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Clinical Cancer Research Vol. 11, 199-205, January 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Overexpression of h-prune in Breast Cancer is Correlated with Advanced Disease Status

Massimo Zollo1,6, Alessandra Andrè1, Antonio Cossu2, Maria C. Sini3, Anna D'Angelo1, Natascia Marino1, Mario Budroni4, Francesco Tanda2, Gianluigi Arrigoni5 and Giuseppe Palmieri3

1 Telethon Institute of Genetics and Medicine, Naples, Italy; 2 Istituto Anatomia Patologica, Università di Sassari; 3 Istituto di Chimica Biomolecolare-Sezione di Sassari, Consiglio Nazionale delle Ricerche, Località Tramariglio; 4 Centro Multizonale di Osservazione Epidemiologica,Azienda U.S.L.1, Sassari, Italy; 5 Ospedale San Raffaele, HSR, Departimento di Patologia, Milan, Italy; and 6 Centro di ricerca per l'ingegneria genetica di Napoli, Naples., Italy

Requests for reprints: Massimo Zollo, Telethon Institute of Genetics and Medicine, Via Pietro Castellino 111, 80131 Naples, Italy. Phone: 39-081-6132218; Fax: 39-081-6132351; E-mail: zollo{at}tigem.it.


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: The h-prune gene is involved in cellular motility and metastasis formation in breast cancer through interacting with the nm23-H1 protein. The aim of this study was to better define the clinical and pathologic role of h-prune in breast cancer patients.

Experimental Design: Using immunohistochemistry, we assessed h-prune and nm23-H1 protein expression in two series of breast cancer patients: (i) in 2,109 cases with pathologic reports on primary tumors and (ii) in 412 cases with detailed clinical information. To assess the role of DNA amplification in gene activation, the h-prune copy number was evaluated by fluorescence in situ hybridization analysis in 1,016 breast cancer cases.

Results: In the patients tested (n = 2,463), 1,340 (54%) had an increased level of h-prune expression; a positive immunostaining for nm23-H1 was observed in 615 of 2,061 (30%) cases. Overexpression of h-prune was associated with multiple gene copy number at chromosome 1q21.3 in a very limited fraction of cases (68 of 1,016; 6.7%), strongly indicating that alternative pathways induce h-prune activation in breast cancer. Multivariate Cox regression analysis showed that neither h-prune overexpression nor decreased nm23-H1 immunostaining is independent prognostic factors. However, a significant association of h-prune overexpression with either advanced lymph node status (P = 0.017) or presence of distant metastases (P = 0.029) was observed.

Conclusions: Although not significantly correlated with overall survival, positive h-prune immunostaining identifies subsets of breast cancer patients with higher tumor aggressiveness. Further investigations using larger collections of advanced breast cancer patients are required for assessing the predictive role of h-prune in breast cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breast cancer is a complex disease, which has a difficult clinical management due to its biological heterogeneity and its wide spectrum of responsiveness to different treatments (1). Over the past few years, knowledge of the molecular mechanisms underlying tumorigenesis have allowed the identification of an increasing number of biomarkers, which have been correlated with cancer prognosis or used as predictors of response to specific treatments, with varying degrees of success (1, 2). The well-established prognostic factors currently used in cases of primary breast cancer include axillary lymph node involvement, histologic subtype, tumor size, nuclear or histologic grade, estrogen and progesterone receptor (ER and PR, respectively) status, and proliferative index (3, 4). Novel tumor markers with potential clinical utility are now awaited, starting from newly identified molecules that are involved in cell transformation, invasion, and metastasis.

The human homologue of Drosophila prune (h-prune) protein belongs to the DHH superfamily of phosphoesterases, which have a cytoplasmic cyclic nucleotide phosphodiesterase activity (5, 6). We reported previously that h-prune is involved in both promoting cellular motility and stimulating expression of genes involved in metastatic pathways (7–9). In this respect, h-prune physically interacts with the nucleoside diphosphate kinase nm23-H1, a known suppressor of cancer metastasis (7). Our recent findings indicate that h-prune may have a role in the metastatic process through specific inhibition of the antimetastasis function of nm23-H1 "in vivo" (8, 9). Indeed, overexpression of h-prune seems to be involved in cancer progression and tumor aggressiveness (8, 9). One potential mechanism leading to h-prune activation may be through the amplification of gene copy number, which has been shown to induce cell proliferation and increased expression levels of h-prune (8, 9). Thus, inhibition of h-prune activity could interfere with the establishment of metastases and represent a new target for future cancer therapy (9).

In the present study, we examined the distribution of both h-prune and nm23-H1 expression in large and well-characterized cohorts of invasive breast carcinomas to determine their association with clinical and pathologic variables, as well as with patients' prognosis.


   PATIENTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients. Patients were collected and considered eligible if they had a histologic diagnosis of invasive breast carcinoma. Cases were retrieved from two archives. The first, from the University Hospital of Basel (Switzerland), which included 1,531 invasive ductal carcinomas, 310 invasive lobular carcinomas, 69 mucinous carcinomas, 65 tubular carcinomas, 48 medullary carcinomas, and 86 other types of invasive carcinomas. Formalin-fixed (4%; buffered), paraffin-embedded tumor samples were thus available from the Institute of Pathology, University Hospital Basel; the Institute for Clinical Pathology, Basel; and the Triemli Hospital, Zurich. The second archive was from the Institute of Pathology at the University of Sassari (Italy) and included 307 invasive ductal carcinomas, 69 invasive lobular carcinomas, 12 mucinous carcinomas, and 24 other types of invasive carcinomas. All of the slides from all of the tumors were reviewed by at least three experienced pathologists who participated in the study (A.C., G.A., and M.C.S.).

All of the clinicopathologic features for each patient, including disease stage at diagnosis, therapy, relapse, disease-free survival, and time of last control (for overall survival), were either obtained from the cancer registry of Basel (10) and Sassari (11) or abstracted from the hospital records. The pathologic stage, the histologic grade (according to Elston and Ellis 12), and the nodal status were obtained from the primary pathology reports.

Disease status at the time of diagnosis was defined depending on the clinical stage, as assessed by medical history, physical examination, blood cell count with white cell differential and biochemistry, and instrumental tests (chest radiography, liver CT scan and/or ultrasound, bone nuclear scan, etc.). Up to 10-year clinical follow-up data were available for 2,299 cases (median = 56 months, range = 2–120 months); 15-year clinical follow-up data were available for 222 patients (median = 138 months, range = 121-176 months).

The use of the specimens and data for this study was approved by the Ethics Committees at both Basel University Hospital and the University of Sassari.

Tissue Microarrays. Tissue microarray construction was as previously described (10, 13). Briefly, tissue cylinders with a diameter of 0.6 mm were punched from representative tumor areas of a "donor" tissue block using a home-made semiautomatic robotic precision instrument. These were positioned in six different recipient paraffin blocks, each of which contained between 342 and 522 individual samples. Four-micrometer sections of the resulting tissue microarray blocks were transferred to an adhesive coated slide system (Instrumedics, Inc., Hackensack, NJ) following standardized procedures (10, 13). The presence of tumor tissue on the multiple-arrayed sample was verified by H&E staining. Tissue microarrays included at least two core sections from different areas of breast cancer from each patient as well as several normal tissues as controls.

Immunohistochemistry. Sections from formalin-fixed, paraffin-embedded tissues were immunostained using an anti-h-prune monoclonal antibody (clone 4G3/4, raised against a recombinant fusion protein of amino acids 1–351; Apotech Co., Switzerland,) and an anti-nm23-H1 antibody (clone K73, specific for the H1 isoform; Apotech). The specificity of both antibodies was previously characterized in detail (8, 9). Immunohistochemistry analysis was done using the Vectastain Elite ABC Kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer's instructions and following a previously described protocol (8, 9). Optimized immunohistochemistry protocols were established by staining representative control histopathology sections of healthy breast tissue. In normal breast tissue, nm23-H1 was homogeneously expressed, whereas expression of h-prune was absent or of low intensity.

Staining was evaluated semiquantitatively, using 54 normal samples randomly positioned in duplicate across the multiple arrays. Intensity and distribution of immunohistochemistry staining was used to classify tumor samples as positive (strong [+++] to moderate [++] staining, homogeneously distributed or presented by large majority of tumor cells) or negative (absent or weak staining [+]) for both h-prune and nm23 expression. For cases with discordant results between the expert pathologists, agreement was achieved after discussion at the microscope.

Fluorescence In situ Hybridization Analysis. Paraffin-embedded tissue microarray sections were treated according to previously reported protocols (13, 14). The PAC 279-H19 clone, spanning the h-prune gene region at chromosome 1q21.3, and a DNA/BAC clone specific for the nm23-H1 gene at chromosome 17q21.3 were labeled by nick translation with dUTP-CY3 (red) and used as probes. The pUC177 clone, corresponding to the pericentromeric region at 1q12, and the pZ17-14 clone, corresponding to the centromeric region of chromosome 17, were labeled by nick translation with dCTP-FluorX (green) and used as controls. The pUC177 clone was kindly provided by Dr. Mariano Rocchi (Istituto di Genetica, Universitá degli Studi di Bari, Bari, Italy). Nuclei were counterstained with 4',6-diamidino-2-phenyl-indole. Two distinct experiments were done for each case. Digital images were captured using an Olympus BX-61 epifluorescence microscope equipped with the appropriate filters for excitation of 4',6-diamidino-2-phenyl-indole, Cy3 (orange) or FluorX (vysis), and with a COHU video and Cytovision software. Hybridization signals on at least 100 intact, well-preserved, and nonoverlapping, nuclei were evaluated by at least two investigators.

Statistical Analyses. The following variables and categories were defined and included in our analyses: pathologic primary tumor size, pathologic nodal status, presence of metastases, ER and PR status, age at diagnosis, histologic tumor type, and overall survival (calculated starting from the time of diagnosis). Some of these variables were missing for some of the patients. In particular, nodal status, presence of metastases, and receptor status were not known in a large fraction of the cases because these variables were not required for the inclusion of the patients in the study.

The survival data analysis was carried out with the statistical package Egret (version 2.0.3). The Cox regression model was done using raw mortality and tumor-specific mortality. The time of overall survival was expressed in months, and the independent variables (h-prune, nm23-H1, pathologic primary tumor size, and pathologic nodal status) were stratified in three age groups: 23 to 44 (n = 252), 45 to 64 (n = 1,146), and 65 to 98 (n = 1,123). Kaplan-Meier estimates were executed through stratifying by h-prune and nm23-H1 immunostaining data. The Pearson's {chi}2 test was used for assessing h-prune and nm23-H1 expression, with the above mentioned pathologic variables (histologic tumor type, pathologic primary tumor size, pathologic nodal status, presence of metastases, ER, and PR). The exact coefficient for sample proportion analysis was calculated to determine all of the significant variables (<0.05 level). All analyses were done with the statistical package SPSS/7.5 for Windows.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemical Analysis. Assessment of h-prune and nm23-H1 expression levels, as well as of h-prune chromosomal copy number, was carried out on tissue microarray sections from the archival tissues of 2,109 patients with both a histologically proven diagnosis of breast carcinoma and available follow-up data. Additional tissue microarray sections from 412 breast cancer patients with a well-assessed stage of the disease (i.e., whose tumor-node-metastasis classification was available) were evaluated for h-prune immunostaining. Overall, the majority of patients had ductal carcinoma as the histologic variant (1,838; 73%) and were >60 years of age (1,425; 57%) at the time of diagnosis. Records of the clinical follow-ups for each patient were available, covering a median period of 59 months (range = 2–176); the majority of the patients were still alive (1,716; 68%), and only a very few cases were lost in the follow-up (12; 0.5%) at the time of this study.

Expression of h-prune and nm23-H1 was evaluated by immunohistochemistry analysis using two specific antibodies (4G3/4 and K73, respectively). Figure 1A and B shows representative examples of immunohistochemistry staining for h-prune in cases from a tissue microarray series. Altogether, a strongly positive cytoplasmic immunostaining for h-prune (referred to as h-prune+) was observed in the majority (1,340; 54%) of the 2,463 tumors tested (58 cases were not assessable); conversely, a positive cytoplasmic immunostaining for nm23-H1 was observed in 615 (30%) of the 2,061 tumors tested. Among the 2,061 tumor tissues evaluated for both h-prune and nm23-H1 expression, an inverse distribution of positive immunostaining was observed (1,180 [57%] breast carcinomas were h-prune+, whereas 615 [30%] were nm23-H1+; Table 1A). No statistical correlation between h-prune and nm23-H1 expression was observed (Table 1A).



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  		Fig. 1 Expression and cytogenetic analysis of h-prune in breast carcinomas. A, IHC analysis of h-prune expression in representative TMA sections; 40x magnification of positive (a) and negative (b) h-prune immunostaining. B, FISH analysis on the same samples using h-prune/PAC279-H19 (red) and control pUC177 (green) as probes.

 

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  		Table 1 Results of immunohistochemistry and FISH analyses for h-prune in the 2,109 series of invasive breast carcinomas

 
Fluorescence In situ Hybridization Analysis. Fluorescence in situ hybridization (FISH) analysis was done on 1,016 assessable tumors from breast cancer patients using as probes a PAC clone (279-H19) corresponding to the h-prune genomic region at chromosome 1q21.3, and a control clone spanning the pericentromeric region at 1q12. Multiple FISH signals at 1q21.3 in >20% of the analyzed nuclei were found in 173 (17%) cases (Table 1B; 1C). Considering the tumors with at least two gene copies per centromere (i.e., at least tetrasomic signals) and even including breast carcinomas with polysomy of entire chromosome 1 due to the simultaneous presence of multiple centromere signals, a very low level of h-prune amplification atchromosome 1q21.3 was observed (68 of 1,016; 6.7%; Table 1B). Using the BAC clone specific for the nm23-H1 gene at chromosome 17q21.3 and the pZ17-14 clone at centromeric region of chromosome 17 as control, no cytogenetic alteration was found at this level in our series. Absence of karyotypic anomalies in cells from normal tissues surrounding the tumors strongly indicated that amplification of the h-prune genomic region is highly specific for breast cancer cells.

The increase in DNA copy number at the h-prune genomic region was significantly associated to the presence of a positive h-prune immunostaining in tumors with at least trisomy (P = 0.027) or tetrasomy (P = 0.033; Table 1B). However, the low frequency of such a cytogenetic alteration strongly suggests that alternative pathogenetic pathways are involved in determining h-prune activation and the increased somatic expression of h-prune in breast cancer.

Correlation between h-prune and Clinicopathologic Variables. To evaluate the clinicopathologic role of h-prune overexpression in breast cancer, immunohistochemistry analyses were done on invasive primary breast tumors using both the series of 2,109 breast cancer cases (with all of the pathologic primary tumor information, and up to 15-year clinical follow-up data; Table 2A) and the series of 412 breast cancer cases (with a complete tumor-node-metastasis (TNM), system for staging cancer classification, and up to 10-year clinical follow-up data; Table 3). Tumor sections from the subset of 2,109 patients were also investigated for nm23-H1 expression by immunohistochemistry analysis (Table 2B). This latter subset was uninformative for the presence of distant metastases; information on nodal status was partially available for such breast cancer patients in terms of presence (N+) or absence (N–) of lymph node involvement (positive h-prune immunostaining was observed in 210 of 482 [44%] N+ cases and 179/496 [36%] N–cases, P = 0.109; analogously, no statistical correlation for nm23-H1+ immunostaining was found in these two groups). Due to this fragmentary information on progression-free survival in our series, the prognostic values of each variable were determined on the basis of the overall survival analysis.


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  		Table 2 Correlation between IHC results and pathologic variables in the series of 2,109 breast cancer patients

 

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  		Table 3 Correlation between h-prune expression and pathological variables in the series of 412 breast cancer patients

 
As of December 2002, 782 (31%) patients have died due to disease, with the median overall survival of the whole sample being 59 months, with a median follow-up of live patients of 72 months. Using Pearson's {chi}2 test, h-prune and nm23-H1 immunostaining were evaluated for association with several pathologic variables: histologic type, tumor grading (as standardized by Elston and Ellis), pathologic primary tumor size, ER, PR, and (when available) pathologic nodal status and presence of metastases. No statistically significant correlations between either h-prune or nm23-H1 expression and tumor type (ductal versus lobular), histologic grade, pathologic primary tumor size, ER, and PR reactivity were observed (Table 2). Positive h-prune immunostaining was instead significantly associated with either the advanced nodal status (N2–N3 group; P = 0.017) or the presence of distant metastases (M1 group; P = 0.029) among the series of 412 breast cancer patients (Table 3). Statistical analysis of this series confirmed the absence of any significant association between h-prune overexpression and primary tumor size (Table 3).Using the Cox model adjusted according to disease stage and age at diagnosis, no prognostic values of either h-prune overexpression [hazard ratio, 0.61; 95% confidence interval (95% CI), 0.31–1.18; P = 0.144] or nm23-H1 immunostaining (risk ratio, 0.98; 95% CI, 0.90–1.07; P = 0.158) were observed (Table 4; Fig. 2). Overall, tumor grading, primary tumor size, and axillary nodal status always remained the variables that were closely correlated to prognosis in this series of breast cancer patients (Table 4).


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  		Table 4 Correlation of histopathological and IHC markers with overall survival

 


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  		Fig. 2 Kaplan_Meier cumulative survival analyses according to h-prune and nm231H1 status. All cases of breast carcinomas evaluated for positive immunostaining of nm23-H1 (A), and h-prune in the series of 2,109 patients (B), or h-prune in the series of 412 patients (C).In A, (dashed line) nm23 positive versus (continous line) nm23 negative. In B, (dashed line) h-prune negative, (continous line) h-prune positive. In C, (dashed line) h-prune negative, (continous line) h-prune positive.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have shown that neither high expression levels of h-prune, which is a member of the DHH superfamily of phosphoesterases, or down-regulated expression of nm23-H1, which physically interacts with h-prune, have a role as independent predictive prognostic factors in the clinical outcome for invasive breast carcinomas. However, h-prune overexpression seems to be associated with the presence of either advanced nodal status (N2–N3 cases) or distant metastases (M1 cases), strongly suggesting that it can be used as a marker for the identification of subsets of breast cancer patients with a more aggressive disease.

The h-prune protein has been previously shown to be expressed at high levels in human sarcomas and breast carcinomas (8). For h-prune function in breast cancer cells, we previously showed the relevance of its cyclic nucleotide phosphodiesterase activity in its inhibition of nm23-H1, the gene for which is known to act as a suppressor of metastasis. This occurs through the direct binding of h-prune to nm23-H1, and the resultant decrease in the amount of free, unbound cellular nm23-H1 molecules (8, 9). This interaction with nm23-H1 greatly increases the h-prune phosphodiesterase activity, and when this is associated with high levels of h-prune, it induces positive effects on cellular motility both in vitro and in vivo (8, 9). These effects have been shown to promote metastasis formation in breast cancer, and seem to be reduced by an anti-h-prune phosphodiesterase inhibitor (9, 15, 16).

Overexpression and increased enzymatic activity of h-prune has thus been suggested to be involved in promoting cancer metastasis through the alteration of nm23-H1 metastasis suppressor activity (17). In particular, it has been suggested that cellular motility may be strongly induced by the amplification ofh-prune in tumor cells in vitro, and that mobilization of neoplastic cells from the site of the primary lesion is necessary, although not sufficient, to produce distant metastases (8, 9). Our findings through FISH analysis have shown that increase of the h-prune copy number is an infrequent event in breast cancer. Such a discrepancy with previous FISH data indicating a higher frequency of h-prune amplification in breast cancer (although these results have been obtained in series with a much more limited number of cases; refs. 8, 9) suggests that different pathogenetic mechanisms may concur for h-prune activation and protein overexpression during breast cancer progression. In this respect, similar observations have been inferred by comparing data from CGH analysis and whole-gene expression in several breast cell lines (18–20). Indeed, a significant number of new potential breast markers are located in chromosomal regions with low levels of DNA gain (2-4 copies per genome) and are sufficient to actively participate in the global deregulation of gene expression that affects cancer progression (a 2-fold change in DNA copy number has been definitively associated with a corresponding 1.5-fold change in mRNA levels; refs. 18–20). On the other hand, there is no clear explanation for the apparently non-specific presence of increased gene copy number in cases with normal expression of h-prune (although, near-diploid karyotypes with few or single structural or numerical anomalies have been reported to be a specific feature in the initial stages of neoplasia; refs. 21, 22).

In the present study, we have analyzed a large series of breast carcinomas and found somatic overexpression of h-prune in the majority of cases tested (1,340 of 2,463; 54%), as determined by immunohistochemistry analysis with tissue microarrays (the advantage of using the tissue microarray approach is the improved standardization, capacity, and speed of the analyses; refs. 10, 13). Our data indicate that strong h-prune immunoreactivity is highly specific for breast cancer cells, since the normal counterpart (epithelial cells) in each case of breast carcinoma consistently failed to express h-prune at comparably high levels (see Fig. 1). However, h-prune+ cases were more or less equally distributed among the different patients' subsets, and no correlations with histologic tumor type, primary tumor size and grading, ER and PR status were observed. Conversely, a significant association of h-prune overexpression with either advanced axillary nodal status or presence of distant metastases was found in our series. Multivariate analyses revealed that both h-prune and nm23-H1 protein levels were not correlated with the overall survival in breast cancer patients, thus not adding precision to the predictive power of the stage of disease (see Tables 2–4). Overall, one could speculate that standardized adjuvant therapies or other, as-yet-unidentified, factors may affect the predictive role of the alterations within the h-prune/nm23-H1 molecular pathway.

Overexpression of h-prune has been shown to interfere with the nm23-H1 antimetastatic activity through a physical protein-protein interaction (7). Increased levels of nm23-H1 expression have been shown to be associated with early disease stages, whereas a lack of nm23-H1 expression has been associated with more advanced stages and more aggressive tumors (8, 9). Conversely, our findings have shown the absence of any correlation between nm23-H1 somatic expression and extent of disease, in terms of primary tumor size and nodal status.

As the presence or absence of distant metastases is a determining factor in the clinical course of breast cancer, the lack of an association between loss of nm23-H1 expression and poor prognosis strongly argues against any association between nm23-H1 expression and distant metastases in our experimental system. Although distal dissemination has been associated with nm23-H1 levels in primary tumors (23), other studies have also failed to identify any prognostic significance of nm23-H1 expression in breast cancer patients (24, 25). Data on distant tumor dissemination are lacking in patients analyzed for nm23-H1 expression from our series, and the establishment of distant metastases has been shown to be particularly associated with the levels of nm23-H1 expression (breast cancer patients with lesions positive for nm23-H1 seem to present significantly reduced distant recurrence-free survival; refs. 23–25). Overall, the real impact of nm23-H1 expression levels on clinical outcome is still a controversial issue in human cancers (23–28) and needs further investigation. On this regard, we started collecting a larger number of additional breast cancer cases with more detailed clinicopathologic information, in order to increase the number of N2-3 and M1 groups, therefore obtaining a clear and definitive result about the role of the nm23-H1 pathway in such a disease.

Identification of new surrogate markers as potential predictive factors will be useful to improve the clinical management of breast cancer patients (i.e., to identify which subset of patients is expected to show either a response or a lack of response to a particular therapy). Nevertheless, the prediction of metastatic potential has been longer indicated as one of the main goals to be pursued to assess the risk subgroups in patients with primary breast cancer (29, 30). Metastasis formation in breast cancer is now continually under investigation, and basic research efforts are under way that are focused on the multistep events occurring in tumor dissemination (i.e., motility, invasion, angiogenesis, and metastatic colonization), in which h-prune seems to participate by increasing metastatic potential (9, 31) . Based on our findings, h-prune represents a new cell-type-specific gene that is associated with disease progression in breast cancer patients. Further studies that must include cohorts of patients undergoing different therapeutic approaches are required to better understand the real clinical significance of the h-prune/nm23-H1 pathway.


   ACKNOWLEDGMENTS
 
We thank Drs. G. Sauter, M. Mirlacher, R. Simon from the Institute of Pathology at the University Hospital of Basel (Switzerland), for providing part of the tumor cases in this study and for helpful discussion.


   FOOTNOTES
 
Grant support: Associazione Italiana Ricerca sul Cancro (M. Zollo and G. Palmieri), Compagnia San Paolo Torino (M. Zollo), Ricerca Finalizzata Ministero della Salute (A. Cossu and G. Palmieri), Regione Autonoma della Sardegna (M. Budroni and F. Tanda), and Telethon Institute of Genetics and Medicine-Telethon Regione Campania (M. Zollo).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/22/04; revised 9/13/04; accepted 10/ 7/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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