Barbara Dockhorn-Dworniczak

Sensitization for death receptor- or drug-induced apoptosis by re-expression of caspase-8 through demethylation or gene transfer

Resistance of tumors to treatment with cytotoxic drugs, irradiation or immunotherapy may be due to disrupted apoptosis programs. Here, we report in a variety of different tumor cells including Ewing tumor, neuroblastoma, malignant brain tumors and melanoma that caspase-8 expression acts as a key determinant of sensitivity for apoptosis induced by death-inducing ligands or cytotoxic drugs. In tumor cell lines resistant to TRAIL, anti-CD95 or TNFα, caspase-8 protein and mRNA expression was decreased or absent without caspase-8 gene loss. Methylation-specific PCR revealed hypermethylation of caspase-8 regulatory sequences in cells with impaired caspase-8 expression. Treatment with the demethylation agent 5-Aza-2′-deoxycytidine (5-dAzaC) reversed hypermethylation of caspase-8 resulting in restoration of caspase-8 expression and recruitment and activation of caspase-8 at the CD95 DISC upon receptor cross-linking thereby sensitizing for death receptor-, and importantly, also for drug-induced apoptosis. Inhibition of caspase-8 activity also inhibited apoptosis sensitization by 5-dAzaC. Similar to demethylation, introduction of caspase-8 by gene transfer sensitized for apoptosis induction. Hypermethylation of caspase-8 was linked to reduced caspase-8 expression in different tumor cell lines in vitro and, most importantly, also in primary tumor samples. Thus, these findings indicate that re-expression of caspase-8, e.g. by demethylation or caspase-8 gene transfer, might be an effective strategy to restore sensitivity for chemotherapy- or death receptor-induced apoptosis in various tumors in vivo.

Comparative genomic hybridization of ductal carcinoma in situ of the breast—evidence of multiple genetic pathways

There is strong evidence that ductal carcinoma in situ (DCIS) represents a precursor lesion of invasive breast cancer. In order to analyse specific chromosomal alterations of DCIS, 38 paraffin‐embedded specimens of DCIS and six associated invasive carcinomas were examined by means of comparative genomic hybridization (CGH). Losses of 16q material were seen almost exclusively in well‐ and intermediately‐differentiated DCIS. These two subgroups differed in the average number of genetic imbalances, 2·5 and 5·5 respectively. Additionally, a higher frequency of gains of 1q and losses of 11q material was seen in intermediately‐differentiated in contrast to well‐differentiated DCIS. Poorly‐differentiated DCIS displayed a higher frequency of amplifications (17q12, 11q13) and a higher average rate of genetic imbalances (7·1). Analysis of adjacent invasive breast carcinoma revealed a genetic pattern almost identical to the one seen in the DCIS counterpart. These data characterize DCIS as a genetically far‐advanced, heterogeneous lesion and as a direct precursor of invasive breast cancer. Copyright

Primary metastatic (stage IV) Ewing tumor: Survival analysis of 171 patients from the EICESS studies

BACKGROUND In the multicenter European Intergroup Cooperative Ewings Sarcoma Studies, localized Ewing tumors of bone were treated by combination chemotherapy with surgery and/or radiotherapy. Patients with primary metastases (pm-pts) were treated in high risk protocols. PATIENTS AND METHODS One hundred seventy-seven pm-pts were registered from January 1990 to December 1995, 171 were evaluable for survival analyses. Thirty-six pm-pts received myeloablative megatherapy with stem cell rescue following conventional treatment. Bilateral whole lung irradiation (WLI) was administered in 57 pm-pts with pulmonary involvement. Event-free survival (EFS) rates were estimated by Kaplan-Meier analysis. Prognostic factors were identified by log-rank statistics, Cox procedures and logistic regression. RESULTS Eighty-nine deaths were recorded by 1 February 1997, EFS four years after diagnosis for all 171 pm-pts was 0.27. EFS for isolated lung metastases was 0.34, for bone/bone marrow (BM) metastases, 0.28, and for combined lung plus bone/BM metastases, 0.14 (P < 0.005). WLI improved outcome in case of isolated pulmonary involvement (0.40 vs. 0.19, P < 0.05). In pm-pts with combined pulmonary/skeletal metastases, intensification by megatherapy and/or WLI improved EFS from 0.00 to 0.27 (P = 0.0001). CONCLUSIONS EFS four years after diagnosis in patients with disseminated Ewing tumors is 0.27. Whole lung irradiation and megatherapy improve outcome in subgroups of patients with disseminated Ewing tumors is 0.27. Whole lung irradiation and megatherapy improve outcome in subgroups of patients with disseminated Ewing disease.

Different genetic pathways in the evolution of invasive breast cancer are associated with distinct morphological subtypes.

Invasive breast cancer shows a wide range of morphological differentiation, associated with differences in prognosis, but as yet, the underlying genetic mechanisms cannot be accounted for. In order to establish a model of the possible progression from the different subtypes of ductal carcinoma in situ (DCIS) to invasive breast cancer, 77 selected cases of invasive breast cancer representing distinct morphological subtypes were investigated by means of comparative genomic hybridization (CGH). There was a high degree of genetic homology between tubular and tubulo‐lobular carcinoma and well‐differentiated DCIS, and between ductal invasive carcinoma G3 and poorly differentiated DCIS. Highly differentiated invasive breast cancers were characterized by a loss of 16q and a low average number of aberrations per case. In high‐grade tumours, losses of this chromosomal region were seen with a much lower frequency in cases with evidence of an aneuploid tumour status. These data demonstrate the close genetic similarity of well‐, intermediately, and poorly differentiated DCIS and distinct morphological types of invasive breast carcinoma, providing further evidence that DCIS is a direct precursor lesion of invasive breast cancer and that various evolutionary genetic pathways exist. Copyright

Ductal invasive G2 and G3 carcinomas of the breast are the end stages of at least two different lines of genetic evolution

Ductal invasive grade (G) 2 and G3 carcinomas represent the majority of invasive breast cancers. Previous morphological and cytogenetic studies have provided evidence that ductal invasive G2 carcinoma may originate from at least two different genetic pathways. The aim of this study was to evaluate further the heterogeneity of G2 breast cancer in comparison with G3 cancers by cytogenetic and quantitative analysis. To this end, 35 cases of ductal invasive G2 and 42 cases of ductal invasive G3 carcinomas were investigated by means of comparative genomic hybridization (CGH) and these findings were correlated with DNA ploidy status, mitotic activity index (MAI), mean nuclear area (MNA), volume per lumen (VPL), and clinico‐pathological parameters. The findings of this study demonstrate that ductal invasive G2 carcinomas, in contrast to ductal invasive G3 carcinomas, have to be interpreted as the morphological end stage resulting from two different cytogenetic and morphological pathways; the loss of 16q material is the cytogenetic key event in the evolution of a subgroup of this entity. By correlating genetic alterations with DNA ploidy status, an extended morphology‐based cytogenetic progression model is presented, with early and late genetic alterations in the pathogenesis of breast cancer. The correlation with MAI gives rise to the hypothesis that these different genetic pathways significantly differ in their proliferation rate. Further studies will be required to elucidate which genes contribute to an altered proliferation rate in these subgroups and to the associated prognosis. Copyright

Alternative lengthening of telomeres is associated with chromosomal instability in osteosarcomas

Telomere maintenance is regarded as a key mechanism in overcoming cellular senescence in tumor cells and in most cases is achieved by the activation of telomerase. However there is at least one alternative mechanism of telomere lengthening (ALT) which is characterized by heterogeneous and elongated telomeres in the absence of telomerase activity (TA). We evaluated the prevalence of TA, gene expression of telomerase subunits and ALT in relation to telomere morphology and function in matrix producing bone tumors and in osteosarcoma cell lines and present evidence of a direct association of ALT with telomere dysfunction and chromosomal instability. Telomere fluorescence in situ hybridization (T-FISH) in ALT cells revealed elongated and shortened telomeres, partly in unusual configurations and loci, dicentric marker chromosomes and signal-free chromosome ends. Free ends give rise to end-to-end associations and may induce breakage-fusion-bridge cycles resulting in an increased number of complex chromosomal rearrangements, as detected by multiplex-FISH (M-FISH). We propose that ALT cannot be seen as an equivalent to telomerase activity in telomere maintenance. Its association with telomere dysfunction and chromosomal instability may have major implications for tumor progression.

Pediatric high-grade astrocytomas show chromosomal imbalances distinct from adult cases.

We studied 23 pediatric high-grade astrocytomas by comparative genomic hybridization. Chromosomal imbalances were found in 10 of 10 anaplastic astrocytomas and 11 of 13 glioblastomas and consisted of +1q (43%), +3q (26%), +1p, +2q, +5q (22%), -22q (34%), -6q, -10q (30%), -9q, -11q, -13q, -16q, and -17p (22%). Anaplastic astrocytomas frequently showed +5q (40%), +1q (30%), -22q (50%), -6q, -9q (40%), and -12q (30%); glioblastomas +1q (54%), +3q (38%), +2q, +17q (23%), -6q, -8q, -10q, -13q, and -17p (31%). Minimal common regions mapped to +1q21-41, +3q27-qter, +2q31-32, +5q14-22, -22q12-qter, -10q23-25, -6q25-qter, -9q34.2, -11q14-22, -16q22-qter, and -17p. High-level gains were located on 1q (7 cases), 2q, 7q (4 cases), 3q (3 cases), 9, 17q (2 cases), 4q, 8q, 18, and 20q (1 case). A significantly shorter survival was found for anaplastic astrocytomas showing +1q (P: < 0.05), MIB-1 proliferation index >25% (P: < 0.001) and glioblastomas (P: < 0.05). Compared with adult cases, +1p, +2q, and +21q as well as -6q, -11q, and -16q were more frequent in pediatric malignant astrocytomas. Among the latter +5q, -6q, -9q, -12q, and -22q were characteristic for pediatric anaplastic astrocytomas and +1q, +3q, +16p, -8q, and -17p for pediatric glioblastomas. Our results show that chromosomal aberrations differ between pediatric anaplastic astrocytomas and glioblastomas as well as between pediatric and adult high-grade astrocytomas, supporting the notion of a different genetic pathway. Furthermore, gains of chromosomal material on 1q might be correlated with a worse prognosis in pediatric anaplastic astrocytomas.

Genetic relation of lobular carcinoma in situ, ductal carcinoma in situ, and associated invasive carcinoma of the breast

Aims—The mutual relation of lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS) of the breast, as accepted precursor lesions of invasive breast cancer, is controversial. Because they display genetic heterogeneity, it is not clear how genetically advanced these entities are and what causes the transition to an invasive carcinoma. Methods—Six cases of LCIS, four of them with associated lobular invasive carcinoma, four cases of intermediately differentiated DCIS with an associated invasive lobular carcinoma, and nine cases of intermediately and poorly differentiated DCIS with associated ductal invasive carcinoma were investigated by means of comparative genomic hybridisation (CGH) after microdissection and immunohistochemical staining of E-cadherin. Results—LCIS was characterised by a low average rate of copy number changes, no evidence of amplifications, and a high rate of gains and losses of chromosomal material at 1q and 16q, respectively. A high degree of genetic homology with well differentiated DCIS was obvious, as reported previously. The cases of intermediately differentiated DCIS with associated lobular invasive components and lobular differentiation revealed striking homologies, and a significant difference of E-cadherin expression. The comparison of preinvasive and invasive breast lesions, irrespective of differentiation within the same patient, revealed no specific alteration that might be associated with invasion. Genetic alterations seen in invasive carcinoma were not necessarily seen in the adjacent precursor lesions. Conclusions—These results provide strong evidence that invasive breast cancer is a disease with multiple cytogenetic subclones already present in preinvasive lesions. Moreover, specific CGH alterations associated with invasion were not observed. Furthermore, the close genetic association between well differentiated and a subgroup of intermediately differentiated DCIS and LCIS led to the hypothesis that LCIS and a subgroup of DCIS are different phenotypic forms of a common genotype.

An immunohistochemical study of the breast using antibodies to basal and luminal keratins, alpha-smooth muscle actin, vimentin, collagen IV and laminin. Part II: Epitheliosis and ductal carcinoma in situ.

A detailed immunohistochemical study has been carried out on 63 breast lesions with epitheliosis, ductal carcinoma in situ and clinging carcinoma (lobular cancerization), using antibodies directed against keratins 5/14 and 14, 15, 16, 18, 19, vimentin, smooth muscle actin, collagen IV and laminin. The results have shown that epitheliosis on the one hand and ductal in situ and clinging carcinoma on the other are immunohistochemically different epithelial lesions. Epitheliosis appears to be epithelial hyperplasia with keratin 5/14 and keratin 14, 15, 16, 18, 19-positive cells. Compared to epitheliotic cells tumor cells of clinging carcinoma, lobular cancerization and ductal carcinoma in situ expressed only luminal keratins 14, 15, 16, 18, 19 in 85% of the cases studied; whereas in 15% there was a basal keratin expression. From our results we conclude that the clinging carcinoma (lobular cancerization) represents the initial morphological step in the development of ductal carcinoma in situ and thus may be interpreted as a minimal ductal neoplasia. With the immunohistochemical demonstration of basal and luminal keratins it may be possible in individual cases to differentiate between benign and malignant in situ lesions of the breast.

Monoclonality in normal epithelium and in hyperplastic and neoplastic lesions of the breast

The clonal nature of neoplastic lesions such as invasive breast cancer and ductal carcinoma in situ (DCIS) has been widely proven by several proliferative, genetic or other malignancy‐associated markers. The aim of this study is to clarify whether benign hyperplastic lesions such as ductal hyperplasia of usual type (DH) and papilloma can be distinguished from neoplastic lesions such as DCIS by X‐chromosome inactivation analysis. Clonal analysis was performed using a polymerase chain reaction‐based assay for non‐random X‐chromosome inactivation of the human androgen receptor gene (HUMARA). Formalin‐fixed and paraffin‐embedded archival tissue of ten DCIS, sixteen DH, nine papillomas, and seven normal terminal ductal lobular units (TDLUs) was laser‐microdissected to avoid contamination with surrounding tissue. All of the cases analysed revealed a monoclonal origin. Furthermore, in one of these cases, opposite X chromosomes were inactivated within the same breast. X‐linked inactivation analysis clearly demonstrates that, at least in the breast, monoclonality is not restricted to neoplastic processes. The data support the hypothesis that the mammary gland is organized into distinct stem cell‐derived monoclonal patches and that TDLUs are monoclonal in origin. Any proliferative lesion arising within such a pre‐existing clonal patch should therefore be clonal, irrespective of whether it originates from one or more patch cells. Thus, X‐chromosome inactivation analysis cannot be considered a valid method for distinguishing between neoplastic and hyperplastic breast lesions. Copyright