Mordechai Weiss

Preferential expression of novel MUC1 tumor antigen isoforms in human epithelial tumors and their tumor-potentiating function.

The human MUC1 gene expresses at least 2 type 1 membrane proteins: MUC1/REP, a polymorphic high m.w. MUC1 glycoprotein often highly expressed in breast cancer tissues and containing a variable number of tandem 20 amino acid repeat units, and the MUC1/Y protein, which lacks this repeat array and, therefore, is not polymorphic. Despite their documented importance in signal transduction processes, the relative expression of the 2 isoforms in epithelial tumors is unknown. Using antibody reagents which recognize different MUC1 domains, the expression of these isoforms in malignant epithelial cells has been evaluated. A comparison of the amounts of the 2 isoforms revealed preferential expression of the novel MUC1/Y protein in breast cancer tissue samples. Furthermore, although the MUC1/REP protein is almost undetectable in HeLa cervical adenocarcinoma epithelial cells, the MUC1/Y isoform is extensively expressed in these cells. The presence of the MUC1/Y sequence as well as that of an additional tandem‐repeat‐array‐lacking isoform, designated MUC1/X, were demonstrated by reverse transcriptase PCR amplification of RNA extracted from HeLa and ovarian carcinoma cells. It has been shown previously that the MUC1 cytoplasmic domain interacts with the SH2 domain containing GRB2 protein, which transduces signals to ras, a protein which in its activated form can lead to cell transformation. We present here data demonstrating that MUC1/Y isoform expression increases the tumorigenic potential of DA3 mouse mammary epithelial cells; in contrast, potentiation of tumorigenicity is not observed with MUC1/REP expression. Our studies thus demonstrate that expression of the MUC1 gene in epithelial tumors can give rise to substantial levels of MUC1 proteins devoid of the tandem repeat array, which are generated by alternative splicing mechanisms. Int. J. Cancer 71:741‐749, 1997.

Basal plasma dehydroepiandrosterone sulfate level: a possible predictor for response to electroconvulsive therapy in depressed psychotic inpatients

BACKGROUND Dehydroepiandrosterone (DHEA) and its sulfate derivative DHEAS are neuroactive steroids. In the brain, they interact with gamma-aminobutyric acid (GABA(A)) receptors, which are involved in the regulation of anxiety and mood. The relevance of circulatory neurosteroids to psychiatric disorders and biological treatment is unknown. METHODS Basal plasma levels of cortisol, DHEA, and DHEAS and the DHEAS-DHEA ratio were determined in 17 psychiatric inpatients before and after six electroconvulsive (ECT) therapy sessions, and all changes were statistically analyzed. For baseline values, 25 healthy individuals served as control subjects. Severity of depression and psychosis in the patients was measured with the Hamilton Depression Rating Scale (HDRS) and the Brief Psychiatric Rating Scale, respectively. RESULTS Both basal and post-ECT levels of cortisol, DHEA, and DHEAS were significantly higher in the patients than in the control subjects. DHEAS levels in responding patients were higher at completion of treatment than at baseline. Patients defined as ECT nonresponders (change in HDRS < 30% from before treatments) exhibited elevated basal DHEAS levels compared with ECT responders. CONCLUSIONS Markedly elevated basal DHEAS levels (mean + 2 SD of control value) are associated with resistance to ECT and may serve as a potential predictive marker of nonresponsiveness to ECT in depressed patients.

MUC1 isoform specific monoclonal antibody 6E6/2 detects preferential expression of the novel MUC1/Y protein in breast and ovarian cancer

The products of the MUC1 gene are known to be highly expressed in human breast cancer cells. The best characterized MUC1 protein is a polymorphic, type 1 transmembrane molecule containing a large extracellular domain composed primarily of a variable number of 20 amino acid tandem repeats. We have recently identified a novel protein product of the MUC1 gene, the MUC1/Y protein, that is also a transmembrane protein but is devoid of the tandem repeat array and its immediate flanking sequences. To analyze its expression in tumor cells we generated monoclonal antibodies directed against the MUC1/Y extracellular domain (anti‐MUC1/Yex MAbs). Epitope mapping identified the MAb, 6E6, which recognized the MUC1/Y isoform with exquisite specificity‐ the repeat‐array‐containing MUC1 isoform could not compete out this immunoreactivity. A 30mer peptide which is unique for MUC1/Y and corresponds to the “join” region generated by the MUC1/Y specific splice, abrogated all 6E6 MAb immunoreactivity towards MUC1/Y. Immunoprecipitation of the MUC1/Y protein with 6E6 MAbs revealed that, in contrast with the proteolytic cleavage of the tandem‐repeat‐array‐containing MUC1 isoform, MUC1/Y is not cleaved. Flow cytometry analyses using the 6E6 MAbs demonstrated that the MUC1/Y isoform is expressed on the cell surface of both MCF‐7 breast cancer cells and malignant epithelial cells present in effusions obtained from breast and ovarian cancer patients. Our results unequivocally establish that the MUC1/Y protein is expressed on the surface of breast cancer cells and cells of other epithelial malignancies. The anti‐MUC1/Y MAbs described here can target MUC1/Y expressing tumor cells in vivo and are likely to be important reagents both for epithelial tumor diagnosis and immunotherapy. Int. J. Cancer 82:256–267, 1999.

Expression of genes coding for pS2, c-erbB2, estrogen receptor and the H23 breast tumor-associated antigen: A comparative analysis in breast cancer

Expression of the gene coding for a new breast tumor‐associated antigen, H23, was compared to expression of genes coding for pS2, c‐erbB2 and estrogen receptor (ER). Comparison involved mRNA expression in normal and malignant breast tissues as well as in non‐breast tumors. Results obtained by RNA dot blot and Northern hybridizations showed that expression of the H23 antigen coding gene is a discriminatory marker in human breast cancer. It is expressed in 92% of breast tumors whereas 69%, 62% and 56% of breast tumors demonstrate significant mRNA levels of c‐erbB2, ER and pS2, respectively. Non‐malignant or normal breast tissue expresses much lower levels of the H23 antigen mRNA. From the comparative analysis presented here it is concluded that the gene coding for H23 antigen furnishes a most useful marker for human breast cancer.

PATE Gene Clusters Code for Multiple, Secreted TFP/Ly-6/uPAR Proteins That Are Expressed in Reproductive and Neuron-rich Tissues and Possess Neuromodulatory Activity

We report here syntenic loci in humans and mice incorporating gene clusters coding for secreted proteins each comprising 10 cysteine residues. These conform to three-fingered protein/Ly-6/urokinase-type plasminogen activator receptor (uPAR) domains that shape three-fingered proteins (TFPs). The founding gene is PATE, expressed primarily in prostate and less in testis. We have identified additional human PATE-like genes (PATE-M, PATE-DJ, and PATE-B) that co-localize with the PATE locus, code for novel secreted PATE-like proteins, and show selective expression in prostate and/or testis. Anti-PATE-B-specific antibodies demonstrated the presence of PATE-B in the region of the sperm acrosome and at high levels on malignant prostatic epithelial cells. The syntenic mouse Pate-like locus encompasses 14 active genes coding for secreted proteins, which are all, except for Pate-P and Pate-Q, expressed primarily in prostate and/or testis. Pate-P and Pate-Q are expressed solely in placental tissue. Castration up-regulates prostate expression of mouse Pate-B and Pate-E, whereas testosterone ablates this induced expression. The sequence similarity between TFP/Ly-6/uPAR proteins that modulate activity of nicotinic acetylcholine receptors and the PATE (Pate)-like proteins stimulated us to see whether these proteins possess analogous activity. Pharmacological studies showed significant modulation of the nicotinic acetylcholines by the PATE-B, Pate-C, and Pate-P proteins. In concert with these findings, certain PATE (Pate)-like genes were extensively expressed in neuron-rich tissues. Taken together, our findings indicate that in addition to participation of the PATE (Pate)-like genes in functions related to fertility and reproduction, some of them likely act as important modulators of neural transmission.

Preoperative diagnosis of thyroid papillary carcinoma by reverse transcriptase polymerase chain reaction of the MUC1 gene

As thyroid nodules are common, it is imperative to recommend operation only to those with a high risk of malignancy. Fine‐needle aspiration (FNA) biopsy, which is widely used for this purpose, is limited by the considerable rate of non‐diagnostic or non‐interpretable conclusions. Therefore, it is highly desirable to acquire a new diagnostic means for thyroid cancer. We have recently described a marked over‐expression of the MUC1 gene in thyroid papillary‐carcinoma tissue, as compared with various benign thyroid pathologies. As the amount of mRNA obtainable by FNA is not amenable to hybridization analysis, we amplified the mRNA sequence of the MUC1‐gene upstream of the variable number tandem repeat array using the reverse‐transcription polymerase chain reaction (RT‐PCR). Seven out of 8 FNA samples obtained from thyroid papillary carcinoma resulted in 336 and 309 base‐pair products. In contrast, in all 13 FNA samples obtained from various benign pathologies, only the smaller RT‐PCR product was observed. Sequence analysis of the RT‐PCR products indicates that alternative splicing of the exon 2 acceptor site accounts for the difference between the 2 amplification products. It is suggested that RT‐PCR of the MUC1‐gene transcript may add a biomolecular diagnostic dimension to the routine cytological preoperative FNA diagnosis.

Early postmenopausal bone loss in hyperthyroidism

OBJECTIVES To evaluate the effect of hyperthyroidism on bone in relation to the menopausal state. METHODS Fifty-nine hyperthyroid (HYPER), 40 hypothyroid (HYPO), and 51 control euthyroid (EUTH) women were studied. Bone mineral density (BMD) was assessed by dual X-rays absorptiometry (DXA) at the lumbar spine, and at the femoral neck. A multi-site QUS device evaluated speed of sound (SOS) at the radius (RAD), tibia (TIB), metatarsus (MTR), and phalanx (PLX). Bone markers used were serum bone specific alkaline phosphatase (BSAP) and urinary deoxypyridinoline (DPD). RESULTS At all sites, SOS was lower in HYPER than in EUTH (RAD P<0.05, TIB P<0.01, MTR P<0.05, PLX P=0.01). The low SOS was only noted at the early postmenopausal period. BMD at the femoral neck but not at the lumbar spine was lower in HYPER as compared to EUTH (P<0.05). Both femoral neck and tibia were the sites with the highest odds ratio for being hyperthyroid (2.3 and 2.04, respectively). There was no correlation between BMD or SOS and FT(4), TT(3) or duration of hyperthyroidism. BSAP and DPD positively correlated with FT(4) and TT(3) (P<0.05). CONCLUSIONS This study suggests that hyperthyroidism affects bone mineralization especially during the early postmenopausal period, and the effect is mainly at the cortical bone.

A Novel Protein Derived from the MUC1 Gene by Alternative Splicing and Frameshifting

Genes that have been designated the name “MUC” code for proteins comprising mucin domains. These proteins may be involved in barrier and protective functions. The first such gene to be characterized and sequenced is the MUC1 gene. Here we report a novel small protein derived from the MUC1 gene by alternative splicing that does not contain the hallmark of mucin proteins, the mucin domain. This protein termed MUC1/ZD retains the same N-terminal MUC1 sequences as all of the other known MUC1 protein isoforms. The common N-terminal sequences comprise the signal peptide and a subsequent stretch of 30 amino acids. In contrast, the MUC1/ZD C-terminal 43 amino acids are novel and result from a reading frameshift engendered by a splicing event that forms MUC1/ZD. The expression of MUC1/ZD at the protein level in human tissues is demonstrated by Western blotting, immunohistochemistry, immunoprecipitation, and an ELISA. Utilization was made of affinity-purified MUC1/ZD-specific polyclonal antibodies as well as two different monoclonal antibodies that are monospecific for the MUC1/ZD protein. The MUC1/ZD protein is expressed in tissues as an oligomeric complex composed of monomers linked by disulfide bonds contributed by MUC1/ZD cysteine residues. MUC1/ZD protein expression did not parallel that of the tandem-repeat array-containing MUC1 protein. Results presented here demonstrate for the first time the expression of a novel MUC1 protein isoform MUC1/ZD, which is generated by an alternative splicing event that both deletes the tandem-repeat array and leads to a C-terminal reading frameshift.

Localization of adrenocorticotropic hormone-secreting bronchial carcinoid tumor by somatostatin-receptor scintigraphy.

Most ectopic adrenocorticotropic hormone (ACTH)-secreting tumors are bronchial carcinoids [1], and many are malignant [2]. Intensive localization studies are therefore warranted. However, frequently, and especially in small centrally located tumors, various imaging modalities are inconclusive. Recently, somatostatin receptor scintigraphic studies using the radiolabeled long-acting somatostatin analog Indium-111-pentetreotide (Mallinckrodt Medical, Pettem, the Netherlands) have enabled the detection of medullary thyroid carcinoma and islet cell tumor, both secreting ACTH. However, a small ACTH-secreting bronchial carcinoid was not similarly identified [3]. We describe a patient with ACTH-producing malignant bronchial carcinoid in whom octreotide scintigraphy led to localization of the tumor and its successful resection. Case Report A 22-year-old woman presented with a 3-month history of postpartum amenorrhea. Physical examination showed a blood pressure of 190/100 mm Hg, moon facies, and truncal obesity. Prominent purple striae covered her abdominal and chest walls. No goiter was palpable. Laboratory data included serum cortisol concentrations of 966 nmol/L at 0800 h (normal, 138 to 552 nmol/L) and 828 nmol/L at 2000 h (normal, 55 to 414 nmol/L). The plasma ACTH level at 0800 h was 60 pmol/L (normal, 2.3 to 13.8 pmol/L). Urinary free cortisol excretion was 1269 nmol/24 h (normal, 30 to 300 nmol/24 h). Dexamethasone administration of up to 16 mg daily in four equally divided doses did not significantly suppress serum cortisol and plasma ACTH levels or urinary free cortisol excretion. Cortisol and ACTH levels were both determined using commercially available kits (cortisol: DPC, Los Angeles, California; ACTH: Nichols Institute, San Juan Capistrano, California). Contrast-enhanced pituitary computed tomographic (CT) scans were normal. Abdominal CT scans and magnetic resonance imaging studies showed that both adrenal glands were diffusely enlarged but that the pancreas seemed normal. An enhanced axial CT scan of the chest with 5-mm-thick slices showed no abnormality. Somatostatin receptor imaging done after intravenous administration of 20 g of Indium-111-pentetreotide (222 MBq) showed a focus of increased activity in the left upper lobe, parahilar in location (Figure 1 a), as early as 4 hours after the injection of the tracer. This focus was evident for at least an additional 20 hours thereafter. Repeat CT scanning of the chest with coronal reconstruction of the left upper lobe showed a central 1-cm rounded mass occluding a subsegmental bronchus of the apicoposterior segment of the left upper lobe. Distal to the mass, a wedge-shaped small zone of air trapping was noted Figure 1 b. Figure 1. Panel a. Panel b The patients hypercortisolism was controlled using ketoconazole, 800 mg/d, and blood pressure was controlled using atenolol, 100 mg/d. Left upper lobectomy showed a typical invasive carcinoid and metastatic foci within one lymph node. Immunocytochemical staining of specimens from both the tumor and the metastatic lymph node was positive for ACTH. The plasma ACTH concentration was 33 pmol/L in the pulmonary artery feeding the tumor and 64 pmol/L in the pulmonary vein draining the tumor. One month after surgery, the patient showed marked clinical improvement. Without any medication, her blood pressure was 120/80 mm Hg. The morning cortisol level after 1-mg overnight dexamethasone administration was 30 nmol/L (normal, <138 nmol/L). Discussion Excess ACTH is usually secreted by pituitary adenomas, which can be distinguished from the ectopic ACTH-producing tumors on the basis of their response to various suppressive and stimulatory agents. Ectopic ACTH-producing tumors commonly have their origin in the neuroendocrine system [4], and more than half are bronchial carcinoid tumors [5]. At least 30% of the bronchial carcinoid tumors eventually develop local and distant metastases [2]. Control of the metastatic potential and the endocrine activity of the tumor necessitates accurate localization, which has proved to be difficult [6]. This difficulty is especially evident in cases of small-sized central bronchial carcinoid tumors, which may easily be confused with blood vessels of the same size. Several neuroendocrine tumors, including those secreting ACTH, have somatostatin receptors [7] and respond clinically and biochemically to administration of the long-acting somatostatin analog octreotide [8]. Nevertheless, some ACTH-secreting tumors do not have these receptors and do not respond to octreotide treatment [3, 9]. Recently, the interaction of Indium-111-pentetreotide with ACTH-secreting bronchial carcinoid has been reported in two cases. In one case, the carcinoid proved to be devoid of somatostatin receptors both on autoradiography and on scintigraphy [3]. In the second case, which has been reported only in abstract form, scintigraphic studies with Indium-111-pentetreotide showed the carcinoid [10]. It thus appears that the inconsistent detection of ACTH-secreting bronchial carcinoids depends on the degree of somatostatin-receptor expression in these tumors. This expression can be identified in vivo by a radiolabeled somatostatin analog. Our findings suggest that in cases of obscure ectopic ACTH-producing tumor, somatostatin-receptor scintigraphic study is warranted because it may localize the tumor for surgical removal or indicate which patients need octreotide treatment. Addendum After submission of our manuscript, a similar case was reported elsewhere [11].

Follicular variant of papillary thyroid carcinoma: clinical-pathological characterization and long-term follow-up.

PURPOSEQuestions arise concerning the behavior and prognosis of the follicular variant of papillary thyroid carcinoma. PATIENTS AND METHODSBetween 1990 and 2003, 92 patients with follicular variant of papillary carcinoma (group A) were enrolled in a long-term study and compared with control groups of follicular thyroid carcinoma (group B, 40 cases) and pure papillary thyroid carcinoma (group C, 99 subjects). RESULTSGender (female/male), age, and follow-up duration (years, mean ± standard error) in groups B, A, and C were 36/4, 43 ± 3, 11 ± 1.1; 79/13, 46 ± 2, 9.5 ± 0.7; and 82/17, 44 ± 1, 10 ± 0.6, respectively. At the time of diagnosis, the rates of extensive extra thyroidal local spread, bilateral lesions, and vascular invasion were higher in group A than in group C. The rate of metastasis tumors was higher in group A than in group C and was comparable in groups A and B. Complete remission was reported in 95% of group B patients, 98% of group C individuals, and in only 77% of group A subjects. Persistent stable lesions and progressive disease rates in groups B, A, and C were 2.5% and 2.5%, 15% and 8%, and 0% and 2%, respectively. The survival rates at the end of the study were 100% in all cohorts, but the cumulative dose of administered radioiodine in group A was higher than in group C and was comparable to that given in group B. Metastases dedifferentiation was observed only in the group A (three patients). DISCUSSIONFollicular variant of papillary thyroid carcinoma may be more aggressive than previously considered and should be clearly distinguished from the two other forms of well-differentiated thyroid carcinoma.