference in oestrogen receptor-alpha or -beta expression between the tumours from both groups. CONCLUSION: Breast carcinomas in women over the age of 85 years have a different morphological spectrum from carcinomas in younger age groups and may have different pathogenesis mechanisms that may be more dependent on androgen and androgen receptor interaction. Differences from the results of the other studies are discussed.

[The examination of usefulness of the disposable pump in case of cancer pain relaxation and the economic problem].

Recently, an increasing number of cancer patients being taken care of at home has been able to use morphine to treat their pain by themselves. The most suitable administration method for individual patients-oral, intravenous, subcutaneous or depository--is being investigated. When oral intake becomes difficult, the subcutaneous via of administration is best option because it is the less dangerous and easier to use compared with the other two options. These are also thought to be less useful because it is difficult to judge the exact dosage. The use of pumps might be an economic problem to some patients. We will examine this problem.

Deficiency of the macrophage migration inhibitory factor gene has no significant effect on endotoxaemia.

By targeted disruption of the MIF gene, we have established a mouse strain deficient in macrophage (Mphi) migration inhibitory factor (MIF). Despite previous reports indicating an essential role of MIF in endotoxaemia, an injection of lipopolysaccharide (LPS) into the MIF-deficient mice (maintained under specific pathogen-free conditions) caused shock. No significant difference was detected between the MIF-deficient mutant and normal mice in susceptibility to LPS for endotoxaemia or tumour necrosis factor-alpha (TNF-alpha) formation upon LPS injection. Peritoneal Mphi from the two strains produced TNF-alpha in response to LPS with similar dose responses. Dexamethasone suppressed the LPS-induced TNF-alpha response of Mphi, but no difference was detected between the Mphi from the two strains. These results suggest that endogenous MIF has no significant effect on the LPS-induced TNF-alpha production and no effect on suppression of the response by glucocorticoids. Thus, MIF is not crucial for LPS-induced immune responses leading to shock.

Immunosuppressive activities of recombinant glycosylation-inhibiting factor mutants.

We have shown previously that glycosylation-inhibiting factor (GIF) in culture supernatants of suppressor T cell (Ts) hybridomas had bioactivity, while the same cells contained a substantial quantity of inactive GIF in cytosol. Mass-spectrometric analysis of GIF in the culture supernatant and cytosol of a Ts hybridoma provided direct evidence that GIF protein was posttranslationally modified in the Ts cells, and that the GIF bioactivity is associated with the posttranslationally modified species. Assuming that conformational changes induced by the posttranslational modifications are responsible for generation of bioactivity, we constructed cysteine mutants of human rGIF (rhGIF) in which cysteine at position 57, 60, or 81 was replaced with Ala, and the mutants were expressed in Escherichia coli. Replacement of Cys57 or Cys60 with Ala resulted in generation of bioactivity, while replacement of Cys81 with Ala failed to do so. It was also found that replacement of Cys57 with Ala and carboxymethylation of a sulfhydryl group in Cys60 synergistically increased the GIF bioactivity of the GIF derivatives. A mutated GIF protein, in which Cys57 and Asn106 in the rhGIF were replaced with Ala and Ser, respectively, had immunosuppressive effects on the IgE and IgG1 Ab responses of BDF1 mice to DNP-OVA, while wild-type rhGIF did not. Evidence was obtained that the mutated GIF suppressed Ag priming of Th cells for the Ab responses and proliferative response.

Phosphorylation of retinoblastoma protein at apoptotic cell death in rat neuroblastoma B50 cells.

Phosphorylation of the retinoblastoma protein (RB) was observed during apoptosis of B50 neuroblastoma cells following induction by dibutyryl cAMP, after differentiation into neurons, or by cycloheximide during proliferation. A weak but distinct increase in a RB and histone H1 kinase activity was detected at the time of RB phosphorylation. However, the RB kinase appeared to correspond to neither p34cdc2 kinase, CDK2 nor CDK5 because it was not inhibited by butyrolactone I, an inhibitor for them. Expression of CDK4 and 6 along with several cyclins also did not coincide with the appearance of phosphorylated RB in the apoptotic process.

Perilla oil prevents the excessive growth of visceral adipose tissue in rats by down-regulating adipocyte differentiation.

We examined the effect of dietary oils with different fatty acid compositions on the growth of visceral adipose tissue in rats. Rats were fed for 4 mo starting at weaning a basal diet containing (12 g/100 g diet) perilla oil rich in (n-3) polyunsaturated fatty acids (PUFA), safflower oil rich in (n-6) PUFA, olive oil rich in monounsaturated fatty acid, or beef tallow rich in saturated fatty acids. The amount of food consumed and body weight gain did not differ among the four dietary groups. The weight of the epididymal fat pad and the serum triglyceride concentration in perilla oil-fed rats were significantly lower (P < 0.05) than those of olive oil- and beef tallow-fed groups. The product of [(volume of individual adipocytes) x (number of adipocytes in epididymal fat pad)], which presumably represents total adipocyte volume in the fat pad, was significantly lower (P < 0.05) in perilla oil-fed rats than in beef tallow- and olive oil-fed groups. Expression of the late genes of adipocyte differentiation, peroxisome proliferator-activated receptor alpha, adipocyte P2 and adipsin, was significantly (P < 0. 05) down-regulated in epididymal fat tissue of rats that had been fed perilla oil rather than beef tallow or olive oil, whereas expression of the early gene, lipoprotein lipase, was not significantly affected. Greater levels (P < 0.05) of (n-3) PUFA in the membrane phospholipid fraction of the fat tissue were observed in perilla oil-fed rats than in the other dietary groups. These results suggest that perilla oil or (n-3) PUFA prevents excessive growth of adipose tissue in rats at least in part by suppressing the late phase of adipocyte differentiation.

Two types of apoptotic cell death of rat central nervous system-derived neuroblastoma B50 and B104 cells: apoptosis induced during proliferation and after differentiation.

We describe here two types of apoptotic cell death observed in the rat CNS-derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 mM DBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatases.

A phage display system for detection of T cell receptor-antigen interactions.

The process of T cell recognition involves a complex set of interactions between the various components of the TCR/MHC/peptide trimolecular complex. We have developed a system for exploring the specific binding interactions contributed by the constituent subunits of TCR complexes for components of their ligands. We utilized an M13 phage display system, designed for multivalent receptor display, to explore specific binding interactions between various TCR alpha chains and specific antigen in the absence of MHC. The multivalent TCR-phage display system was sensitive enough to reveal some TCR alpha chains capable of binding directly to antigen with the same fine specificity shown by the MHC-restricted T cells from which the alpha chains were derived. Cross-specificity analysis using two antigen-binding TCR alpha chains derived from T cells with different polypeptide antigen specificities confirmed the fidelity of this binding. In mixtures of antigen-binding and non-binding TCR alpha-displaying phage, specific selection was achieved at a starting frequency of 1/1000, suggesting that this system can be employed for selection and analysis of TCR-displaying phage libraries. While the binding specificities exhibited by these TCRs are unusual, they provide a novel perspective from which to study the specific binding interactions that constitute TCR antigen binding.

Molecular cloning of a novel actin-binding protein, p57, with a WD repeat and a leucine zipper motif.

A 57 kDa protein (p57) was obtained during the study on phosphatidylinositol-specific phospholipase C. Its cDNA was isolated from calf spleen and human leukemia cell line HL60 libraries and cloned. In the primary structures of p57, they have two unique amino acid sequence motifs, a WD repeat and a leucine zipper motif. Furthermore, p57 shared sequence similarity (40%) with coronin, an actin-binding protein responsible for chemotaxis, cell motility, and cytokinesis of Dictyostelium discoideum, which has only the WD repeat. p57 also showed an actin-binding activity and was mainly expressed in immune tissues. From these results, we conclude that p57 is a coronin-like novel actin-binding protein in mammalian cells but may also have a different function from coronin.

Electron-microscopic and immunohistochemical study of beta-2-microglobulin-related amyloidosis.

beta 2-Microglobulin (beta 2-MG)-related amyloidosis has been reported as a complication in long-term hemodialysis patients. We observed beta 2-MG amyloid deposits in synovial sheaths, bone cysts and gastric mucosa. They showed unique ultrastructural features, that is bundles or nodules consisting of curved or linear amyloid fibrils, associated with various cell reactions. The electron-microscopic histochemical study showed that they strongly stained with periodic acid-silver methenamine stain. A similar phenomenon was noticed in the spicules or bundles of amyloid fibrils in primary and secondary renal amyloidosis. With the cationic reagent toluidine blue 0, proteoglycan-like structures were observed around amyloid bundles and nodules, but not on each fibrils. Based on these results, we postulate that there is a close relationship between ultrastructural features and histochemical characteristics in beta 2-MG amyloid fibrils.

Beta 2-microglobulin: a new form of amyloid protein associated with chronic hemodialysis.

Carpal tunnel syndrome (CTS) has been associated with amyloid deposits and is now regarded as a major complication in chronic hemodialysis patients. While this new syndrome has been receiving increasing attention, its etiology has not been clarified. We have isolated amyloid fibrils from amyloid laden tissues inside the carpal tunnel in four different hemodialysis patients with CTS. After solubilization in guanidine HCl, a significant amount of the protein was located in a homogeneous, low molecular weight fraction. Each protein was found to be identical to beta 2-microglobulin with regard to its molecular weight of 11,000 on SDS-PAGE, amino acid composition and N-terminal amino acids: Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu. In direct immunofluorescent study, anti-beta 2-microglobulin did react positively with amyloid deposits. These results demonstrate that the amyloid associated with chronic hemodialysis contains as major component a new form of amyloid fibril protein that is homologous to beta 2-microglobulin. It is postulated that beta 2-microglobulin cannot be removed from the blood by conventional hemodialysis, and accumulates in tissues causing the formation of amyloid fibrils, which, having a relatively high affinity to the carpal tunnel area, thus causes CTS.