Human Milk Fat Globule Research Articles

Generation of murine monoclonal antihuman milk fat globule membrane antibodies using immunoprecipitation and BIAcore analyses.

A selection of monoclonal antibodies was developed against deoxycholine-solubilized human milk fat globule membranes (HMFG). The antibodies were selected for their ability to immunoprecipitate 125I-labeled HMFG and then further analyzed by surface plasmon resonance on the BIAcore for their reactivity with HMFG and with a fusion protein containing a 4-mer of the muc-1 tandem repeat. Both the HMFG and the fusion protein proved to be robust surfaces for the analysis of crude supernatants. The BIAcore evaluation was useful in identifying true positives. BIAcore analyses of purified antibody preparations were used to determine binding characteristics such as affinity and intensity. The latter proved useful in selecting a panel for evaluation by immunohistochemistry for breast tissue reactivity. Four of 6 antibodies appeared to react more intensely with tumor compared with normal breast tissues. One of those antibodies reacted with the fusion protein 4-mer of the muc-1 tandem repeat.

Tubular adenoma of the breast: An immunohistochemical study of ten cases

Ten cases of so-called "tubular" adenoma and six cases of fibroadenoma of the breast have been investigated with an immunohistochemical technique with the aim of providing both more details on their immunophenotype and of ascertaining the possible relationships between tubular adenoma and fibroadenoma. Smooth Muscle Actin, Cytokeratin 14, GFAP, S-100 Protein and Vimentin immunoreactivity have clearly demonstrated that cells with myoepithelial immunophenotype are one of the major cell components in breast adenomas. Epithelial Membrane Antigen (EMA), Human Milk Fat Globule II (HMFG II), Estrogen and Progesterone receptors have been detected in adluminal epithelial cells exclusively. Furthermore, Smooth Muscle Actin and Vimentin highlighted an abundant myofibroblastic component, intermingled with tubular structures in both tumor types. A low percentage (10-22%) of adluminal cells and of myofibroblasts showed Ki-67 immunoreactivity in tubular adenomas and in fibroadenomas, whereas only rare myoepithelial cells demonstrated Ki-67 positivity in both tumor types. These data seem to indicate that several cell components of both epithelial and mesenchymal origin (epithelial cells, myoepithelial cells, myofibroblasts) are involved in the genesis of tubular adenomas. The morphological and immunohistochemical features of tubular adenomas closely resemble, in some areas of the tumors, those of fibroadenoma. Therefore, they may represent histogenetically related neoplasms with exuberant ductular component in tubular adenomas and predominant stromal component in fibroadenoma.

Pre-treatment serum levels of tumour markers in metastatic breast cancer: a prospective assessment of their role in predicting response to therapy and survival

The value of pre-treatment serum tumour marker levels in 85 consecutive patients of newly diagnosed metastatic breast cancer was prospectively assessed for predicting response to therapy and survival. The markers studied were carcinoembryonic antigen (CEA), orosomucoid (ORO), erythrocyte sedimentation rate (ESR), C reactive protein (CRP), ferritin (FERR), human milk fat globule membrane 1 and 2 (HMFG1 and 2), CA 15-3 and NCRC-11. There was no correlation between serum marker levels and response to therapy. Only serum concentrations of CRP (P = 0.02), FERR (P = 0.001), HMFG1 (P = 0.004) and HMFG2 (P = 0.04) were predictive for survival. The prognostic significance of HMFG1 was restricted to a minority of patients (7%) with extreme values of these serum markers.

Characterisation of a recombinant Fv fragment of anti-MUC1 antibody HMFG1

A recombinant Fv (variable fragment) has been produced for the murine monoclonal antibody HMFG1. This antibody was raised against human milk fat globules and reacts with an epitope (PDTR) in the protein core of MUC1 mucins, which are up-regulated in human breast and other carcinomas. Binding specificity of the Fv fragment has been demonstrated through immunoaffinity purification, and by radioimmunoassay. The affinity constants for this Fv fragment and for the proteolytically produced Fab (antigen binding fragment) of the related humanised antibody HuHMFG1 were determined by monitoring the fluorescence quenching of the antibody fragments whilst adding aliquots of MUC1 related antigenic peptides KAPDTRPAPG and VTSAPDTRPAPG. Using these techniques it has been demonstrated that the products of these different methods of antibody fragmentation are comparable, and suitable for solution structure analysis using nuclear magnetic resonance (NMR) spectroscopy.

Distinguishing primary and metastatic carcinomas of the liver: Immunohistochemical updates and reminders

Distinguishing primary hepatocellular carcinoma (HCC) from metastatic carcinomas to the liver is often difficult, if not impossible, particularly in needle biopsy and fine-needle aspiration specimens. In an attempt to identify a specific immunohistochemical profile that would distinguish HCC from metastatic carcinomas, we studied 56 HCCs, 8 cholangio-carcinomas, and 24 metastatic adenocarcinomas with monoclonal antibodies to alpha-fetoprotein (AFP), keratin (AE1, AE3, and CAM5.2), Leu-M1, human milk fat globule (HMFG-2), tumor-associated glycoprotein-72 (B72.3), epithelial specific membrane antigen (Ber-EP4), and BCA-225 (CU-18). Both monoclonal and polyclonal (mCEA and pCEA) antibodies to carcinoembryonic antigen also were used. Metastatic adenocarcinomas were often positive for CU-18 (71%), Leu-M1 (75%), B72.3 (50%), HMFG-2 (67%), Ber-EP4 (83%) and mCEA (71%). Using these antibodies, the frequency of positivity for HCC was 9%, 16%, 11%, 20%, 36%, and 11%, respectively. CU-18 was the only monoclonal antibody in which there was a significant difference in positive rates between HCC and metastatic adenocarcinomas. Most HCCs (71%) revealed a bile canalicular staining pattern with pCEA. Because this staining pattern was absent in metastatic carcinomas, pCEA appears to be useful in confirming a diagnosis of HCC, AE1, AE3 and CAM5.2 antibodies were not useful in distinguishing HCC from metastatic carcinomas. Each cholangiocarcinoma shared a staining profile similar to that of metastatic carcinomas. Hepatocyte paraffin 1 is a monoclonal antibody that has been developed specifically to react with hepatocytes in routine formalin-fixed and paraffin-embedded surgical pathology tissues. It results in a distinct, granular cytoplasmic staining of hepatocytes but fails to react with bile ducts and nonparenchymal liver cells. The antibody decorates a majority of hepatocellular carcinomas, including fibrolamellar variants. It fails to react with a wide variety of other adult malignancies, with the exception of focal staining in a few gastrointestinal malignancies, including a sub-population of gastric carcinomas.

Distinguishing primary and metastatic carcinomas of the liver: Immunohistochemical updates and reminders

Distinguishing primary hepatocellular carcinoma (HCC) from metastatic carcinomas to the liver is often difficult, if not impossible, particularly in needle biopsy and fine-needle aspiration specimens. In an attempt to identify a specific immunohistochemical profile that would distinguish HCC from metastatic carcinomas, we studied 56 HCCs, 8 cholangio-carcinomas, and 24 metastatic adenocarcinomas with monoclonal antibodies to alpha-fetoprotein (AFP), keratin (AE1, AE3, and CAM5.2), Leu-M1, human milk fat globule (HMFG-2), tumor-associated glycoprotein-72 (B72.3), epithelial specific membrane antigen (Ber-EP4), and BCA-225 (CU-18). Both monoclonal and polyclonal (mCEA and pCEA) antibodies to carcinoembryonic antigen also were used. Metastatic adenocarcinomas were often positive for CU-18 (71%), Leu-M1 (75%), B72.3 (50%), HMFG-2 (67%), Ber-EP4 (83%) and mCEA (71%). Using these antibodies, the frequency of positivity for HCC was 9%, 16%, 11%, 20%, 36%, and 11%, respectively. CU-18 was the only monoclonal antibody in which there was a significant difference in positive rates between HCC and metastatic adenocarcinomas. Most HCCs (71%) revealed a bile canalicular staining pattern with pCEA. Because this staining pattern was absent in metastatic carcinomas, pCEA appears to be useful in confirming a diagnosis of HCC, AE1, AE3 and CAM5.2 antibodies were not useful in distinguishing HCC from metastatic carcinomas. Each cholangiocarcinoma shared a staining profile similar to that of metastatic carcinomas. Hepatocyte paraffin 1 is a monoclonal antibody that has been developed specifically to react with hepatocytes in routine formalin-fixed and paraffin-embedded surgical pathology tissues. It results in a distinct, granular cytoplasmic staining of hepatocytes but fails to react with bile ducts and nonparenchymal liver cells. The antibody decorates a majority of hepatocellular carcinomas, including fibrolamellar variants. It fails to react with a wide variety of other adult malignancies, with the exception of focal staining in a few gastrointestinal malignancies, including a sub-population of gastric carcinomas.

Response of normal and oncogene-transformed human mammary epithelial cells to transforming growth factor β1 (TGF-β1): Lack of growth-inhibitory effect on cells expressing the simian virus 40 large-t antigen

The relationship between the expression of selected oncogenes having different modes of action and the loss of the capacity to respond in vitro to transforming growth factor-beta I (TGF-beta I) was analyzed in human mammary epithelial cells (MEC). Primary MEC cultures from healthy donors and the spontaneously immortalized MCF-10A cell line were used as normal controls. Various assays (employing both complete and chemically defined media) were used: short-term DNA synthesis, long-term cell proliferation under anchorage-dependent and -independent conditions, expression of surface-differentiation molecules. Whereas primary MEC and the MCF-10A cell line were fully responsive to the growth-inhibitory activity of TGF-beta I under different test conditions, MEC transformed by c-Ha-ras, c-erbB2, int-2, or SV40-large-T antigen were not inhibited by TGF-beta I in a short-term DNA-synthesis assay. However, in anchorage-dependent conditions TGF-beta I inhibited the proliferation of all lines investigated, with the exception of SV40-T-antigen-transformed MEC. The colony-formation assay in soft agar revealed that all lines, but not those expressing the int-2 or the SV40-T-antigen genes, were inhibited by TGF-beta I. Neutralizing antibody to TGF-beta had no significant effects on oncogene-transformed lines, suggesting that the endogenous production of an active form of this growth factor is not a major determinant in MEC transformation by the oncogenes investigated. The only observed effect of TGF-beta I on selected surface-differentiation molecules was that normal MEC produced increased levels of the human milk fat globule antigen-I. Thus it appears that the response of MEC to TGF-beta I is consistently attenuated by the insertion of a variety of oncogenes and that it is abolished only by the expression of the SV40-large-T antigen. Whereas no single in vitro assay was capable of accurately reflecting the actual responsiveness of different lines, the growth-curve assay in anchorage-dependent conditions was the best single predictive test.

Multivariate prognostic model for infiltrating ductal carcinoma of the breast in the axillary node-free patient.

Antigens present on the human milk fat globule (HMFG) were originally detected with a polyclonal antibody1, that identified several specific breast glycoproteins2, some of them later detected in the sera of breast cancer patients3. Later, monoclonal antibodies (MoAbs) we created against HMFG first identified a heavy molecular weight glycoprotein4, later called the breast epithelial mucin. This molecule, recently sequenced5 possesses heterogeneous amino and carboxyterminal moieties linked by a central moiety constituted by a dodecamer tandem repeat. This tandem repeat sequence is the area of the molecule to which all anti-breast mucin MoAbs are directed to6 (also see article by Peterson et al, in this publication). The reason for the differential specificity of these anti-mucin MoAbs could be ascribed to diverse glycosylation at the core of these tandem repeat sequences6.KeywordsBreast CancerHuman Mammary Epithelial CellTraditional VariableInfiltrate Ductal CarcinomaCytoplasmic IntensityThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Digestion of triacylglycerols containing long-chain polyenoic fatty acids in vitro by colipase-dependent pancreatic lipase and human milk bile salt-stimulated lipase

To assess the role of human milk bile salt-stimulated lipase (BSSL) in the digestion of polyunsaturated ester bonds of triacylglycerols, hydrolysis of docosahexaenoic acid (22:6( n − 3)) ester bonds was compared to that of oleic acid (18:1( n − 9)) or arachidonic acid (20:4( n − 6)) esters. As model substrates, we used rat chylomicrons obtained after feeding human milk fat globules and radiolabeled fatty acids. Radiolabeled chylomicrons were incubated with colipase-dependent pancreatic lipase, with BSSL, or with both enzymes in combination. Both enzymes hydrolyzed 18:1 more efficiently than 22:6 esters. With colipase-dependent lipase there was a large accumulation of 22:6 in diacylglycerol whereas with BSSL it accumulated mainly in monoacylglycerol. Esters containing 20:4 were hydrolyzed by BSSL as efficiently as 18:1 but this fatty acid also accumulated as diacylglycerol with colipase-dependent lipase. At low bile salt concentrations, as found in duodenal contents of newborns, colipase-dependent lipase was virtually unable to hydrolyze esters of 20:4 and 22:6 whereas BSSL hydrolyzed these esters at appreciable rates. Combining the two enzymes gave the most efficient hydrolysis of all fatty acids tested regardless of bile salt concentrations. BSSL may thus have a physiological role in completing duodenal hydrolysis of milk triacylglycerols containing 22:6- or 20:4-esters to free fatty acids and monoacylglycerol.

Analysis of a heterogeneous group of human breast carcinoma associated glycoproteins bearing the Tn determinant

The Tn determinant (GalNAc alpha-O-Ser/Thr) is expressed by about 90% of human carcinomas, but is cryptic in most normal human tissues. A murine monoclonal antibody (MAb) 83D4, developed following immunization with human breast carcinoma cells, reacts with a Tn-related epitope. In the present study we characterized the glycoprotein antigen identified by 83D4 in the human breast carcinoma cell line MCF-7. We further showed that the 83D4 antigenic determinant is masked in human milk fat globule membranes (HMFGM), and can be exposed upon mild m-periodate treatment after desialylation. Western-blot analysis resolved the 83D4 antigen from MCF-7 into two main components of 120-190 kD and > 500 kD respectively. Non equilibrium pH gradient electrophoresis/SDS PAGE revealed the acidic nature of the reactive glycoproteins (pI 4.43-4.70). 83D4 antigenic activity resolved by CsCl gradient ultracentrifugation layered on a wide range of densities (1.30-1.46 g/ml) including typical densities of mucin-like glycoproteins but also lower densities. The amino acid composition of the antigen, relatively rich in serine but poor in threonine and proline, confirmed the divergence from other mucin-like carcinoma-associated glycoproteins. Dicarboxylic amino acids were abundant, accounting in part for the acidic nature of the molecules. ELISA and Western-blot analysis of the subcellular fractions from MCF-7 cells revealed that the 83D4 antigen is mainly contained in plasma membranes (85%) from which it may be resolved into two broad bands (slow and fast migrating components). These results provide information on a group of breast carcinoma associated glycoproteins related to but different from typical mucins, and provide data on alteration of O-glycosylation in tumor cells.

The value of the human milk fat globule membrane antigen HMFG2 in epithelial ovarian cancer monitoring: comparison with CA125

We assayed serum HMFG2 in serial samples from 215 primary epithelial ovarian cancer patients using an 'in-house' single determinant ELISA, 45% of patients with stage I, 54% with stage II, 61% with stage III and 75% with stage IV disease had elevated serum HMFG2. Post-operative levels were significantly related with residual tumour volume (P < 0.005), and fell in the majority of responders, although the association with response to first-line chemotherapy was not significant. HMFG2 had a sensitivity of 50% specificity of 83%, accuracy of 61%, PVP of 86% and PVN of 45% for disease at second-look laparotomy. Serial levels gave a lead time to clinical relapse in 47% of patients who responded to therapy, including one patient with negative CA125 levels. HMFG, paralleled CA125 in many respects, although it was elevated in fewer patients. In a stepwise discriminant analysis, HMFG2 added to the discrimination of CA125 (r = 0.183, P < 0.005), although additional accurate information was only given in patients with advanced poorly differentiated serous cystadenocarcinoma. Given that HMFG2 is expressed in few patients who are CA125 negative it is unlikely that it will have a significant clinical impact upon patient management.

Radiation doses obtained from 99mTc-labelled human milk fat globule monoclonal antibodies

Human milk fat globule (HMFG) monoclonal antibodies labelled with 123I and 111In are used for the diagnosis of ovarian and breast cancer. Methods have been developed to label HMFG with 99mTc and this study reports on the subsequent radiation dose delivered. The 99mTc HMFG1 distribution was obtained in 5 normal female baboons up to 71 h after administration. The liver, kidneys, bladder and whole body were identified as source organs and total absorbed doses for the target organs were 0.054 (kidneys), 0.013 (liver), 0.140 (bladder), 0.010 (ovaria), 0.004 (bone marrow), 0.002 (thyroid), 0.003 (heart wall), 0.017 (uterus) and 0.005 mGy/MBq (whole body). The effective dose was 0.014 mSv/MBq.

Expression of epithelial and extracellular matrix protein markers in meningiomas.

In an attempt to characterize the dual mesenchymal and epithelial differentiating potential of meningiomas, cryostat sections from 50 meningiomas of diverse histological subtypes were examined immunohistochemically with a panel of markers for epithelial and mesenchymal differentiation. The overall positivities were: keratins 50%, epithelial membrane antigen 94%, human milk fat globules 38%, carcinoembryonic antigen 4% (secretory meningiomas only), desmoplakins 64%, collagen I 82%, procollagen I 96%, collagen III 74%, collagen IV 60%, laminin 54%, fibronectin 98% and vimentin 98%. Such production of keratins was not found in many previous immunohistochemical analyses of meningiomas with paraffin sections. The extracellular matrix proteins were present in a pericellular distribution suggestive of their being produced by the tumour cells. The potential of dual epithelial and mesenchymal differentiation in meningiomas was further examined in seven cases established on short-term cultures. Morphologically, subcultured tumour cells resembled fibroblasts and in five cases revealed similar epithelial and mesenchymal immunohistochemical profiles as for direct tumour immunostaining. In two cases, cells from the primary cultures revealed a fine skeleton of intercellular matrix proteins stainable by immunohistochemical methods, providing further proof that meningioma cells possess the capability to elaborate extracellular matrix proteins, a major mesenchymal function akin to that of fibroblasts.

A histochemical and immunohistochemical study of extra‐ocular sebaceous carcinoma

A histochemical and immunohistochemical study of five cases of extra-ocular sebaceous carcinoma was performed using formalin-fixed and paraffin-embedded tissue specimens. Histochemically, the clear cells of sebaceous carcinomas were negative with periodic acid-Schiff and alcian blue staining. Immunohistochemically, the tumour cells of sebaceous carcinomas showed positive reactions for epithelial membrane antigen, human milk fat globules subclass 1, human milk fat globules subclass 2 and Leu M1, but did not express carcinoembryonic antigen, breast carcinoma associated antigen, S-100 protein, gross cystic disease fluid protein-15 or Dako M1. These histochemical and immunohistochemical findings were compared with those of other skin cancers which must be distinguished histopathologically from sebaceous carcinoma. We conclude that sebaceous carcinoma can be distinguished from eccrine porocarcinoma, malignant clear cell hidradenoma, extramammary Paget's disease, malignant trichilemmoma, squamous cell carcinoma and basal cell carcinoma by histochemical and immunohistochemical techniques using formalin-fixed and paraffin-embedded tissue specimens.

IMMUNOHISTOCHEMICAL EVALUATION OF MAM-3 AND MAM-6 ANTIGENS IN SQUAMOUS-CELL CARCINOMA OF THE ORAL CAVITY

MAM-3 and MAM-6 antigens of human milk fat globule membrane designated as human polymorphic epithelial mucin (PEM) were detected immunohistochemically in a total of 86 specimens from 39 cases of oral squamous cell carcinoma (SCC) and 6 cases of normal oral mucosa. The two antigens were identified by MoAbs 67D11 and 115G3 (MAM-3 antigen) and by MoAbs 115D8 and 115F5 (MAM-6 antigen) in paraffin sections. MoAb 115G3 staining was found in well keratinized foci; whereas, MoAb 115D8 and 115F5 staining was very low in keratinized tumor cells. The cytochemical distribution of the antigens was classified into 3 types; (i) cell membrane positive type, (ii) whole cell positive type and (iii) antigen negative type. Reduction and disappearance of the two antigens were correlated to the mode of tumor invasion and suggested that glycocalyx complex in cell membrane properties was changed for malignant transformation.

Immunohistochemical characteristics of human milk-fat globule antibodies in predicting chest wall and distant metastasis after mastectomy for localized cancer of the breast.

Because postoperative radiation reduces chest wall metastasis after mastectomy, it is important to identify patients in whom it might develop. Pathologic and immunohistochemical features in 59 patients with chest wall metastasis were compared with characteristics in disease-free patients and patients with systemic metastases without chest wall metastasis. Immunohistochemical studies with human milk-fat globule (HMFG)-2 were not predictive, but a membrane reaction with HMFG-1 was associated with fewer systemic metastases and tumors, in which most of the cells reacted with the antibody had significantly decreased likelihood of chest wall metastasis. Antigenic concordance between the primary breast cancer and the chest wall metastasis was demonstrated. Neither tumor size nor axillary node metastasis predicted development of chest wall metastasis, but systemic metastases were less common when fewer than four nodes were involved, the primary tumor was well differentiated, and estrogen receptors were positive.

Prognostic significance of the immunohistochemical reaction to human milk fat globule antibodies in node-negative and node-positive breast cancer

The status of axillary lymph nodes in patients with breast cancer is important prognostically but does not identify all patients who will have long or short survival. To determine if the immunohistochemical reaction of tumor cells to human milk fat globule (HMFG) antibodies will define prognosis more specifically, we examined patients with node-negative disease and patients with more than three positive nodes, the extremes of the prognostic spectrum. In both node-negative and node-positive groups, patients who relapsed were matched by age at diagnosis, tumor size, histologic type, and receptor status, with patients who remained disease-free for a minimum of 48 months. Patients with poor survival had a higher proportion of poorly differentiated cancers. The ability to generate antigens recognized by HMFG antibodies was decreased in patients with recurrent disease, but this was significant only in patients with node-positive tumors. Tumors of patients who remained disease-free were more likely to have a pattern of membrane staining, while cytoplasmic staining was more frequent in those who relapsed. The results suggest that immunohistologic response to HMFG antibodies may assist in identifying cancers with poor prognosis, supplementing the prediction derived from node status.

Inhibition of adhesion of S-fimbriated Escherichia coli to buccal epithelial cells by human milk fat globule membrane components: a novel aspect of the protective function of mucins in the nonimmunoglobulin fraction.

We investigated the presence of factors in human milk that inhibit invasion of pathogenic bacteria. The effect of human milk fat globule membrane (HMFGM) components on adhesion of cloned S-fimbriated Escherichia coli to human buccal epithelial cells was analyzed. S fimbriae are a common feature of E. coli strains causing sepsis and meningitis in newborns and are bound to epithelia via sialyl-(alpha-2-3)galactoside structures. Human milk fat globules (HMFG) could be agglutinated by the above-mentioned bacteria. Agglutination could be inhibited by fetuin, human glycophorin, and alpha 1-acid glycoprotein. In addition, pretreatment of HMFG with Vibrio cholerae neuraminidase markedly reduced bacterium-induced agglutinations, indicating the involvement of neuraminic acid-containing glycoproteins. In contrast, lipid droplets of infant formula or artificial lipid emulsions (Intralipid) could not be agglutinated. HMFG were present in stools of breast-fed neonates as shown by indirect immunofluorescence staining with a monoclonal antibody directed against carbohydrate residues present on HMFGM. These HMFG could be agglutinated by bacteria. HMFG inhibited E. coli adhesion to buccal epithelial cells. To further characterize relevant E. coli binding structures, HMFGM components were separated by gel chromatography. The mucin fraction showed the most pronounced inhibitory effect on adhesion of S-fimbriated E. coli to human buccal epithelial cells. Our data suggest that HMFG inhibit bacterial adhesion in the entire intestine and thereby may provide protection against bacterial infection.

Differential diagnostic patterns of lung neuroendocrine tumours. A clinico-pathological and immunohistochemical study of 122 cases.

A series of 3 tumourlets (TLs), 81 typical carcinoids (TCs), 14 atypical carcinoids (ACs) (well-differentiated neuroendocrine carcinomas, WDNCs) and 24 small cell-intermediate cell carcinomas (SCC-ICCs) of the lung were studied. Histopathological features were correlated with amine and peptide hormone immunoreactivity and with clinical data. All types of tumours expressed general neuroendocrine (NE) markers: Grimelius positivity and chromogranins were detected more frequently in well-differentiated (TLs, TCs) than in less well differentiated tumours [ACs (WDNCs) and SCC-ICCs] whereas neuron specific enolase (NSE) was prominent in the latter tumours. TLs and peripheral TCs were benign, often showing a paraganglioid pattern and frequently expressing gastrin-releasing peptide (GRP), which is present in the peripheral airways of normal lung. Central TCs were associated with lymph node metastases in 8.5% of the cases, frequently had a trabecular architecture, often associated with human milk fat globule 2 (HMFG2)-positive acinar and rosette-like structures, and were mainly immunostained for the alpha-subunit of human chorionic gonadotrophin (alpha-hCG) and serotonin. ACs (WDNCs) were associated with intrathoracic and/or extrathoracic metastases in 57.1% of the cases with a mortality rate of 35.7%. Their histological and cytological features were intermediate between those of TCs and SCC-ICCs. ACs (WDNCs) expressed serotonin and alpha-hCG less frequently than TCs. All SCC-ICCs were surgically treated and displayed a mortality rate of 91.6% with a mean survival of 10.2 months after operation. These tumours were characterized by high expression of HMFG2 and NSE, while the expression of both orthotopic (serotonin, GRP) and ectopic (ACTH) specific NE substances was very low. Since all TCs (either central or peripheral) had a favourable outcome, while about 36% of ACs (WDNCs) were fatal, the latter seem more appropriately designated "well-differentiated NE carcinomas". The differential diagnosis between different NE tumours of the lung is important and is mainly based on morphology. Both panendocrine and specific immunohistochemical markers are helpful in distinguishing the less aggressive, mostly benign varieties from the more malignant varieties.

High level expression in E. coli of an alternate reading frame of pS2 mRNA that encodes a mimotope of human breast epithelial mucin tandem repeat.

The high molecular weight mucin found in human milk fat globule and on the surface of mammary and other epithelial cells contains a 20 amino acid tandem repeat sequence that is highly immunogenic. We have immunoscreened lambda gt11 cDNA expression libraries from MCF7 cells and lactating breast tissue with 5 anti-mucin monoclonal antibodies. We isolated a group of cDNA clones that had the repeat sequence (HB11-2, HB11-6, HB11-10) and a group that had little or no homology with the repeat sequence (NP4, NP5, HB11-4). A fusion protein produced by NP4 bound preferentially BrE2, while HB11-4 bound only BrE2 and BrE3, NP5 produced a fusion protein that bound only Mc5 and not the other MAbs. Sequencing of the NP5 cDNA revealed it to be distinct from the mucin sequence and instead to have 97% identity with the estrogen induced transcript, pS2. An alternate reading frame was translated by the lambda gt11 fusion gene yielding a 44 amino acid protein having no homology with pS2 protein. Only a short region of homology (5 amino acids) with the breast mucin tandem repeat was found which was shown to be a mimotope for the Mc5 epitope on the breast mucin. High level expression of the NP5 cDNA was achieved by subcloning it into pEX2. The NP5 fusion protein has been useful for developing an assay for the presence of mucin derived antigen in patient serum.