Variability in the diagnosis of dysplasia in ulcerative colitis by dynamic telepathology.

Telepathology (TP) is the practice of evaluating pathology cases by the digital transmission of diagnostic slides as either static pictures (static TP) or by a continuous flow of pictures from a robotic microscopy (dynamic TP). The diagnostic efficacy of dynamic TP-based consultation services has not been widely tested. Dysplasia arising in association with chronic ulcerative colitis (CUC) is, at present, the most important marker for an increased risk of malignancy in patients with this disease. However, the diagnosis of dysplasia suffers from a significant degree of intra- and interobserver variability which usually necessitates a second opinion prior to definitive treatment. Thus, it is often necessary to obtain expert consultation of potential dysplasia cases by dedicated gastrointestinal pathologists. The aim of this study was to evaluate the utility and interobserver variability of diagnosing dysplasia in CUC with the use of dynamic TP. Dynamic TP was used to evaluate digitalized images of 38 CUC cases with areas considered negative, indefinite, or positive for dysplasia (low or high grade) independently by seven pathologists. Subsequently, all cases were graded by each of the pathologists by light microscopic examination of the H&E-stained glass slides. The degree of intra- and interobserver variability was determined by Kappa statistics. Overall, there was a poor degree of interobserver agreement (K=0.32) among the seven pathologists after analysis of the cases by dynamic TP. The poorest level of agreement was in the indefinite and low-grade dysplasia categories, whereas the highest level was in the negative and high-grade dysplasia categories. Grouping together several diagnostic categories (for instance: Indefinite and low, or low- and high-grade dysplasia) had no significant effect on the level of agreement. The degree of variability in interpretation of cases by microscopic slide analysis was similar (K=0.35). After reviewing all the cases by microscopic analysis of the glass slides, the diagnosis was changed in 51% of the observations; in the majority of these (61%), the grade of dysplasia was decreased. In summary, the use of dynamic TP for consultation in CUC-associated dysplasia has a poor level of interobserver agreement, but does not differ significantly from that obtained by the evaluation of the cases by microscopic slide analysis. Diagnoses rendered by dynamic TP tend to be of a higher grade compared to that obtained by microscopic slide analysis. Thus, although dynamic TP may be used for the consultation of CUC dysplasia cases, more specific criteria are needed in the general categorization of dysplasia in CUC.

Constitutive activation of neuregulin/ERBB3 signaling pathway in clear cell sarcoma of soft tissue.

Clear cell sarcoma of soft tissue (CCSST) represents a highly malignant tumor of the musculoskeletal system that is characterized by the chromosomal translocation t(12;22)(q13;q12) of the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1). In a former microarray expression study, we identified ERBB3, a member of the epidermal growth factor receptor (EGFR) family, as a promising new diagnostic marker in the differential diagnosis of CCSST. Here we show that, besides ErbB3, all CCSST cell lines (n = 8) also express the ErbB2 receptor or the ErbB4 receptor, representing an adequate coreceptor of ErbB3. The phosphorylation status of ErbB3 revealed these receptor pairs to be either constitutively activated in CCSST cells with high neuregulin-1 (NRG1) expression (n = 4) or activatable by exogenic NRG1 in cells showing low amounts of NRG1 mRNA (n = 4). Exogenous NRG1 stimulated the growth of a subset of CCSST cells but did not affect the kinetics of another subset. This difference was not strictly dependent on endogenous NRG1 expression; however, the growth-inhibiting effect of the pan-ErbB tyrosine kinase inhibitor CI-1033 or PD158780 clearly correlated with NRG1 expression indicating an autocrine growth stimulation loop which may constitute an interesting target of new therapeutic strategies in this tumor entity.

Central giant cell granuloma of the jaws: a clinical, radiologic, and histopathologic study of 26 cases.

The clinical behavior of central giant cell granuloma (CGCG) of the jaws is variable and difficult to predict. Clinical data and follow-up information of 26 patients with CGCG were analyzed. Histologic features were correlated with the clinical course of the disease. In 16 patients the CGCGs were asymptomatic; 10 lesions presented with aggressive growth, pain, massive swelling, root resorption, cortical perforation, and/or recurrence. These patients were younger and the lesions were larger than in the nonaggressive group. The histomorphometric analysis proved a significant increase in large giant cells, fractional surface area, and mitotic activity in aggressive CGCG lesions. Immunohistologic investigation (Ki-67 and p53 stain) revealed no significant differences. After surgical treatment, 3 patients with aggressive lesions developed a recurrence. The data show that clinical and histomorphometric features may be reliable indicators for the differentiation between aggressive and nonaggressive CGCG. This should be accounted for to improve the individual planning of the treatment and follow-up.

C-KIT expression in ductal carcinoma in situ of the breast: co-expression with HER-2/neu.

The proto-oncogene c-KIT (CD117) is highly expressed in normal breast epithelium and is decreased in invasive breast cancer. In this study, we analyzed the protein expression and the mutational status of c-KIT in ductal carcinoma in situ (DCIS) of the breast and correlated these findings with nuclear grade, architectural pattern, and expression of HER-2, estrogen receptor (ER)-alpha, and progesterone receptor (PR). C-KIT, HER-2, ER, and PR expression were analyzed immunohistochemically in 106 cases of paraffin-embedded DCIS (85 pure DCIS and 21 DCIS with concurrent carcinoma). Direct sequencing of exons 9 and 11 of the c-KIT gene was performed to analyze the hot spot mutational regions in representative cases. C-KIT expression was found in 55 (52.8%) of all DCIS, correlating with high nuclear grade (P < .0001), comedonecrosis (P < .0001), and solid growth pattern (P = .001). Furthermore, c-KIT expression was strongly associated with HER-2 positivity (P < .0001) and was significantly lower in ER- or PR-positive cases (P = .001 and P = .006, respectively). C-KIT expression alone or co-expression with HER-2 in pure DCIS did not differ significantly from DCIS with invasive component (P = .09). Mutational analysis in 6 c-KIT-positive DCIS revealed no activating mutations in exons 9 or 11. Our findings suggest that the expression of c-KIT protein might define a subset of poorly differentiated, HER-2-positive DCIS with decreased expression of steroid hormone receptors, comedonecrosis, and a solid growth pattern. The implications of c-KIT and HER-2 co-expression for breast carcinogenesis should be further evaluated.

Doxorubicin modulates telomerase activity in Ewing's sarcoma in vitro and in vivo.

Since most tumours escape replicative senescence by re-activation of the enzyme telomerase, telomerase is a promising target in the treatment of cancer and a promising marker for diagnosis and therapeutic response. We evaluated the effects of doxorubicin, one of the most active drugs in the treatment of Ewing's sarcoma, on telomerase in the human Ewing's sarcoma cell line STA-ET-1 in vitro and in STA-ET-1 xenografts in vivo. Telomerase activity (TA) was examined by TRAP-assay and real-time PCR. Real-time PCR was also used to quantify the mRNA expression of the catalytic subunit of telomerase (hTERT). In vitro growth inhibition was determined by the MTT-assay. Tumour xenografts were analyzed for tumour volume, apoptosis, necrosis, and proliferation. Doxorubicin concentrations that inhibited in vitro growth of STA-ET-1 by 50% compared to untreated controls ranged between 0.14 microM after 24 h and 0.01 microM after 72 h. Compared to untreated controls doxorubicin reduced TA in STA-ET-1 at toxic concentrations, but increased TA at non-toxic concentrations. In comparison with untreated xenografts, TA was reduced to 65% and hTERT expression dropped to 25% within 72 h in xenografts treated with 17.5 mg/kg doxorubicin i.p.; both recovered to initial values after 264 h. The rate of proliferating cells dropped to 70% within 96 h and increased thereafter. The highest rates of necrosis and apoptosis were seen after 96 h. hTERT expression co-varied significantly with proliferation but not with TA, apoptosis, and necrosis. No correlation was observed between TA, proliferation, apoptosis and necrosis. The results suggest doxorubicin induces down-regulation of hTERT gene expression that at least in part modulates TA in these tumours.

Androgen receptor expression in ductal carcinoma in situ of the breast: not a helpful marker for classification such as estrogen receptor alpha and progesterone receptor.

Androgen receptor (AR) is known to be expressed in approximately 70 to 90% of invasive breast cancers, but there are still conflicting data in terms of AR expression in ductal carcinoma in situ (DCIS). The aim of this study was to evaluate AR expression in DCIS and to compare these results with nuclear grading and with other common endocrine-related markers. On this basis the authors performed immunohistochemical staining for estrogen receptor (ER)-alpha and ER-beta, progesterone receptor (PR), pS2, her-2/neu, and AR in 59 cases of DCIS (24 low grade, 5 intermediate grade, 30 high grade). They found a strong correlation of expression of ER-alpha (P=0.003), PR (P<0.0001), and nuclear grading. For AR expression, 44.1% of all DCIS were positive, but there was no correlation between nuclear grading (P=0.535) and the expression of the other factors. The authors conclude that AR expression in DCIS is not correlated with nuclear grading and with the expression of other known endocrine-related markers such as ER-alpha and -beta, PR, pS2, and her-2/neu. The immunohistochemical assessment of AR status, therefore, may not help in providing a more objective way of classifying DCIS.

Estrogen receptor alpha and beta, progesterone receptor, pS2 and HER-2/neu expression delineate different subgroups in ductal carcinoma in situ of the breast.

Antiestrogen therapy of ductal carcinoma in situ (DCIS) has become a more common option in reducing risk of developing invasive cancer. Previously, estrogen receptor alpha (ER-alpha) was evaluated as the only predictive factor. Immunohistochemical staining was performed for ER-alpha, estrogen receptor ER-beta, progesterone receptor (PR), pS2 and her-2/neu in 59 cases of ductal carcinoma in situ (DCIS). We observed a positive correlation between the expression of ER-alpha (p=0.003), PR (p<0.001) and histopathological grading. Of the DCIS, 61.5% of ER-beta positive (p=0.046) and 35.5% of PR positive samples showed a coexpression with pS2, whereas 72.1% of pS2 negative DCIS were also negative for ER-beta and 92.9% of PR negative DCIS were negative for pS2 (p=0.012). In contrast, 50.0% of her-2/neu negative DCIS expressed ER-beta receptor (p=0.052). We propose that there are subpopulations of DCIS which can be described by distinct endocrine-associated expression patterns.

Changed adhesion molecule profile of Ewing tumor cell lines and xenografts under the influence of ionizing radiation.

BACKGROUND: Adhesion molecules are involved in cell-cell and cell-matrix interactions and may be informative to characterize intercellular mechanisms of invasion and metastasis. This study was performed to characterize radiation-induced changes in the adhesion molecule profile of Ewing tumor subpopulations on a single cell level. MATERIALS AND METHODS: In the present study, two Ewing tumors were characterized in vitro 4, 24 and 72 hours after radiation with 5 Gy and in vivo in a xenograft model 4, 6 and 15 days after radiation with 30 Gy, together with non-irradiated controls, by five parameter flow cytometry. Directly fluorescence-conjugated antibodies that were directed against adhesion molecules (LFA-1 (CD11a), HCAM (CD44), VLA-2 (CD49b), ICAM-1 (CD54), NCAM (CD56), LECAM-1 (CD62L) and CD86) were used. Annexin V and 7-AAD were used to characterize radiation-induced apoptosis. RESULTS: Tumor cell subpopulations were identified by the expression of adhesion molecules, apoptotic markers and DNA content. Heterogeneous changes of the adhesion molecule profile were identified on tumor cell subpopulations after radiation. The expression of CD11a and CD62L correlated with the expression of apoptosis-associated markers. CONCLUSION: The changes of flow cytometric profile under radiation may potentially correlate with a changed metastatic potential of tumor cell subpopulations.

BC200 RNA in invasive and preinvasive breast cancer.

BC200 RNA, a small functional RNA that operates as a translational modulator, has been implicated in the regulation of local synaptodendritic protein synthesis in neurons. Cell type-specific expression of BC200 RNA is tightly controlled such that the RNA is not normally detected in somatic cells other than neurons. However, the neuron-specific control of BC200 expression is deregulated in a number of tumors. We here report that BC200 RNA is expressed at high levels in invasive carcinomas of the breast. In normal breast tissue or in benign tumors such as fibroadenomas, in contrast, we found that the RNA is not detectable at significant levels. The difference in expression levels between invasive carcinomas and normal/benign tissue was statistically highly significant. Receiver Operating Characteristics analysis of sensitivity and specificity confirmed the diagnostic power of BC200 RNA as a molecular marker of invasive breast cancer. In ductal carcinomas in situ, furthermore, significant BC200 expression was associated with high nuclear grade, suggesting that the presence of BC200 RNA in such tumors may be used as a prognostic indicator of tumor progression. The combined results demonstrate the potential of BC200 expression to serve as a molecular tool in the diagnosis and/or prognosis of breast cancer.

Expression profiling of t(12;22) positive clear cell sarcoma of soft tissue cell lines reveals characteristic up-regulation of potential new marker genes including ERBB3.

Clear cell sarcoma of soft tissue (CCSST), also known as malignant melanoma of soft parts, represents a rare lesion of the musculoskeletal system usually affecting adolescents and young adults. CCSST is typified by a chromosomal t(12;22)(q13;q12) translocation resulting in a fusion between the Ewing sarcoma gene (EWSR1) and activating transcription factor 1 (ATF1), of which the activity in nontransformed cells is regulated by cyclic AMP. Our aim was to identify critical differentially expressed genes in CCSST tumor cells in comparison with other solid tumors affecting children and young adults to better understand signaling pathways regulating specific features of the development and progression of this tumor entity. We applied Affymetrix Human Genome U95Av2 oligonucleotide microarrays representing approximately 12,000 genes to generate the expression profiles of the CCSST cell lines GG-62, DTC-1, KAO, MST2, MST3, and Su-CC-S1 in comparison with 8 neuroblastoma, 7 Ewing tumor, and 6 osteosarcoma cell lines. Subsequent hierarchical clustering of microarray data clearly separated all four of the tumor types from each other and identified differentially expressed transcripts, which are characteristically up-regulated in CCSST. Statistical analysis revealed a group of 331 probe sets, representing approximately 300 significant (P < 0.001) differentially regulated genes, which clearly discriminated between the CCSST and other tumor samples. Besides genes that were already known to be highly expressed in CCSST, like S100A11 (S100 protein) or MITF (microphthalmia-associated transcription factor), this group shows an obvious portion of genes that are involved in cyclic AMP response or regulation, in pigmentation processes, or in neuronal structure and signaling. Comparison with other expression profile analyses on neuroectodermal childhood tumors confirms the high robustness of this strategy to characterize tumor entities based on their gene expression. We found the avian erythroblastic leukemia viral oncogene homologue 3 (ERBB3) to be one of the most dramatically up-regulated genes in CCSST. Quantitative real-time PCR and Northern blot analysis verified the mRNA abundance and confirmed the absence of the inhibitory transcript variant of this gene. The protein product of the member of the epidermal growth factor receptor family ERBB3 could be shown to be highly present in all of the CCSST cell lines investigated, as well as in 18 of 20 primary tumor biopsies. In conclusion, our data demonstrate new aspects of the phenotype and the biological behavior of CCSST and reveal ERBB3 to be a useful diagnostic marker.

Endothelin-1-, endothelin-A-, and endothelin-B-receptor expression is correlated with vascular endothelial growth factor expression and angiogenesis in breast cancer.

PURPOSE: Endothelin-1 (ET-1) and its receptors (ET(A)R and ET(B)R), referred to as the endothelin (ET) axis, are overexpressed in breast carcinomas, and influence tumorigenesis and tumor progression by various mechanisms, including angiogenesis. The objective of the study was to clarify if expression of the ET axis participates in angiogenesis of breast carcinoma EXPERIMENTAL DESIGN: We analyzed expression of ET-1, ET(A)R, ET(B)R, and vascular endothelial growth factor (VEGF) immunohistochemically in 600 tissue array specimens from 200 paraffin-embedded breast carcinomas performing tissue microarray technology. Microvessel density (MVD) was determined by counting microvessels (identified by factor VIII) in each core specimen. RESULTS: Moderate or strong immunostaining was observed for ET-1 in 25.4%, for ET(A)R in 43.7%, and for ET(B)R in 22.2% of breast carcinomas. Of all cases, 44.7% showed significant expression of VEGF. MVD varied between different tumor specimens (range, 0-80; median, 17). We observed a statistically significant correlation between MVD and ET expression status with higher MVD in ET-positive tumors. Moreover, expression of VEGF was found more frequently in tumors with overexpression of the ET axis (each P < 0.001). Staining of VEGF was correlated positively with MVD CONCLUSIONS: These results indicate that increased ET-1, ET(A)R, and ET(B)R expression is associated with increased VEGF expression and higher vascularity of breast carcinomas and, thus, could be involved in the regulation of angiogenesis in breast cancer. Our findings provide evidence that the expression pattern of the ET-axis and in particular of ET(A)R may have clinical relevance in future antiangiogenic targeted therapies for breast cancer.

Endothelin-1, Endothelin-A- and Endothelin-B-receptor expression in preinvasive and invasive breast disease.

Endothelin-1 (ET-1) is overexpressed in breast carcinomas and influences via its receptors (ETAR and ETBR) transformation, differentiation and growth processes in the human breast, but little is known about the ET expression in breast cancer precursors. On this basis we evaluated the expression of ET-1, ETAR and ETBR in a series of breast carcinomas, ductal (DCIS) and lobular carcinoma in situ (LCIS) and normal breast tissue by immunohistochemical (IH) methods. IH staining of ET-1, ETAR and ETBR was performed in 88 invasive breast carcinomas, with adjacent carcinoma in situ and concomitant normal breast tissue. Moderate or strong cytoplasmic immunostaining was observed for ET-1 in 33.3%, for ETAR in 45.3% and for ETBR in 55.7% of invasive breast carcinomas. Comparative analysis of invasive cancer (CA), concomitant carcinoma in situ (CIS) and normal breast epithelium (NBE) revealed a stepwise increase of ET-1 and ETAR expression in the sequence NBE < CIS < CA. ETBR expression tended to be slightly higher in CIS than in CA (NBE versus CIS and NBE versus CA, for ETAR and ETBR, p<0.001, respectively; NBE versus CA for ET-1, p=0.035). Our data suggest that the expression of ET-1, ETAR and ETBR correlates with the acquisition of malignant potential and may be used as a prognostic indicator of aggressive behaviour and invasive potential of premalignant breast lesions.

Secretory carcinoma of the breast: a distinct variant of invasive ductal carcinoma assessed by comparative genomic hybridization and immunohistochemistry.

Secretory carcinomas (SCA) are distinguished from infiltrating ductal carcinomas (IDC) of the breast by their characteristic histomorphology and more favorable prognosis and by the expression of a chimeric tyrosine kinase that is encoded by the ETV6-NTRK3 fusion gene. On this basis, we evaluated 13 SCAs (12 of them with ETV6-NTRK3 gene fusion) by molecular and immunohistochemical (IHC) methods. DNA was obtained from 8 of 13 microdissected SCAs and was analyzed for genetic alterations (GA) by comparative genomic hybridization (CGH). IHC staining was performed for estrogen receptor (ER), progesterone receptor (PR), HER2/neu, and Ki-67 (MIB1) in all 13 cases. Molecular and immunohistochemical results in SCAs were compared with previous data regarding immunohistochemical and molecular characteristics of IDCs. An average of 2.0 GAs (range: 0 to 6) were detected, including recurrent gains of chromosome 8q (37.5%) and 1q (25%) and losses of 22q (25%). Four of 13 (31%) SCAs were positive for ER, and 2 were positive for PR. The mean MIB1-labeling index was 11.4% (range: <1 to 34%). Her-2/neu protein overexpression was detected in 2 cases, including 1 with strong (score 3+) and 1 with weak HER2/neu expression (score 2+). Fluorescence in situ hybridization analysis of the latter case showed no evidence of HER-2/neu-gene amplification. Compared with previous findings in IDCs, SCAs are characterized by a relatively low number of GAs, a low proliferative rate, infrequent HER2/neu protein overexpression, decreased steroid hormone receptor expression, and expression of ETV6-NTRK3 fusion gene. These results support the hypothesis that SCAs have immunohistochemical and genetic features that distinguish them from IDCs of the usual type.

Expression of endothelin-1, endothelin-A, and endothelin-B receptor in human breast cancer and correlation with long-term follow-up.

PURPOSE: Endothelin-1 (ET-1) is overexpressed in breast carcinomas and stimulates tumor cell growth in an autocrine and paracrine fashion via its receptors, ET(A)R and ET(B)R. In this study, we evaluated the expression of ET-1 and ET receptors in breast carcinomas and determined its clinical and prognostic significance. EXPERIMENTAL DESIGN: We analyzed expression of ET-1, ET(A)R, and ET(B)R in 176 breast carcinomas using a semiquantitative immunohistochemical approach. Statistical analysis of clinicopathological variables such as pT stage, pN stage, hormone receptor status, Her-2/neu amplification, histological grade, and long-term follow-up data were performed. RESULTS: We observed a moderate to strong cytoplasmic staining for ET-1 in 69 (43.1%), for ET(A)R in 74 (46.5%), and for ET(B)R in 86 (53.4%) cases of primary breast cancer. A correlation was found between increased ET-1 expression and its receptors with several clinicopathological parameters that characterize aggressive types of breast cancer, with the exception of increased ET(A)R and ET(B)R expression with positive estrogen receptor status. Elevated expression of ET-1, ET(A)R, and ET(B)R was more common in breast carcinomas of patients with lower disease-free survival time and overall survival. In addition, a statistically significant correlation was observed between ET(A)R expression and reduced disease-free survival time (P = 0.041). Interestingly, the prognostic impact of ET(A)R expression was shown to be more pronounced in the subgroup of patients with a putative favorable prognosis according to classic prognostic factors. CONCLUSIONS: Therefore, analysis of ET(A)R expression may improve the prediction of relapse and death and facilitate an individually based risk-directed adjuvant therapy in breast cancer patients.

Relationship between HER2 status and proliferation rate in breast cancer assessed by immunohistochemistry, fluorescence in situ hybridisation and standardised AgNOR analysis.

Lack of standardisation and inaccuracy in HER2 test results may adversely influence patient evaluation and therapy selection. In the present study we applied immunohistochemistry (IHC) using the A0485 and CB11 antibodies and fluorescence in situ hybridisation (FISH) for detection of HER2 in 74 routinely processed breast carcinoma specimens. The rapidity of cellular proliferation was assessed by standardised AgNOR analysis and compared with HER2 status. Protein over-expression was found in 30/74 cases by A0485 and in 20/74 by CB11 antibodies, while amplification was detected in 22/74 carcinomas by FISH. Twenty-seven of 74 tumours were high-level AgNOR expressors (mean AgNOR area >3.369 microm2), 19 of which revealed amplification. The highest concordance between results was achieved by FISH and CB11-IHC (97%), the concordance between FISH and A0485-IHC was 89 and 84% between FISH and AgNOR quantity, respectively. The overall concordance between A0485 and CB11-IHC was 85% with 10 incongruent cases, all scored 2+ by A0485 and 0/1+ by CB11. Eight of the discordant tumours were non-amplified by FISH and 7 were low AgNOR-expressors. Our results indicate that using CB11 antibody, a nearly complete agreement can be achieved between HER2 IHC and FISH in diagnostic paraffin material. Moreover, in 2+ positive IHC cases, the AgNOR analysis may represent an additional tool to select patients as candidates for Herceptin therapy due to the strong negative predictor value.

Analysis of cyclooxygenase-2 expression in human breast cancer: high throughput tissue microarray analysis.

PURPOSE: The objective of this study was to evaluate breast carcinomas for the expression of cyclooxygenase-2 (Cox-2) using a tissue microarray (TMA) and to determine its clinical and prognostic relevance. METHODS: We analyzed Cox-2 expression in 600 samples from 200 breast carcinomas immunohistochemically performing TMA technology and semiquantitative analysis. Results were correlated with various clinicopathological variables and follow-up data. Expression of estrogen receptor, progesterone receptor, Ki-67, and Her-2/neu-oncogene was analyzed and correlated with Cox-2 status. RESULTS: We observed a moderate or strong cytoplasmic staining for Cox-2 in 78 (40.6%) of breast carcinomas. Increased Cox-2 expression corresponded to higher pT stage ( P=0.038), amplification of Her-2/neu ( P=0.032), lymphovascular invasion ( P=0.006), a high MIB-1 labeling index (LI) ( P<0.001), and histological grading ( P=0.013). We also observed an inverse relationship between strong Cox-2 expression and estrogen and progesterone receptor content of tumors ( P=0.037 and P=0.010). However, we could not demonstrate a significant association between Cox-2 staining and overall survival or disease free survival time. CONCLUSIONS: These results suggest that Cox-2 expression is significantly associated with less differentiated and more aggressive breast carcinomas and might therefore be a useful prognostic indicator as well as a target for therapy.

Radiation-induced changes of telomerase activity in a human Ewing xenograft tumor.

AIM: The effect of ionizing irradiation on telomerase activity and further associated biological factors was evaluated in a human Ewing tumor xenograft model on nude mice. MATERIAL AND METHODS: The human Ewing tumor cell line STA-ET-1 was established in a nude mouse model. Initially, the dose-response relationship for the tumor model was established. For the radiation experiments two dose levels were chosen: 5 Gy and 30 Gy. After 5 Gy, there was no significant growth delay whereas after 30 Gy there was a marked growth delay without the induction of a complete remission. Tumors were examined 6, 12, 24, 48, 72, and 96 hours post irradiation. After irradiation with 30 Gy further time points were 6, 9, 12 and 15 days. For each dose and time group, three tumors were evaluated. RESULTS: There was a reduction of telomerase activity after 5 Gy to 50% (not statistically significant) after 3 days; however, after 30 Gy there was a reduction of telomerase activity to 23% of the initial value after 6 days (p = 0.001). Telomerase activity correlated with the expression of human telomerase reverse transcriptase (hTERT), but not with the expression of telomerase-associated protein (TP1) and human telomerase RNA (hTR). The maximal amounts of necrosis or apoptosis after 30 Gy were 19% and 6.9%, respectively. CONCLUSIONS: Ionizing radiation reduces telomerase activity and the expression of hTERT which cannot be explained by the induction of necrosis or apoptosis alone. The reduction of telomerase activity may contribute to delayed cell death after radiotherapy. The combined use of radiation and specific telomerase inhibitors may be a potentially synergistic treatment strategy.

Usual ductal hyperplasia of the breast is a committed stem (progenitor) cell lesion distinct from atypical ductal hyperplasia and ductal carcinoma in situ.

Current classification systems in proliferative mammary gland pathology are based on a two-cell system, recognizing only glandular and myoepithelial lines of differentiation. A third cell type has recently been characterized in normal breast tissue by double-immunofluorescence analysis to express cytokeratin 5 (Ck5) only. These cells were shown to represent progenitor or adult stem cells that give rise to the glandular and myoepithelial cell lineage. The double-labelling technique has been applied to characterize a spectrum of intraductal epithelial proliferations, namely benign usual ductal hyperplasia, atypical ductal hyperplasia, and ductal carcinoma in situ, all of which are thought to represent the gradual steps of a sequence in the development of breast cancer. Immunofluorescence studies with specific antibodies against Ck5, Ck8/18/19, and smooth muscle actin were complemented by western blotting analysis of Ck5 and Ck8/18/19 expression in normal breast tissue and in proliferative lesions. Usual ductal hyperplasia appears to be a Ck5-positive committed stem (progenitor) cell lesion with the same differentiation potential as seen in the normal breast. This is in sharp contrast to atypical ductal hyperplasia/ductal carcinoma in situ, which display the differentiated glandular immunophenotype (Ck8/18/19-positive, but Ck5-negative). These data require the abandonment of the idea of an obligate biological continuum of intraductal proliferations from benign to malignant. This study provides evidence that cells undergoing malignant transformation tend to be fairly advanced in the glandular lineage of differentiation. The committed stem (progenitor) cell model may contribute to a better understanding of both benign proliferative breast disease and breast cancer development.

Genetic imbalances revealed by comparative genomic hybridization in osteosarcomas.

Osteosarcomas are the most frequent bone sarcomas. The molecular chromosomal aberrations in osteosarcomas were analyzed by comparative genomic hybridization (CGH). We studied 47 frozen tumors (41 primary samples, 6 relapses) in osteosarcoma patients registered in the Cooperative Osteosarcoma Study (COSS) protocol. Genomic imbalances were detected in 40 of 41 primary tumors and 6 of 6 relapsed tumors. Gains were more frequent than losses (ratio of 1.3:1). The median number of changes was 16 and 12 in primary and relapsed osteosarcomas, respectively. The median number of aberrations in primary high-grade osteosarcomas (17.0) was significantly higher than in low- or intermediate-grade osteosarcoma subtypes (3.0) (p = 0.038). The most frequent gains included 8q, 1p21-p31 and 1q21-q24, and the most frequent losses were 10q, 5q and 13q. High-level gains were observed on 8q23-q24, 17p13 and 1q21-q24. A gain of 19p (p < 0.001) or loss of 9p (p = 0.027) was more frequent in poor responders than in good responders. Univariate analysis revealed that patients with primary metastases (p = 0.002), poor histologic responses (p = 0.005), high-level gains of 19p (p = 0.012) or losses of 13q14 (p = 0.042) had significantly lower event-free survival (EFS), whereas patients with a loss of 5q (p = 0.007) or a loss of 10q21-22 (p = 0.017) had significantly higher EFS than patients without these aberrations. Multivariate analysis demonstrated that primary metastasis, loss of 13q14 and loss of 5q were independent prognostic factors. The findings of our study seem to be useful for evaluating the prognosis of patients and may finally lead to treatment strategies based on genetic background of osteosarcoma.