Francisco X. Real

MUC6 apomucin shows a distinct normal tissue distribution that correlates with Lewis antigen expression in the human stomach

BACKGROUND & AIMS Among the human mucin complementary DNAs thus far identified, two (MUC5AC and MUC6) were cloned from stomach libraries. This study examines the distribution of MUC6 in normal tissues and compares it with that of MUC5AC as well as with the expression of Lewis blood group antigens. METHODS Affinity-purified rabbit antibodies detecting epitopes within the repetitive sequence of MUC5AC and MUC6 were used in enzyme-linked immunosorbent assays and immunohistochemical assays. RNA expression was analyzed by in situ hybridization. Double-labeling immunofluorescence was used to study apomucin and Lewis antigen coexpression. RESULTS MUC6 is detected in the stomach, colon, gallbladder, and endocervix. Two patterns of staining are observed, perinuclear and diffuse cytoplasmic, possibly reflecting differences in MUC6 glycosylation. Using both immunohistochemical assays and in situ hybridization on stomach tissue sections, MUC6 is expressed mainly in antral mucous cells, whereas MUC5AC is detected mainly in the superficial epithelium and neck glands. In antral mucosa, MUC6+ cells express Lewis(y), whereas MUC5AC+ cells express Lewis(b) and sialyl-Lewis(a). CONCLUSIONS It was concluded that MUC6 has a distinct tissue distribution pattern, different from that of MUC1-MUC5; MUC5AC and MUC6 are expressed by different cellular populations in normal stomach; and in this tissue, MUC5AC+ cells and MUC6+ cells show different patterns of Lewis antigen expression.

Differential apomucin expression in normal and neoplastic human gastrointestinal tissues.

BACKGROUND/AIMS The cloning of genes encoding human mucins is the basis for the study of their normal tissue distribution and the alterations associated with cancer. The aim of this study was to determine the normal and tumor tissue expression of MUC1, MUC2, MUC5B, and MUC5C. METHODS The reactivity of apomucin-specific antibodies with fresh normal and tumor tissues was analyzed using immunohistochemical techniques. RESULTS Anti-MUC1 antibodies reacted with most glandular epithelia. Anti-MUC2 antibody was mainly reactive with intestinal goblet cells and cervical mucous cells. Anti-MUC5B was reactive with a wide range of epithelial tissues whereas anti-MUC5C was reactive with stomach, trachea, and endocervix. Double-labeling experiments showed coexpression of MUC1/MUC2 and MUC2/MUC5C in colonic tissue. Multiple apomucins were detected in colon cancers, but no relationship to histochemical mucus stains was observed. CONCLUSIONS It is concluded that (1) each apomucin shows a distinct tissue expression pattern; (2) multiple apomucins are present in a single tissue and at the single cell level; and (3) altered apomucin expression takes place in pathological colonic tissue.

Altered expression of MUC2, MUC4, and MUC5 mucin genes in pancreas tissues and cancer cell lines.

BACKGROUND/AIMS Neoplastic transformation of epithelial cells is commonly associated with altered synthesis and structure of mucin glycoproteins. The aim of the study was to determine if altered mucin gene expression takes place in pancreas cancer. METHODS To examine mucin gene expression in normal pancreas and pancreas cancer, antibodies detecting the MUC1, MUC2, MUC5B, and MUC5C apomucins were used in immunohistochemical techniques and complementary DNA probes specific for the MUC1-MUC5 genes were used in Northern blots. RESULTS MUC1 is the major apomucin expressed in normal pancreas, whereas MUC2-MUC5 are weakly expressed or undetectable. In pancreas cancer tissues and cell lines, increased expression of MUC2, MUC4, and MUC5C is shown. The cytoplasmic expression of MUC2 and MUC5C in tumor cells suggests that these apomucins are underglycosylated and abnormally compartmentalized. CONCLUSIONS Enhanced expression of MUC2, MUC4, and MUC5C genes is a frequent event in pancreas cancer and may contribute to the alterations in the biochemical structure of pancreas cancer mucins.

In situ hybridization shows distinct patterns of mucin gene expression in normal, benign, and malignant pancreas tissues

BACKGROUND & AIMS Northern blotting and immunohistochemistry have shown cell-specific patterns of mucin gene expression in the pancreas and alterations associated with neoplastic transformation. The aim of this study was to determine the presence of mucin transcripts at the single cell level in tissue samples from normal pancreas and benign and malignant pancreatic proliferative lesions. METHODS In situ hybridization with 35S-labeled oligonucleotides was performed on sections of paraffin-embedded tissues. RESULTS Acinar cells express only MUC1. Normal pancreatic ducts show homogeneous expression of MUC1 and MUC5B and heterogenous expression of MUC3. MUC2, MUC4, and MUC5AC are generally undetectable in all cells of normal pancreas tissue. Pancreas cancers generally express MUC1, MUC3, MUC4, MUC5B and MUC5AC. Obstructive chronic pancreatitis adjacent to pancreas cancers shows the same pattern of mucin gene expression as normal ducts. In areas of papillary hyperplasia, altered expression of MUC3, MUC5B, and MUC5AC is observed. CONCLUSIONS In situ hybridization has confirmed that neoplastic transformation of the exocrine pancreas is accompanied by changes in mucin gene expression. Although this type of change is not restricted to cancer cells, the findings of this study suggest that analysis of mucin gene expression may be of value in the differential diagnosis of pancreatic lesions.

Exocrine pancreatic cancer: symptoms at presentation and their relation to tumour site and stage

Introduction. The need to detect pancreatic cancer at earlier stages is undisputed. We recorded the signs and symptoms of patients presenting with exocrine pancreatic cancer and evaluated their association with clinical characteristics such as tumour site and disease stage.Patients and methods. All patients (n=185) with exocrine pancreatic cancer newly diagnosed at five general hospitals in Eastern Spain were prospectively recruited over 3 years. Symptoms were elicited through personal interviews and signs were recorded by the attending physician on admission.Results. At diagnosis, one third of tumours of the pancreas head were in stage I and another third in stage IV. None of the tumours of the body and tail were in stage I, and over 80% were in stage IV (p<0.001). At presentation, the most frequent symptoms were asthenia (86%), anorexia (83%), weight-loss (85%), abdominal pain (79%), and choluria (59%). Cholestatic symptoms were more common in tumours affecting only the pancreatic head (p<0.001). There was a clear trend towards more localized tumours with increasing numbers of cholestatic signs (p<0.001). Asthenia, anorexia and weight-loss were unrelated to stage. An increased symptom-to-diagnosis interval was associated with more advanced stage (p=0.048).Conclusions. Proper attention to signs and symptoms, especially cholestasis, may help identify patients with pancreatic cancer at an earlier stage. Results also provide a current picture of the semiology of pancreatic cancer which could be of use in studies on the potential of proteomic tests in the early detection of this neoplasm.

Ki-ras mutations in exocrine pancreatic cancer: Association with clinico-pathological characteristics and with tobacco and alcohol consumption

The aims of this study were (i) to assess the prevalence and spectrum of codon 12 Ki‐ras mutations in patients diagnosed with exocrine pancreatic cancer (EPC) in 2 general hospitals between 1980 and 1990, (ii) to analyze the association of this genetic alteration with clinical and pathological characteristics, and (iii) to determine the association of Ki‐ras mutations with tobacco and alcohol consumption. DNA was amplified from paraffin‐embedded tissue samples and mutations in codon 12 of Ki‐ras were detected using the artificial RFLP technique. Cox proportional‐hazards regression and unconditional logistic regression were applied. Codon 12 Ki‐ras mutations were detected in 30 of 51 cases for which molecular results were available. The amino‐acid substitutions were Asp (8), Val (6), and Arg (3). A double mutation, including always a Val, was detected in 5 cases. None of the 4 non‐ductal pancreatic neoplasms were mutated. The mutation prevalence was 79% in metastases and 54% in primary tumors. The risk of a mutated tumor was 3 times higher in alcohol drinkers than in non‐drinkers, and a linear trend was apparent. When age, gender, hospital, and tobacco and alcohol consumption were taken into account, a high risk for mutations was detected in patients who only smoked and in patients who only drank, but less so in patients who both smoked and drank. These results raise novel hypotheses regarding the role of tobacco and alcohol in EPC. Int. J. Cancer, 70:661–667, 1997.

Validity of the hospital discharge diagnosis in epidemiologic studies of biliopancreatic pathology

Background: The aim was to analyse the magnitude, direction and predictors of change in the main hospital discharge diagnosis (HDD) after a clinical expert review, among patients included in a multicentre molecular epidemiologic study of biliopancreatic diseases. Methods: A total of 602 patients with a suspicion diagnosis of pancreas cancer (PC), cancer of the extrahepatic biliary system (CEBS) or benign biliopancreatic pathologies (BPP) were prospectively recruited at five general hospitals. A structured form was used to collect information from medical records. A panel of experts revised all diagnostic information and established the main clinico-pathological diagnosis (CPD) by consensus. Results: Of the 204 cases with a HDD of PC, 176 (86%) were deemed to have a CPD of PC, eight of CEBS, twelve a neoplasm of different origin, four BPP and four syndromic diagnoses. Thus, 28 cases (14%) were false positives. Of the 129 patients with a HDD of CEBS, 15 (12%) were false positives. Nine of the 396 cases with a HDD of non-PC (2%) had a CPD of PC (false negatives), whilst 14 of 471 patients with a HDD of non-CEBS (3%) were deemed to have CEBS. Overall, sensitivity and specificity of HDD for PC were, respectively, 95 and 93%, and for CEBS, 89 and 97%. Cytohistological confirmation and laparotomy were independent predictors of diagnostic change. Conclusions: Validity of the HDD was high, but its association with some clinical variables suggests that sole reliance on HDD can significantly bias results, and highlights the need to review all HDDs. Alternatively, only patients at high risk of misdiagnosis could be reviewed: primarily, those lacking a cytohistological diagnosis or a laparotomy. No exclusions appear warranted solely on the basis of age, gender or tumour spread.

Generalizing molecular results arising from incomplete biological samples: expected bias and unexpected findings.

PURPOSE In molecular epidemiology, obtaining biological samples for all subjects targeted for study is frequently hampered by ethical, clinical, and logistic factors. The extent to which the incompleteness of biological samples could cause bias is rarely analyzed in depth. Here we report some expected bias and some unexpected findings during a study on mutations in the K-ras gene in exocrine pancreatic cancer (EPC). METHODS In this case-case study, all patients registered with EPC between 1980 and 1990 at two general hospitals were retrospectively identified from the hospital tumor registries. Their clinical records were abstracted and paraffin-embedded samples retrieved from pathology records. DNA was amplified, and mutations in codon 12 of the K-ras gene were detected using the artificial RFLP technique. RESULTS Results on the mutations (RM) were obtained for 51 of the 149 cases of EPC (34.2%). There were no significant differences on the availability of RM by age, gender, and tumor stage at diagnosis, but RM were over five times more likely to be available from one of the hospitals. Subjects with RM were more likely to have received a treatment with curative intent (OR = 11.56, 95% CL: 2.88-46.36). The existence of RM was positively associated with the availability of information on alcohol use and family history of cancer. Subjects with RM tended to belong to higher occupational groups and to smoke less than subjects without RM. Unexpectedly--given that in EPC K-ras mutations have consistently been found unrelated to age, gender, tumor stage, and other clinical factors-, cases with a K-ras mutation were more likely than wild-type cases to have information on tobacco and alcohol use (OR = 3.29, p = .21), medical history (OR = 4.46, p = .41), and family history of cancer (OR = 4.80, p = .01). The relationship between completeness of clinical records and K-ras mutations among cases with RM could not be accounted by age, gender, and occupational group. CONCLUSIONS Simple tests of age and gender distributions among subjects with and without available clinical information and molecular results may not rule out selection and information bias. Studies using biologic specimens are even more in need than classic studies to explain clearly the process followed to include and exclude subjects. Additional caution is needed when generalizing molecular results arising from incomplete biological specimens.

Detection of HuD transcripts by means of reverse transcriptase and polymerase chain reaction: implications for the detection of minimal residual disease in patients with small cell lung cancer

Small cell lung cancer (SCLC) expresses neuroectodermal markers including HuD, the best characterized member of the Hu gene family. The aim of this study is to optimize a simple and sensitive reverse transcriptase-polymerase chain reaction assay to detect circulating HuD-expressing cells for the early detection of SCLC recurrences. HuD-specific primers that selectively amplify the three HuD isoforms allowed the detection of one tumor cell/10(6) non-tumor cells. However, HuD transcripts were also detected in the mononuclear fraction of all samples from normal individuals (n=6) and patients with SCLC (n=5). By contrast, HuD protein was not detected in these fractions using Western blotting. More quantitative assays are necessary to examine the value of HuD transcript detection for the identification of tumor recurrences.