Carlos Gamallo

The transcription factor SNAIL represses vitamin D receptor expression and responsiveness in human colon cancer

Several non-hypercalcemic analogs of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) show antitumor activity in a subset of cancer patients. High vitamin D receptor (VDR) expression, which is associated with good prognosis but is lost during tumor progression. We show that the SNAIL transcription factor represses VDR gene expression in human colon cancer cells and blocks the antitumor action of EB1089, a 1,25(OH)2D3 analog, in xenografted mice. In human colon cancers, elevated SNAIL expression correlates with downregulation of VDR.

Characterization of human PA2.26 antigen (T1α-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas

We report the full cDNA sequence encoding the human homologue of murine PA2.26 (T1α‐2, podoplanin), a small mucin‐type transmembrane glycoprotein originally identified as a cell‐surface antigen induced in keratinocytes during mouse skin carcinogenesis. The human PA2.26 gene is expressed as 2 transcripts of 0.9 and 2.7 kb in several normal tissues, such as the placenta, skeletal muscle, heart and lung. Using a specific polyclonal antibody raised against a synthetic peptide of the protein ectodomain, PA2.26 was immunohistochemically detected in about 25% (15/61) of human early oral squamous cell carcinomas. PA2.26 distribution in the tumours was heterogeneous and often restricted to the invasive front. Double immunofluorescence and confocal microscopy analysis showed that PA2.26 colocalized with the membrane cytoskeleton linker ezrin at the surface of tumour cells and that its presence in vivo was associated with downregulation of membrane E‐cadherin protein expression. Ectopic expression of human PA2.26 in HeLa carcinoma cells and immortalized HaCaT keratinocytes promoted a redistribution of ezrin to the cell edges, the formation of cell‐surface protrusions and reduced Ca2+‐dependent cell‐cell adhesiveness. These results point to PA2.26 as a novel biomarker for oral squamous cell carcinomas that might be involved in migration/invasion.

β-Catenin Expression Pattern in Stage I and II Ovarian Carcinomas : Relationship with β-Catenin Gene Mutations, Clinicopathological Features, and Clinical Outcome

The immunohistochemical expression pattern of β-catenin has been correlated with β-catenin gene mutations, clinicopathological features, and disease outcome in 69 stage I and II ovarian carcinomas. β-Catenin expression was localized in the nuclei, in addition to the cytoplasm and membrane, in 11 tumors (16%). nine endometrioid carcinomas with widespread nuclear expression and two serous carcinomas with focal nuclear expression. The remaining 58 carcinomas (84%) only had membranous β-catenin expression. All but one of the endometrioid carcinomas with nuclear β-catenin expression had considerable squamous metaplasia, and five of these cases had large areas of endometrioid tumor of low malignant potential. In addition, β-catenin nuclear expression was observed in atypical epithelial cells in endometriotic glands adjacent to an endometrioid carcinoma. Sequencing was performed on 25 tumors and corresponding normal tissue: all 13 endometrioid tumors as well as 12 carcinomas of other histological types (four serous, two clear cell, two mucinous, and two mixed). There were oncogenic mutations in the phosphorylation sequence for GSK-3β in exon 3 of the β-catenin gene in seven endometrioid carcinomas with β-catenin nuclear expression. Three mutations affected codon 32 (D32G, D32Y, and D32Y), one affected codon 33 (S33C), two affected codon 37 (S37C and S37F), and one affected codon 41 (T41A). No mutations were observed in the other 18 carcinomas analyzed, comprising two endometrioid and two serous carcinomas with β-catenin nuclear expression, and 14 carcinomas of different histological types with only membranous expression. In the univariate and multivariate survival analyses, β-catenin nuclear expression was selected as an indicator of good prognosis, because no patient whose tumor expressed β-catenin in the nuclei showed relapses or died, in contrast to the 19 relapses and deaths among patients with tumors that only had β-catenin membranous expression, including three of the four patients with endometrioid carcinomas. Oncogenic β-catenin mutation is characteristic of a group of endometrioid carcinomas with a good prognosis, most of which originate from previous benign or borderline lesions. Endometrioid carcinomas with exclusively membranous expression of β-catenin seem to represent a different subgroup of carcinomas that probably have a worse prognosis. In early-stage ovarian cancer, determination of the β-catenin expression pattern could prove to be a useful marker for selecting low-risk patients.

Electrocardiographic findings in acute right ventricular infarction: Sensitivity and specificity of electrocardiographic alterations in right precordial leads V4R, V3R, V1, V2and V3

To determine the sensitivity, specificity, predictive value and diagnostic efficiency of electrocardiographic alterations in the diagnosis of acute right ventricular infarction, 43 autopsy patients with acute myocardial infarction and an electrocardiogram including 12 leads plus leads V3R and V4R were studied. Group A included 21 patients with right ventricular infarction, of whom 14 (group AI) had posterior and 7 (group AII) had anterior right ventricular infarction. Group B included 22 patients without right ventricular infarction. Excluding group AII patients, the sensitivity of the presence of a Q wave reached 78.6% in lead V4R and decreased in leads V1 to V3; its specificity was low in all the leads. The sensitivity of ST segment elevation reached 100% in lead V4R and decreased in leads V1 to V3; its specificity was highest (68.2%) in leads V4R and V3R, its negative predictive value was 100% and its diagnostic efficiency was 80.6%. The criterion of ST segment elevation in lead V4R being higher than that in leads V1 to V3 was less sensitive (78.6%) than ST segment elevation in lead V4R alone, but its specificity reached 100%, its positive predictive value 100% and its diagnostic efficiency 91.7%. In conclusion, there are no electrocardiographic criteria to identify anterior right ventricular necrosis, but posterior right ventricular necrosis may be identified by the presence of a Q wave or ST segment elevation in the right precordial leads, reaching the highest sensitivity and specificity in lead V4R. The criterion of ST segment elevation in lead V4R being higher than that in leads V1 to V3 offers the highest specificity and efficiency in the diagnosis.

Cytoplasmic localization of p120ctn and E-cadherin loss characterize lobular breast carcinoma from preinvasive to metastatic lesions

Accumulating evidences indicate that p120 catenin, a member of the E-cadherin (E-CD)/catenin adhesion complex, plays a role in tumor invasion. To establish the expression pattern of p120 in breast cancer, we analysed 326 breast tissue biopsies by tissue microarray. Most of the lobular tumors (88%) showed exclusive cytoplasmic localization, and 6% of them also had p120 nuclear staining. Cytoplasmic p120 strongly associated with complete loss of E-CD and β-catenin not only in lobular carcinoma and its metastases but also in atypical lobular hyperplasias. In the latter, loss of heterozygosity of E-CD gene was also observed. Complete loss of E-CD and cytoplasmic and nuclear p120 staining was also observed in primary lobular cancer cell cultures generated by us. In ductal tumors, by contrast, reduction of p120 and E-CD in membrane was very common (57 and 53%, respectively), whereas cytoplasmic p120 staining was rarely seen. This simultaneous reduction of membranous E-CD and p120 was not associated with increased Src kinase activity. To demonstrate that cytoplasmic p120 localization was a consequence of the absence of E-CD, the endogenous E-CD was re-expressed in MDA-231 cells by 5-Aza-2′-deoxycytidine (5Aza) treatment. After treatment, p120 shifted from the cytoplasm to the membrane, where it colocalized with endogenous E-CD. Additionally, suppressing E-CD expression in Madin–Darby canine kidney cells by stable transfection of the transcriptional repressors Snail, E47 or Slug, provokes p120 cytoplasmic localization and p120 isoform switching. In conclusion, abnormal cytoplasmic and nuclear localization of p120, which are mediated by the absence of E-CD, characteristically occur in the early stages of lobular breast cancer and are maintained during tumor progression to metastasis. Consequently, p120 may be an important mediator of the oncogenic effects derived from E-CD inactivation, including enhanced motility and invasion, in lobular breast cancer.

Epigenetic and genetic alterations of APC and CDH1 genes in lobular breast cancer: Relationships with abnormal E-cadherin and catenin expression and microsatellite instability

The causes and functional consequences of E‐cadherin (E‐CD) loss in breast cancer are poorly understood. E‐CD loss might act in concert with alterations in the APC/β‐catenin pathway to permit oncogenic β‐catenin signaling. To test this hypothesis, we have analyzed the presence of genetic and epigenetic alterations affecting E‐CD (CDH1), APC and β‐catenin (CTNNB1) genes and the immunohistochemical expression of E‐CD, β‐ and γ‐catenin in a series of 46 infiltrating lobular breast carcinomas (ILCs). Since 80% of ILCs featured complete loss of E‐CD expression, we analyzed the molecular alterations responsible for E‐CD inactivation in these tumors. We found that 10 of 46 (22%) cases harbored mutations in CDH1, including 1 case with 2 different mutations (1 of which was germline). CDH1 was also inactivated by loss of heterozygosity (LOH; 30/41, 73%) and promoter hypermethylation (19/46, 41%). Interestingly, LOH and mutations were also detected in the corresponding in situ lesions of the ILCs, implying that these alterations are early events in lobular cancer tumorogenesis. Additionally, the presence of a polymorphism in the CDH1 promoter was found to be inversely correlated with CDH1 mutations, but not with E‐CD levels. We next examined whether alterations in the APC/β‐catenin pathway also occurred in the same series of ILCs. Although no CTNNB1 or APC mutations were detected, promoter methylation (25/46, 52%) and LOH (7/30, 23%) of APC were found. Moreover, methylation of APC and CDH1 occurred concordantly. However, β‐ and γ‐catenin were severely reduced or absent in 90% of these tumors, implying that alterations in CDH1 and APC genes do not promote β‐catenin accumulation in ILC. These molecular alterations were not associated with microsatellite instability. In summary, several different mechanisms (mutations, LOH, methylation) are involved in the frequent CDH1 inactivation in invasive and in situ lobular breast cancer. The same tumors also show genetic and epigenetic alterations of APC gene. However, altered CDH1 and APC genes do not promote β‐catenin accumulation in this tumor type.

Elective neck dissection in early-stage oral squamous cell carcinoma—does it influence recurrence and survival?

This study investigates the influence on survival and regional control rates of neck dissection therapy at the time of surgery of the primary tumor in early stages of squamous cell carcinoma (SCC) of the oral cavity.

β-catenin Expression Pattern, β-catenin Gene Mutations, and Microsatellite Instability in Endometrioid Ovarian Carcinomas and Synchronous Endometrial Carcinomas

&bgr;-Catenin gene mutations and microsatellite instability (MI) have been reported in endometrioid ovarian carcinomas. In colon but not endometrial cancer, &bgr;-catenin gene mutations are associated with a replication error phenotype and MI. In this study the authors investigate whether &bgr;-catenin mutations and MI are two independent oncogenic pathways in endometrioid ovarian carcinomas. They also evaluate the usefulness of these molecular markers in determining the primary origin of simultaneous tumors in the ovary and endometrium. This study was performed on 26 patients diagnosed with primary endometrioid ovarian carcinoma, five of whom also had pathologically diagnosed primary synchronous endometrioid endometrial carcinoma. Immunohistochemical and molecular analyses indicated that there were 25 primary ovarian tumors with four primary synchronous endometrial cancers and one ovarian metastasis of a primary endometrial carcinoma. All studies were performed on formalin-fixed, paraffin-embedded tissue samples. The &bgr;-catenin expression pattern (nuclear vs. membranous) was analyzed immunohistochemically. Mutations in exon 3 of the &bgr;-catenin gene were studied by polymerase chain reaction, single-strand conformational polymorphism, and direct sequencing. MI status was established by studying BAT-26 and BAT-25 mononucleotide repeats. In the group with 21 single ovarian tumors, 18 (85%) had &bgr;-catenin nuclear expression, eight (38%) had &bgr;-catenin gene mutations (always associated with &bgr;-catenin nuclear expression), and four (19%) had MI. Only one case (5%) had both &bgr;-catenin gene mutations and MI. The mutations affected one of the serine/threonine residues targeted for phosphorylation by glycogen synthase kinase-3&bgr; or adjacent residues. At codon 32, a GAC-to-TAC (D32Y) change was found; at codon 33, two TCT-to-TGT (S33C) changes were found; at codon 37, three TCT-to-TTT (S37F) changes and one TCT-to-TGT (S37C) change were found; and, lastly, one ACC-to-GCC change at codon 41 (T41A) was detected. Four of the 25 endometrioid ovarian carcinomas (16%) had an associated synchronous endometrial carcinoma. There was a higher percentage of &bgr;-catenin mutations (n = 3, 75%) in synchronous ovarian carcinomas than in single ones, although with a similar percentage of MI (n = 1, 25%). &bgr;-catenin mutations were S37C in two cases and D32G in one. One of the four endometrial carcinomas showed an S33C &bgr;-catenin mutation, and two carcinomas had MI. None of the four tumors had both &bgr;-catenin gene mutation and MI. &bgr;-catenin gene mutations were always associated with a nuclear &bgr;-catenin expression pattern, whereas MI was associated with a membranous pattern. In one patient both the ovarian and the endometrial carcinomas had &bgr;-catenin gene mutations, in another patient both tumors showed MI, whereas in the remaining two patients the ovarian carcinomas showed &bgr;-catenin gene mutations and the endometrial carcinomas showed MI. To summarize, the results of this study suggest that &bgr;-catenin mutations and MI could represent two independent pathways in endometrioid ovarian carcinomas because they occur simultaneously very infrequently (in 5% of these cases). &bgr;-catenin mutations are always associated with a nuclear &bgr;-catenin expression pattern, whereas cases with a replication error -plus phenotype showed no abnormal &bgr;-catenin subcellular localization. The study of the &bgr;-catenin expression pattern, &bgr;-catenin mutations, and MI, together with conventional clinicopathologic findings, could aid in distinguishing between the metastatic or independent origin of simultaneous endometrioid ovarian and endometrial carcinomas. Tumors with identical immunohistochemical and molecular features should therefore be considered to have a common origin.

Immunohistochemical characterization of fibroblast subpopulations in normal peritoneal tissue and in peritoneal dialysis-induced fibrosis

Peritoneal fibrosis is one of the most common morphological changes observed in continuous ambulatory peritoneal dialysis (CAPD) patients. Both resident fibroblasts and new fibroblast-like cells derived from the mesothelium by epithelial-to-mesenchymal transition are the main cells involved fibrogenesis. In order to establish markers of peritoneal impairment and pathogenic clues to explain the fibrogenic process, we conducted an immunohistochemical study focused on peritoneal fibroblasts. Parietal peritoneal biopsies were collected from four patient groups: normal controls (n=15), non-CAPD uremic patients (n=17), uremic patients on CAPD (n=27) and non-renal patients with inguinal hernia (n=12). To study myofibroblastic conversion of mesothelial cells, α-smooth muscle actin (SMA), desmin, cytokeratins and E-cadherin were analyzed. The expression of CD34 by fibroblasts was also analyzed. Fibroblasts from controls and non-CAPD uremic patients showed expression of CD34, but no myofibroblastic or mesothelial markers. The opposite pattern was present during CAPD-related fibrosis. Expression of cytokeratins and E-cadherin by fibroblast-like cells and α-SMA by mesothelial and stromal cells supports that mesothelial-to-myofibroblast transition occurs during CAPD. Loss of CD34 expression correlated with the degree of peritoneal fibrosis. The immunophenotype of fibroblasts varies during the progression of fibrosis. Myofibroblasts seem to derive from both activation of resident fibroblasts and local conversion of mesothelial cells.

The prognostic significance of P-cadherin in infiltrating ductal breast carcinoma.

We have immunohistochemically investigated P-cadherin (P-CD) expression in a series of 210 infiltrating ductal carcinomas (IDC) in an attempt to assess the biological and prognostic relevance of P-CD in patients harboring IDCs. Although only 74/210 (35%) of IDCs expressed P-CD in >5% of tumor cells (P-CD–positive carcinomas), categorical analyses revealed that P-CD–positive IDCs were larger (26 ± 21 cm versus 22 ± 11 cm, P = .0568), of higher histological grade (P = .0001), and had more lymph node metastases (P = .0327) than P-CD–negative breast carcinomas. In addition, P-CD–positive tumors were negative for estrogen (P = .0001) and progesterone receptors (P = .0001) and showed reduced E-cadherin expression (P = .0276) more frequently than P-CD–negative tumors. Univariate analysis carried out in 171 patients demonstrated that P-CD expression was also an indicator of poor prognosis (χ2 = 8.292, P = .004), extent of lymph node metastasis (χ2 = 20.854, P = .0000), histological grade (χ2 = 12.908, P = .0016), and negative progesterone receptors (χ2 = 4.116, P = .042). However, only histological grade and nodal metastases emerged as independent prognostic markers in the multivariate analysis. These results suggest that although P-CD expression may be involved in the progression of IDCs, its value as an independent prognostic factor remains to be established.