Pathological and molecular characteristics distinguishing contralateral metastatic from new primary breast cancer.

BACKGROUND: Breast cancer patients have a cumulative lifetime risk of 2%-15% of developing a contralateral metastatic or ex novo primary cancer. From prognostic and therapeutic viewpoints, it is important to differentiate metastatic from second primary. To distinguish these entities, we investigated whether the pattern of X chromosome inactivation could determine whether the two tumors derived from different progenitor cells. MATERIALS AND METHODS: The clonality of bilateral breast cancer was evaluated through the X-inactivation analysis using the human androgen receptor gene (HUMARA) polymorphism and the histopathologic and molecular results were compared. A different or an identical pattern of X inactivation was considered as indicator of a second primary cancer or not informative, respectively. We considered morphological indicators of a new primary cancer the absence of concordance in the histological type or a better histological differentiation. RESULTS: Ten patients with bilateral breast cancer were evaluated. Morphological criteria indicated that eight were second primary, a conclusion confirmed by the X-inactivation analysis. Two cases classified as recurrence according to morphological criteria were classified as second tumor by molecular analysis. CONCLUSION: Our results show that the HUMARA clonality assay can improve the histological parameters in differentiating metastatic cancer from second primary cancer.

Biological and clinical role of p73 in neuroblastoma.

The p73 gene is a p53 homologue localized at 1p36.3, a chromosomal region frequently deleted in neuroblastoma. p73 was originally considered an oncosuppressor gene. However, it was soon realized that its mode of action did not resemble that of a classic anti-oncogene. The recent discovery of N-terminal truncated isoforms, with oncogenic properties, showed that p73 has a 'two in one' structure. Indeed, the full-length variants are strong inducers of apoptosis while the truncated isoforms inhibit the pro-apoptotic activity of p53 and of the full-length p73. This review summarizes some aspects of p73 biology with particular reference to its possible role in neuroblastoma.

Expression of DeltaNp73 is a molecular marker for adverse outcome in neuroblastoma patients.

The p73 gene is a p53 homologue which induces apoptosis and inhibits cell proliferation. Although p73 maps at 1p36.3 and is frequently deleted in neuroblastoma (NB), it does not act as a classic oncosuppressor gene. In developing sympathetic neurons of mice, p73 is predominantly expressed as a truncated anti-apoptotic isoform (DeltaNp73), which antagonizes both p53 and the full-length p73 protein (TAp73). This suggests that p73 may be part of a complex tumor-control mechanism. To determine the role of DeltaNp73 in NB we analyzed the pattern of expression of this gene in vivo and evaluated the prognostic significance of its expression. Our results indicate that DeltaNp73 expression is associated with reduced apoptosis in a NB tumor tissue. Expression of this variant in NB patients significantly correlates with age at diagnosis and VMA urinary excretion. Moreover it is strongly associated with reduced survival (HR=7.93; P<0.001) and progression-free survival (HR=5.3; P<0.001) and its role in predicting a poorer outcome is independent from age, primary tumor site, stage and MYCN amplification (OS: HR=5.24, P=0.012; PFS: HR=4.36, P=0.005). In conclusion our data seem to indicate that DeltaNp73 is a crucial gene in neuroblastoma pathogenesis.

Preparation of phage antibodies to the ED-A domain of human fibronectin.

The fibronectin (FN) isoform containing the alternative spliced ED-A domain is much more expressed in fetal, tumoral, and regenerating tissues than in normal adult tissues. The ED-A containing FN is up-regulated by numerous cytokines, such as TGF-beta, and, although in normal adult liver the ED-A domain is undetectable, in regenerating rat liver the expression of ED-A is increased and mediates the conversion of fat storing cells to myofibroblasts. Here we describe the selection from a phage display library and the characterization of human antibody fragments directed against the ED-A sequence of FN. As they can be easily radiolabeled with 32P, these antibodies are very highly sensitive reagents for the determination of ED-A levels in tissues and biological fluids; in fact, use of these scFv induced a more than 10-fold increase in sensitivity with respect to the murine monoclonal IST-9. The possibility of preparing a range of human engineered antibodies should facilitate the development of antibody reagents with suitable pharmacokinetics, valency, functional affinity, and effector functions and that could be useful for clinical purposes.

Features of odontogenesis and expression of cytokeratins and tenascin-C in three cases of extraosseous and intraosseous calcifying odontogenic cyst.

To characterize further the nature of calcifying odontogenic cyst (COC), we studied histologically and immunohistochemically an extraosseous and two intraosseous lesions. The extraosseous COC was in continuity with the stratified squamous epithelium of the alveolar mucosa. Immunostaining with monoclonal antibodies showed reactivity of both low- and high-molecular-weight cytokeratins, the degree of coexpression decreasing with the increasing morphological diversity of the cyst/tumour epithelium. Staining for the matrix glycoprotein tenascin-C was seen not only in the connective tissue, where its distribution patterns corresponded to the stage of hard tissue formation, but also in epithelial elements. The staining patterns were analogous to those described during normal tooth formation. Both the morphological characteristics and expression patterns of the various cytokeratin types and tenascin-C implied that COC represents a pathological counterpart of normal odontogenesis. In the case of the extraosseous COC, the correspondence could be traced back to early stages of tooth development.

Extracellular pH controls pre-mRNA alternative splicing of tenascin-C in normal, but not in malignantly transformed, cells.

In cultured normal human fibroblasts, 2 main tenascin-C (TN-C) isoforms are generated by alternative splicing of the single TN-C primary transcript, 8 type III repeats being included or omitted in the mRNA. In these cultured cells, small pH variations of the culture medium (from 7.2 to 6.8) strikingly modify the alternative splicing pattern of the TN-C primary transcript. We report that malignantly transformed cells do not respond to extracellular pH variations as normal cells do. Indeed, malignantly transformed cells kept in culture media at pH values from 6.6 to 7.6 show no variations in the splicing pattern of the TN-C primary transcript and accumulate almost exclusively the large TN-C mRNA. These observations may explain the preferential accumulation in vivo of the large TN-C isoform in the extracellular matrix of different types of neoplasia.

Different human tenascin-C variants in the extracellular matrix of cultured human fibroblasts.

Using an immunoadsorbent prepared with a monoclonal antibody specific for the high molecular mass isoform of human tenascin-C, we purified tenascin-C molecules containing at least one large subunit from the extracellular matrix of cultured normal human fibroblasts. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting analyses have shown that both high and low molecular mass subunits are present in these tenascin-C preparations. Because the monoclonal antibody used is able to bind only the high molecular mass isoform, the present data show that part of the tenascin-C present in the fibroblast extracellular matrix is made up of heterohexameric molecules.

Immunoreactivity of tenascin-C in dentin matrix in dentinogenesis imperfecta associated with osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heterogeneous group of heritable connective tissue disorders, assigned to different mutations in type I collagen genes. A variety of structural abnormalities of dentin have been described in dentinogenesis imperfecta (DI) associated with OI. To clarify further the constitution of the dentin matrix in OI, we immunostained frozen and paraffin sections of deciduous teeth from four patients, each from a different family, with two monoclonal antibodies (MAbs) to the matrix glycoprotein tenascin-C (TN-C). One of the MAbs recognizes an epitope common to all TN-C isoforms (BC-4), and the other is specific for a splicing variant (BC-2). Normal teeth, oral mucosa, and skin were analyzed for comparison. Staining patterns with the two MAbs did not differ markedly. Normal dentin matrix and odontoblasts were lacking reactivity, but the pulp stained clearly. TN-C reactivity was present in the dentin matrix of all teeth obtained from two patients with different OI phenotypes and DI, and in one out of three teeth from one patient who also had DI. The reactivity was distributed in layers, but the staining patterns varied from one patient to another and from tooth to tooth. Intratubular staining seen in a tooth from the patient with clinically and histologically normal teeth was comparable with that present in normal deciduous teeth. The variation in TN-C expression suggests that, besides genetic heterogeneity, epigenetic factors could influence the composition of the dentin matrix in OI.

The alternative splicing pattern of the tenascin-C pre-mRNA is controlled by the extracellular pH.

Alternative splicing of primary transcripts is an ubiquitous and reversible mechanism for the generation of multiple protein isoforms from single genes. Here we report that in cultured normal human fibroblasts, small pH variations of the culture medium (from 7.2 to 6.9) strikingly modify the alternative splicing pattern of the tenascin-C primary transcript. Since such extracellular pH variations occur in many normal and pathological conditions, microenvironmental pH may be an important element for the regulation of RNA alternative splicing in vivo.

Distribution of oncofetal fibronectin isoforms in normal, hyperplastic and neoplastic human breast tissues.

Two different oncofetal fibronectins (FN) have been reported: one, generated by O-glycosylation in the splicing region IIICS that is recognized by monoclonal antibody (MAb) FDC-6, and another, recognized by MAb BC-I, generated by the alternative splicing of the FN pre-mRNA which includes an extra type-III repeat called ED-B. Using these and 2 other MAbs (IST-4 which recognizes all different FN isoforms and IST-6 which recognizes only the FN molecules that do not include the ED-B sequence) we have immunohistochemically studied 171 normal, hyperplastic and neoplastic breast-tissue specimens. Although all normal specimens reacted strongly with MAbs IST-4 and IST-6, they did not show the presence of oncofetal FNs as established by the use of BC-I and FDC-6. In contrast, out of the 97 cases of invasive ductal carcinomas studied, 90 (93%) and 96 (99%) reacted positively with BC-I and FDC-6, respectively, the reaction being observed in the tumoral stroma connective tissue and in tumoral vessels. Furthermore, invasive lobular carcinoma showed less intense and less frequent staining with BC-1 and FDC-6 (10 and 11 out of 14, respectively). We found differences in the distribution of the 2 oncofetal fibronectin isoforms within the same specimens. The most remarkable difference was observed in the tumoral vessels: in invasive ductal carcinoma MAb BC-1 revealed a positive reaction with vessels in 78% of cases while FDC-6 showed such a reaction in only 59% of cases.

The inclusion of the type III repeat ED-B in the fibronectin molecule generates conformational modifications that unmask a cryptic sequence.

We have previously reported an anti-fibronectin monoclonal antibody (mAb) (BC-1) which reacts with an ED-B-containing beta-galactosidase-fibronectin fusion protein but not with an identical beta-galactosidase-fibronectin fusion protein in which the ED-B sequence is omitted. In further experiments aimed at localizing more precisely the epitope recognized by this mAb, we demonstrate that 1) the mAb BC-1 is indeed specific for ED-B-containing fibronectin (FN) molecules even though the epitope recognized by this mAb is localized on the type III homology repeat 7 (the one which precedes the ED-B sequence) and 2) in fibronectin molecules lacking the ED-B sequence, this epitope is masked. We further demonstrate that, to mask the epitope recognized by the mAb BC-1, the presence of at least half of the FN type III homology repeat 9 is necessary. We also report the production of the mAb IST-6 which recognizes only FN molecules in which the ED-B sequence is lacking. These data clearly demonstrate that the presence of the ED-B sequence within FN molecules generates conformational modification in the central part of the molecules that unmasks previously cryptic sequences and masks others.

Differential expression of the fibronectin isoform containing the ED-B oncofetal domain in normal human fibroblast cell lines originating from different tissues.

Fibronectin (FN) polymorphism is due both to alternative splicing of three sequences (ED-A, ED-B, and IIICS) of the primary transcript and to post-translational modifications. The FN isoform containing the ED-B sequence (B-FN), while having an extremely restricted distribution in normal adult tissues, has a high expression in fetal and tumor tissues. On a panel of non-fetal skin, fetal skin, and fetal lung fibroblast cell lines we have studied, through S1-nuclease protection analysis, the expression of the ED-B containing FN mRNA as well as the expression of the ED-B containing FN isoform through immunoblotting and immunofluorescence techniques, using domain specific monoclonal antibodies. The results show that the expression of B-FN in the different fibroblast cell lines has an extremely great variability depending on the developmental stage of the donor and on the tissue of origin. Moreover, we found that SV-40-transformed fibroblasts present a higher expression of B-FN mRNA with respect to their normal counterparts. An increase in the relative amount of the B-FN isoform in normal human fibroblasts was also obtained by treatment with transforming growth factor-beta.

Transforming growth factor beta regulates the levels of different fibronectin isoforms in normal human cultured fibroblasts.

Fibronectin (FN) polymorphism is caused by alternative splicing patterns in at least three regions of the primary transcript of a single gene. Using a monoclonal antibody (Mab) specific for an FN segment (ED-A), that can be included or omitted from the molecule depending on the pattern of splicing, we have examined whether transforming growth factor beta (TGF-beta) and dexamethasone, which are both known to increase the level of total FN, regulate the levels of different FN isoforms. We found that, while dexamethasone does not significantly change the ratio between the total FN and the ED-A containing FN, TGF-beta preferentially increases the expression of the FN isoform containing the ED-A sequence.

Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells.

Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing have offered an explanation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using these monoclonals, we have quantitatively analyzed the expression of the ED sequence in human fibronectin from different sources. The results demonstrated that, at the protein level, the ED segment is not expressed in plasma fibronectin and that, in fibronectin from the tissue culture medium of tumor-derived or simian virus-40-transformed human cells, the percentage of fibronectin molecules containing the ED segment is about 10 times higher than in fibronectin from normal human fibroblasts. These results suggest that in malignant cells the mechanisms that regulate the splicing of mRNA precursors are altered.

Structural differences in the cell binding region of human fibronectin molecules isolated from cultured normal and tumor-derived human cells.

Fibronectins isolated from human plasma (pFN) and from the conditioned media of normal (N-cFN) and tumor (T-cFN) human cells were compared by cathepsin D digestion followed by immunostaining of released fragments with the monoclonal antibody 3E3, specific for the cell binding site. Two different staining patterns were obtained, one specific for pFN and N-cFN, the second common to fibronectins from the 3 different kinds of tumors studied. This indicates structural differences between N-cFN and T-cFN in the cell binding region of the fibronectin molecule.