The art of diagnosis in head and neck tumors.
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Biomarkers, Tumor↗
Biomedical subjects
C Doglioni
Publications and source records attributed to C Doglioni.
p63, a p53 homologue, is a selective nuclear marker of myoepithelial cells of the human breast.
Myoepithelial cells (MCs) constitute the basal cell layer of normal mammary epithelia, and their identification is of particular diagnostic value because they are retained in most benign lesions while being lost in malignancy. Several MC immunocytochemical markers are currently available for diagnostic purposes, with special reference to smooth muscle-related antigens. p63 is a member of the p53 gene family, and its germline mutations are associated with severe mammary developmental defects in both rodents and humans. Different p63 isoforms have been identified, some of which (DeltaNp63) are preferentially expressed in the epithelial basal cells of different organs and have been considered as possible markers of stem cells/reserve cells. We investigated immunohistochemically 384 samples of normal and diseased human breast, including 300 invasive carcinomas, using four antibodies recognizing all p63 isoforms, or the DeltaNp63 isoforms. Twenty cytologic specimens were also investigated. Furthermore, snap-frozen tissue samples from three fibroadenomas and 10 invasive ductal carcinomas with their paired non-neoplastic tissues and three corresponding lymph node metastases were evaluated for the expression of p63 mRNA by RT-PCR. In normal breast tissue p63 immunoreactivity was confined to the nuclei of MCs. In all benign lesions p63-immunoreactive cells formed a continuous basal rim along the epithelial structures. Stromal cells, and in particular myofibroblasts, were consistently unreactive. Adenomyoepitheliomas showed nuclear staining in most neoplastic cells. A peripheral rim of p63-immunoreactive cells was retained surrounding lobular and ductal carcinoma in situ, although it was discontinuous as opposed to the normal structures. Invasive breast carcinomas were consistently devoid of nuclear p63 staining, with the exception of the two adenoid-cystic carcinomas, of the two ductal carcinomas with squamous metaplasia, and of 11 (4.6%) ductal carcinomas not otherwise specified, showing p63 immunoreactivity in a minor fraction (5-15%) of the neoplastic cells. In comparison with other MC markers, p63 was the most specific, being restricted exclusively to MCs, whereas antibodies to smooth muscle actin and, to a lesser extent, calponin also decorated stromal myofibroblasts. In the cytologic preparations p63 immunoreactivity was a consistent feature of "naked nuclei" and of a subset of cells surrounding benign epithelial clusters. RT-PCR experiments with primers specific for different p63 isoforms documented that normal tissues and fibroadenomas preferentially expressed the DeltaNp63 isoforms. Our study demonstrates that in normal and pathologic breast tissues MCs consistently express the DeltaNp63 isoforms. We suggest p63 as a reliable, highly specific, and sensitive MC marker in both histologic and cytologic preparations. Furthermore, because p63 immunoreactivity in adult epithelia is normally restricted to progenitor cells, it can be speculated that it might be a clue for the identification of the still elusive breast progenitor cells.
Biomarkers, Tumor↗
[Stomach lymphomas: minimum diagnostic requirements for gastrointestinal histopathological diagnosis].
Gastric lymphomas have been the subject of intensive studies in the last years and important progress has been made regarding their etiopathogenesis and therapy. Diagnosis of gastric lymphoma is usually made on bioptic material taken at endoscopy. Histopathologic diagnosis is frequently difficult. This paper summarizes the main diagnostic criteria of this setting. It analyses the morphological, immunohistochemical and molecular features to be considered for the differential diagnosis between: reactive process vs low grade B-cell gastric MALT lymphoma, B-cell MALT lymphoma vs other low grade lymphomas involving the stomach and low grade vs high grade gastric lymphoma. The histopathological aspects of gastric biopsies after antibiotic treatment for Helicobacter pylori eradication and the role of molecular analysis in the follow-up of these patients are also considered.
Algorithms↗
Constitutive p63 expression in airway basal cells. A molecular target in diffuse lung diseases.
The p63 gene has high homology with p53, but more complex physiologic functions, including the regulation of the maintenance of basal cells in stratified epithelia. These cells in fact express high levels of the deltaN-terminal truncated isoforms of the p63 gene that can act as dominant-negative inhibiting the activity of p53. Basal cells in human bronchi and bronchioli seem to use the same strategy, since they constitutively express high levels of p63, at variance with alveolar pneumocytes. Over-expression of these isoforms in airway basal cells can inhibit important functions of the p53-pathway, including cell cycle arrest and apoptosis. This finding underlines the key role of p63 in epithelial renewal in human lung, with important implications in the understanding of the mechanisms of tissue remodelling occurring in diffuse lung diseases.
Apoptosis↗
[Herceptin: a new drug finding new life in the determination of c-erb-B2 in breast cancer].
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Antibodies, Monoclonal↗
p27(kip1) expression in breast carcinomas: an immunohistochemical study on 512 patients with long-term follow-up.
p27(Kip1) (p27), a cyclin-dependent kinase inhibitor, has an important role in the progression of cells from G(1) into S phase of the cell cycle. p27 may act as a tumor suppressor, and several reports suggest that loss of its expression in breast carcinoma is related to tumor progression and poor prognosis. We evaluated p27 immunohistochemical expression in 512 consecutive cases of breast carcinoma with 9 years of median-term follow-up. p27 expression was heterogeneous and frequently less intense than in normal cells. Low p27 expression (<50% of reacting cells) was associated with grade III tumors, N0 status, estrogen receptor-negative status, and low cyclin D1 expression. In the whole series of cases, p27 expression did not predict outcome. In node-negative cases (249 patients), high p27 expression indicated poor prognosis. p27 was not prognostically relevant in the group of 223 patients with pT1 disease or in the group of 154 patients <50 years of age. We also investigated the prognostic value of the combined expression of p27 and cyclin D1, but no differences in survival were seen in this bivariate analysis.
Adult↗
Bidirectional induction of the cognate receptor-ligand alpha4/VCAM-1 pair defines a novel mechanism of tumor intravasation.
Engagement of cell surface adhesion receptors with extracellular constituents and with cellular counter-receptors is crucial for the extravasation of blood-borne neoplastic cells and their seeding at distant sites; however, the early events of tumor dissemination-ie, the intravasation step(s)-have been largely neglected. A role for the alpha4beta7 integrin was hypothesized to explain the high leukemogenicity exhibited by one (NQ22) among several T-cell lymphomas studied. To clarify the mechanisms of early aggressivity, the behavior of highly and poorly leukemogenic cell lines were compared in vitro. Cocultivation of physically separated leukemic cells with resting endothelial cells resulted in the up-regulation of VCAM-1 expression. NQ22 cells expressed mRNA of different cytokines that up-regulate VCAM-1 and at higher levels than cells of a nonaggressive lymphoma, and they migrated more efficiently through an activated endothelial cell layer. With the use of neutralizing antibodies against interferon-gamma, granulocyte macrophage colony-stimulating factor, and tumor necrosis factor (TNF)-alpha, it was determined that TNF-alpha is one of the soluble factors released by NQ22 cells involved in the up-regulation of VCAM-1. The finding that vascular cells within the early local growth were strongly positive for VCAM-1 indicated that NQ22 cells could activate endothelial cells also in vivo. Finally, cocultivation of preleukemic alpha4(-)NQ22 cells with TNF-alpha-activated endothelial cells induced the expression of alpha4 integrins on the former cells. Reciprocal up-regulation and engagement of alpha4/VCAM-1 pairs determined the sequential transmigration and intravasation steps, and similar mechanisms might affect the aggressivity of human T lymphoblastic lymphomas.
Animals↗
Coordinated expression and amplification of the MDM2, CDK4, and HMGI-C genes in atypical lipomatous tumours.
Atypical lipomatous tumours (ALTs) represent a distinctive subset of mesenchymal neoplasms featuring mature adipocytic differentiation. Most ALTs are characterized cytogenetically by the presence of supernumerary ring and/or long marker chromosomes derived from the chromosomal region 12q13-15. The 12q13-15 chromosome region contains several genes which may play an important role in human tumorigenesis. A series of ALTs was analysed by investigating the MDM2, CDK4, and HMGI-C genes and their proteins. The study was extended to a series of ordinary lipomas, to determine whether the immunohistochemical investigation of these gene products might play any diagnostic role. Cytogenetic analysis revealed the presence of various cytogenetic aberrations involving the 12q13-15 region in 11/18 (61%) lipomas and of ring chromosomes in all ALTs. Overexpression of mdm2 protein was observed in 6/12 (50%) atypical lipomatous tumours. All lipomas were mdm2-negative. cdk4 overexpression was present in 100% of ALTs. Weak cdk4 immunopositivity was detected in 2/18 (11%) ordinary lipomas in a minority of cells. HMGI-C immunopositivity was observed in 10/12 (83%) ALTs. Positive immunoreactivity was also observed in 8/18 (44%) lipomas. Southern blot analysis revealed amplification of the CDK4 and MDM2 genes in 3/5 ALTs analysed. HMGI-C was amplified in 3/5 cases and was deleted in one case. Mutation analysis of the CDK4 gene did not demonstrate any mutation. These data support the hypothesis that ordinary lipomas may form a molecular genetic and morphological continuum with ALT. At one end of the spectrum are lipomas characterized by 12q13-15 rearrangements and HMGI-C activation and at the other end are ALTs with ring chromosomes, 12q13-15 amplification with overrepresentation of the HMGI-C, CDK4 or MDM2 genes, and aberrant cdk4, mdm2, and HMGI-C protein expression. These findings not only provide insights into the molecular pathogenesis of lipomatous tumours, but also indicate that the immunohistochemical analysis of mdm2 and cdk4 may help to increase diagnostic accuracy.
Adult↗
Low expression of p27 and low proliferation index do not correlate in hairy cell leukaemia.
The molecular basis accounting for the peculiar clinical and biological features of hairy cell leukaemia (HCL) is currently unknown. Deregulation of cell cycle genes plays a significant role in oncogenesis and there is considerable evidence suggesting that Cdk inhibitors (Ckis) function as tumour suppressors. We and others have recently demonstrated low expression of Cki p27 in very aggressive neoplasms and high-grade lymphomas. To investigate whether HCL cases express normal p27 protein, as in other low-grade lymphomas with a low proliferation index, 58 cases of HCL were characterized using a sensitive biotin-streptavidin-immunoperoxidase technique and specific antibodies against p27. All HCL cases showed either no or very weak reactivity, in contrast to other types of low-grade B-cell lymphoma [22 cases of chronic lymphocytic leukaemia (CLL), 12 cases of gastric marginal B-cell lymphoma (MALT), 16 cases of follicular lymphomas and two cases of splenic marginal zone lymphomas]. To investigate the possible mechanism(s) accounting for the low p27 expression observed in hairy cells, multiple approaches were used. According to these molecular studies, low levels of p2 7 are not as a result of (1) increased ubiquitin-mediated degradation, (2) decreased levels of p27 transcription or (3) p27 somatic mutations and/or allelic loss. These findings suggest that low p27 protein expression in HCL may be achieved through post-transcriptional regulation. Finally, our data demonstrate that p27 expression in HCL does not correlate with either cell cycle progression or proliferation index, suggesting that low levels of p27 in hairy cells may be associated with their unique stage of B-cell differentiation and/or the activation of as yet unknown pathways.
Blotting, Western↗
Cytolytic mechanisms of intraepithelial lymphocytes in coeliac disease (CoD).
The effector arm of the mucosal immune system comprises lymphocytes scattered at intraepithelial and lamina propria levels. Intraepithelial lymphocytes (IEL) are a large population of oligoclonal resting cells which exhibit phenotypic and functional characteristics of cytolytic T cells when activated. Several mechanisms have been demonstrated to account for their cytotoxicity. Among them, one is mediated by perforin and granzyme molecules, another is mediated by Fas ligand (FasL) which delivers apoptotic signals through Fas receptor on target cells. There is good evidence that a flat intestinal mucosa may be produced by activated T cells. The aim of our study was to evaluate FasL and perforin expression by IEL, and its possible correlation with the increased enterocyte apoptosis in coeliac mucosa. Endoscopic duodenal biopsy specimens from 10 untreated coeliac patients, 10 treated coeliac patients, and 10 biopsied controls were evaluated for enterocyte apoptosis by terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick end label method, for perforin expression by immunohistochemistry, and for FasL expression by immunocytochemistry. In untreated CoD there was a significant increase of percentage of both FasL+ and perforin+ IEL which positively correlated with enterocyte apoptosis in comparison with controls. All these parameters were significantly lower in treated CoD, even though they did not normalize. Our study demonstrates that in untreated CoD FasL and perforin expression by IEL is increased, and significantly correlates with the level of enterocyte apoptosis.
Adolescent↗
Calretinin expression in ameloblastomas.
AIMS: Calretinin is a 29-kDa calcium-binding protein which is expressed in the central and peripheral nervous systems as well as in many other normal and pathological human tissues. The objective of this study was to determine whether calretinin was expressed in the epithelium of ameloblastomas. METHODS AND RESULTS: Twenty-seven cases of unicystic ameloblastoma and 31 cases of solid and multicystic ameloblastoma were studied. Five-micron sections were immunohistochemically stained using either antiserum 7696 from Swant, or antiserum 18-0211 from Zymed with a standard ABC method. The results were identical with the two antisera. Positive staining was seen in 22 cases (81.5%) of unicystic ameloblastomas. This generally consisted of diffuse, intense nuclear and cytoplasmic staining of several cell layers of the more superficial cells both in the characteristic and nondescript areas of the cyst linings. In the solid and multicystic ameloblastomas, staining occurred in 29 (93. 5%) of the cases. The staining was almost always restricted to the stellate reticulum-like epithelium, was generally intense and diffuse in distribution. Areas of squamous metaplasia stained particularly intensely as did the cells surrounding micro- and macro-cysts. In both groups of lesions, most of the cases that did not stain were intensely inflamed. CONCLUSIONS: The biological significance of calretinin expression in ameloblastomas is not known and its use as a distinctive, specific immunohistochemical marker for ameloblastic tissues remains to be confirmed. However, the results of this study raise the possibility that calretinin may be an important diagnostic aid in the differential diagnosis of cystic and solid ameloblastic tumours.
Ameloblastoma↗
Parvalbumin is expressed in normal and pathological human parathyroid glands.
The parathyroid glands are of major importance in calcium homeostasis. Small changes in the plasma calcium (Ca2+) concentration induce rapid changes in parathyroid hormone (PTH) secretion to maintain the extracellular Ca2+ levels within the physiological range. Extracellular Ca2+ concentration is continuously measured by a G-protein-coupled Ca2+-sensing receptor, which influences the expression and secretion of PTH. The mechanism of signal transduction from receptor sensing to PTH secretion is not well understood, but changes in PTH secretion are tightly linked to changes in the cytosolic Ca2+ concentration. Using immunohistochemistry and Western blot analysis, we detected the EF Ca2+ binding protein parvalbumin (PV) in normal and in hyperplastic and adenomatous human parathyroid glands. The strongest PV signal was present in chief cells and water clear cells, whereas in oxyphilic cells only a weak signal was observed. Immunohistochemistry and in situ hybridization of the PTH indicated a co-localization of PV and PTH in the same cell types. Because changes in the cytosolic Ca2+ concentration are believed to influence the process of PTH secretion, a possible role of PV as a modulator of this Ca2+ signaling is envisaged.
Adenoma↗
The role of immunohistochemistry in the differential diagnosis of serous effusions.
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Biomarkers↗
Prognostic markers in malignant mesothelioma.
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Biomarkers↗
Twist is a potential oncogene that inhibits apoptosis.
Oncogene activation increases susceptibility to apoptosis. Thus, tumorigenesis must depend, in part, on compensating mutations that protect from programmed cell death. A functional screen for cDNAs that could counteract the proapoptotic effects of the myc oncogene identified two related bHLH family members, Twist and Dermo1. Both of these proteins inhibited oncogene- and p53-dependent cell death. Twist expression bypassed p53-induced growth arrest. These effects correlated with an ability of Twist to interfere with activation of a p53-dependent reporter and to impair induction of p53 target genes in response to DNA damage. An underlying explanation for this observation may be provided by the ability of Twist to reduce expression of the ARF tumor suppressor. Thus, Twist may affect p53 indirectly through modulation of the ARF/MDM2/p53 pathway. Consistent with a role as a potential oncoprotein, Twist expression promoted colony formation of E1A/ras-transformed mouse embryo fibroblasts (MEFs) in soft agar. Furthermore, Twist was inappropriately expressed in 50% of rhabdomyosarcomas, a tumor that arises from skeletal muscle precursors that fail to differentiate. Twist is known to block myogenic differentiation. Thus, Twist may play multiple roles in the formation of rhabdomyosarcomas, halting terminal differentiation, inhibiting apoptosis, and interfering with the p53 tumor-suppressor pathway.
Animals↗
Activation of infiltrating cytotoxic T lymphocytes and lymphoma cell apoptotic rates in gastric MALT lymphomas. Differences between high-grade and low-grade cases.
In this study, we have characterized infiltrating T lymphocytes from 13 low-grade and 17 high-grade primary gastric MALT lymphomas by immunohistochemistry, with particular regard to the presence, activation, and topographic distribution of cytotoxic effectors. Although the prevalence of CD4+ and CD8+ cells was similar in low- and high-grade lymphomas, higher numbers of TIA-1+ cytotoxic effectors were found in this latter group of cases (11.6 versus 7. 8%; P = 0.004). Activation of CD8+ cytotoxic T lymphocytes (CTLs) was significantly more pronounced in high- than in low-grade lymphomas, as shown by immunostaining for perforin (8.7 versus 4.0%; P = 0.001) and granzyme-B (GrB) (8.7% versus 3.0%; P < 0.0001). Of note, CD20/GrB double labeling showed that high-grade lymphomas carried a markedly higher content (about ninefold) of activated CTLs relative to the number of CD20+ lymphoma B cells (0.081 +/- 0.076 versus 0.009 +/- 0.011; P < 0.0001). Moreover, high-grade lymphomas showed significantly increased apoptotic rates compared to low-grade cases (5.3 and 1.1% of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, respectively; P < 0.0001). In the whole series, the percentage of GrB+ cells and the GrB+/CD20+ ratio showed a strong linear correlation with the number of TUNEL-labeled cells. These findings, together with the frequent colocalization of CTLs and TUNEL+ neoplastic cells, suggested that apoptotic death of lymphoma cells may be due at least in part to the killing by cytotoxic effectors. Our results are consistent with the occurrence of host antitumor cell-mediated immune responses in gastric MALT lymphomas. Moreover, the finding of stronger cytotoxic responses in high- than in low-grade cases is of potential usefulness in the design of more effective therapeutic strategies for the management of these disorders.
Antigens, CD20↗
High prevalence of activated intraepithelial cytotoxic T lymphocytes and increased neoplastic cell apoptosis in colorectal carcinomas with microsatellite instability.
Microsatellite instability (MSI) characterizes colorectal carcinomas (CRCs) in hereditary nonpolyposis colorectal cancer (HNPCC) syndrome and a proportion of sporadic CRCs. These MSI+ CRCs share several clinicopathological features, including a reputation for better survival rates than MSI- cases and a pronounced stromal inflammatory reaction of still undefined nature. In the present study, the presence, spatial distribution, and activation status of infiltrating cytotoxic effectors were investigated comparatively in 18 MSI+ and 37 MSI- CRCs by immunohistochemistry. The frequency of apoptosis was also evaluated by morphology and in situ end-labeling. MSI+ cases carried significantly higher numbers of cytotoxic lymphocytes infiltrating within neoplastic epithelial structures, as shown by immunostaining for CD3 (15.1 +/- 6.2 versus 4.6 +/- 4.1, P < 0.001), CD8 (13 +/- 6.4 versus 3.7 +/- 3.8, P < 0.001), and TIA-1 (11.2 +/- 6.5 versus 1.9 +/- 1.7, P < 0.001). These cytotoxic effectors were globally more activated in MSI+ than in MSI- tumors, as revealed by the expression of granzyme B (5.3 +/- 4.5 versus 0.6 +/- 1.3, P < 0.001). In MSI+ CRCs, the number of intraepithelial activated cytotoxic lymphocytes was significantly correlated with the proximal location of the tumor, a poorly differentiated phenotype, and the presence of peritumor lymphoid nodules. Multivariate analysis revealed that MSI was the major determinant of the presence of activated cytotoxic intraepithelial lymphocytes. Moreover, MSI+ CRCs also showed a significantly higher percentage of tumor cells undergoing apoptotic cell death (4.1 +/- 2.1 versus 2.6 +/- 1.1, P < 0.0001, by the TUNEL method), often located in close proximity of activated cytotoxic lymphocytes. These results are consistent with the presence of anti-tumor cytotoxic immune responses in most of MSI+ CRCs, a phenomenon that may at least in part contribute to the survival advantage ascribed to these patients.
Adult↗