Daniel Reimer

Clinical Relevance of Dominant-Negative p73 Isoforms for Responsiveness to Chemotherapy and Survival in Ovarian Cancer: Evidence for a Crucial p53-p73 Cross-talk In vivo

Purpose: We aimed to determine the clinical role of the p53 family members p53 and p73 in the responsiveness to platinum-based chemotherapy and survival in ovarian cancer, considering their cross-talk and the p53 polymorphism at codon 72. Experimental Design: A detailed analysis of p53 and p73 in a series of 122 ovarian cancers was done. We used a functional yeast-based assay to determine the p53 mutational status. Red yeast colonies, indicating mutant p53, were subsequently sequenced to determine the specific p53 alteration. p53 mutations were divided into two groups according to their previous characterization in the literature: those that efficiently inhibit transcriptionally active TAp73 function and those that do not. A p53 polymorphism at codon 72 was determined in corresponding normal tissue or blood of ovarian cancer patients. Isoform-specific p73 expression analysis using real-time reverse transcription-PCR has previously been done in the majority of ovarian cancers included in this study. In a retrospective chart review, responsiveness to chemotherapy was assessed, and survival data with long follow-up times were collected. Results: Eighty of 122 (65.6%) of ovarian cancers harbored p53 mutations. p53 mutational status was an important determinant of responsiveness to platinum-based chemotherapy in all patients with a residual tumor of <2 cm in diameter after initial surgery (wild-type versus mutant, P = 0.029). In addition, p53 mutational status was a strong prognosticator for recurrence-free and overall survival (P < 0.0001 and P = 0.003, respectively) in univariate analyses. High expression levels of dominant-negative p73 isoforms (ΔNp73 and ΔN′p73) significantly correlated with chemotherapeutic failure (P = 0.048) and with worse recurrence-free and overall survival in patients with p53 mutant cancers (P = 0.048 and P = 0.005, respectively). Eight p53 mutations, present in 19 cases, were found that efficiently inhibit TAp73 (i.e., 175H, 220C, 245S, 245D, 248W, 248Q, 266E, and 273H). Patients with p53 mutations that efficiently inhibit TAp73 function had a significantly shorter overall survival than patients with p53 mutations of unknown effect on TAp73 (P = 0.044). The p53 polymorphism at codon 72 had no influence on responsiveness to chemotherapy or survival. Conclusion: We provide the first clinical evidence that dominant-negative p73 isoforms contribute to drug resistance in vivo, underscoring the importance of a p53-p73 cross-talk. NH2-terminally truncated p73 isoforms were of significant clinical effect by providing an additional unfavorable factor for response to platinum-based chemotherapy and survival in p53 mutant ovarian cancers.

Clinical Relevance of E2F Family Members in Ovarian Cancer—An Evaluation in a Training Set of 77 Patients

Purpose: The major obstacle in treating ovarian cancer is the rapid development of platinum resistance during therapy. Deregulation of members of the E2F family of transcription factors is crucially involved in carcinogenesis and probably in mechanisms underlying platinum resistance. We therefore investigated the relevance of the whole set of E2F family members in predicting clinical outcome and their significance in predicting platinum resistance. Experimental Design: Real-time PCR of all E2F family members was done from 77 ovarian carcinomas, defined as our training set, and 8 healthy control samples. The correlation with clinicopathologic characteristics, platinum resistance, and survival was investigated. Furthermore, the cross-talk of E2F family members was assessed for its value in predicting survival and platinum resistance. Results: The proliferation-promoting E2F1 and E2F2 were associated with grade 3 tumors and residual disease >2 cm in diameter after initial surgery. Survival analyses showed low expression of E2F1 or E2F2 to be significantly associated with favorable disease-free and overall survival (E2F1, P = 0.039 and 0.047, respectively; E2F2, P = 0.009 and 0.006, respectively). In contrast, high expression of inhibiting E2F4 or E2F7 predicted favorable disease-free and overall survival (E2F4, P = 0.047 and 0.042, respectively; E2F7, P = 0.048 and 0.042, respectively). A high E2F2 to E2F4 ratio was the most valuable prognostic variable for disease-free survival in multivariate analysis (hazard ratio, 6.494; P = 0.002). Tumors considered platinum resistant were associated with lower E2F4 and E2F7 expression (P = 0.012 and 0.009, respectively) compared with platinum-sensitive tumors. Again, ratios of E2F1 or E2F2 to E2F7 were the most favorable variables in predicting platinum resistance. Conclusions: We here show that deregulation of both proliferation-promoting and proliferation-inhibiting E2F transcription factors and their cross-talk is crucially involved in the tumor biology of ovarian cancer and influences clinical outcome. Furthermore, down-regulation of E2F7 may contribute to mechanisms underlying platinum resistance, and calculation of ratios of proliferation-promoting E2F1 to E2F7 could serve as a putative predictor of platinum resistance.

Alternative splicing of p53 and p73 : the novel p53 splice variant p53δ is an independent prognostic marker in ovarian cancer

Similar to p73, the tumor suppressor gene p53 is subject to alternative splicing. Besides p53ΔE6 and p53β, we identified p53ζ, p53δ and p53ɛ, arising from alternative splicing of exon 6 and intron 9, respectively. p53 splice variants were present in 18 of 34 ovarian cancer cell lines (52.9%) and 134 of 245 primary ovarian cancers (54.7%). p53δ expression was associated with impaired response to primary platinum-based chemotherapy (P=0.032). Also, p53δ expression constituted an independent prognostic marker for recurrence-free and overall survival (hazard ratio 1.854, 95% confidence interval 1.121–3.065, P=0.016; and hazard ratio 1.937, 95% confidence interval 1.177–3.186, P=0.009, respectively). p53β expression was associated with adverse clinicopathologic markers, that is, serous and poorly differentiated cancers (P=0.002 and P=0.008, respectively), and correlated with worse recurrence-free survival in patients exhibiting functionally active p53 (P=0.049). ΔN′p73 constituted the main N-terminally truncated p73 isoform and was preferentially found in ovarian cancer cell lines showing functionally active p53, supporting our hypothesis that N-terminally truncated p73 isoforms can alleviate the selection pressure for p53 mutations by the inhibition of p53 protein function.

Toll-Like Receptor 9 expression in breast and ovarian cancer is associated with poorly differentiated tumors

Toll‐like receptor 9 (TLR9) activates the innate immune response when exposed to non‐methylated CpG‐DNA. TLR9 was recently shown to be expressed by cancer cells which have been previously characterized by global hypomethylation. We set out to examine the expression and molecular activity of TLR9 in breast and ovarian cancer cells. Firstly, we confirmed higher levels of hypomethylated DNA in the serum of patients with metastatic breast cancer (n = 18) versus age‐matched tumor‐free women (n = 18). In breast cancer cell lines and tissues, TLR9 mRNA expression was associated with estrogen‐receptor (ER) status (n = 124, P = 0.005). Expression also correlated with increasing tumor grade in both breast (P = 0.03) and ovarian cancer specimens (n = 138, P = 0.04). Immunohistochemical analysis of formalin‐fixed paraffin‐embedded (FFPE) breast cancer tissues revealed higher TLR9 protein expression in hormone‐receptor (HR)‐negative specimens (n = 116, P < 0.001). Using an in vitro scratch assay, we observed that cell lines transfected to overexpress TLR9 demonstrated increased cellular migration when stimulated with CpG‐DNA. When assessing the molecular activity of TLR9 in breast cancer, we found a strong positive correlation of nuclear factor‐kappa B (NF‐κB) activity with TLR9 mRNA expression (correlation coefficient r = 0.7, P < 0.001). Finally, immunofluorescence analysis of BT‐20 and Hs578T breast cancer cell lines showed partial colocalizations of CpG‐DNA with TLR9, which diminished when the cells were exposed to methylated CpG‐DNA (mCpG‐DNA) or control GpC‐DNA. In summary we demonstrate that TLR9 expression is associated with poor differentiation in breast and ovarian cancer specimens, and that TLR9 overexpression and stimulation with hypomethylated DNA augments the migratory capacity of cancer cell lines.

Expression of the E2F Family of Transcription Factors and Its Clinical Relevance in Ovarian Cancer

Abstract:  The E2F family of transcription factors plays a pivotal role in the regulation of cellular proliferation. On the basis of sequence homology and function, eight distinct members of E2F transcription factors (E2F‐1 to E2F‐8) have been distinguished to date. The regulation of E2F transcription factors is closely associated with the function of the retinoblastoma family of tumor suppressors (RB pathway). In the last decade various alterations of distinct components of the RB‐E2F pathway were found to be associated with tumor progression. However, no data on the role of E2F family members are available in tumor biology of ovarian cancer. Here we describe an expression study of E2F transcription factors in various human ovarian cancer cell lines; its clinical relevance was examined in a training set of 77 ovarian cancer patients. Expression levels of E2F‐1, E2F‐2, and E2F‐8 were elevated in all the ovarian cancer cell lines studied when compared with human peritoneal mesothelial cells (HPMCs). Interestingly, EGF treatment showed a time‐dependent upregulation of the activating transcription factor E2F‐3 and a simultaneous increase of DP‐1, the heterodimeric partner of E2F‐3. High expression of E2F‐1, E2F‐2, and E2F‐8 was found to be associated with histopathologic grade 3 tumors and residual tumor over 2 cm in diameter after primary debulking surgery in ovarian cancer patients. Taken together, these data suggest that the proliferation‐promoting E2F transcription factors E2F‐1 and especially E2F‐2 play a pivotal role in tumor biology of ovarian cancer and may be candidates for specific therapeutic targets.

DNA ploidy, nuclear size, proliferation index and DNA-hypomethylation in ovarian cancer

OBJECTIVE The present study was undertaken to analyze the impact of epigenetic alterations with a main focus on nuclear area, aneuploidy, hyperploidy, and proliferation in 70 ovarian cancer specimens. METHODS Morphometric changes and somatic chromosomal ploidy status were assessed by Feulgen spectrophotometry. DNA-hypomethylation of LINE1 repeats was analyzed by means of MethyLight PCR, and methylation levels of satellite 2 (Sat2) and satellite alpha (Satα) DNA sequences in chromosome 1 were measured by Southern blot analysis. These parameters were analyzed with regard to correlations as well as to recurrence and survival. RESULTS We identified a significant association between LINE1 DNA-hypomethylation and patient age (p=0.029). Furthermore, LINE1 DNA-hypomethylation was positively correlated with the nuclear area (r=0.47; p<0.001) and the proliferation index (r=0.36; p<0.001). Univariate survival analysis showed that the nuclear area and LINE1 DNA-hypomethylation were prognostic factors for overall (p=0.015 and =0.006, respectively) and progression-free survival (p=0.020 and p=0.001 respectively), the percentage of aneuploidy only for overall survival (p=0.031). Subgroup survival analyses revealed that the prognostic value of these factors is strictly confined to mucinous cancers. In serous cancers no prognostic value could be pointed out for any analyzed parameter. Multivariate analysis of the entire cohort showed that the percentage of hyperploidy was an independent prognostic parameter for overall survival (p=0.003) and LINE1 DNA-hypomethylation for progression-free survival (p=0.03). In mucinous cancers nuclear area and LINE1 DNA-hypomethylation were found to be independent predictors of progression-free and overall survival. CONCLUSIONS In this study we identified the correlations between early cancer-associated genome DNA-hypomethylation, nuclear morphometric changes, somatic chromosomal ploidy status and the proliferation index. Prognostic relevance of nuclear area and LINE1 DNA-hypomethylation was revealed exclusively in mucinous ovarian cancers.

Regulation of transcription factor E2F3a and its clinical relevance in ovarian cancer

Recently we showed an integral epidermal growth factor receptor (EGFR)–E2F3a signaling path, in which E2F3a was found to be essential in EGFR-mediated proliferation in ovarian cancer cells. The present work evaluates the clinical relevance of this novel axis and of E2F3a itself in a large set of 130 ovarian cancer specimens. For this purpose E2F3a and its counterpart, E2F3b, were measured by RT–PCR and activated EGFR was assessed by immunohistochemistry. When compared with healthy control tissue, both E2F3 isoforms were overexpressed in the cancers, but only E2F3a expression correlated with tumor stage (ρ=0.349, P=0.0001) and residual disease (ρ=0.254, P=0.004). Univariate survival analyses showed E2F3a and activated EGFR to be associated with poor PFS and OS. Furthermore, a strong, positive correlation between activated EGFR and E2F3a expression was shown (P=0.0001). We further identified two EGFR-independent mechanisms that regulate E2F3a expression, namely one, acting by promoter methylation of miR-34a, which by its physical interaction with E2F3a transcripts causes their degradation, and the second based on 6p22 gene locus amplification. MiRIDIAN-based knockdown and induction of miR-34a evidenced a direct regulatory link between miR-34a and E2F3a, and the tumor-suppressive character of miR-34a was documented by its association with improved survival. Although, 6p22 gene locus amplification was detected in a significant number of ovarian cancer specimens, 6p22 ploidy was not relevant in predicting survival. In Cox regression analysis, E2F3a, but not activated EGFR or miR-34a expression, retained independent prognostic significance (PFS: hazards ratio 3.785 (1.326–9.840), P=0.013; OS: hazards ratio 4.651 (1.189–15.572), P=0.013). These clinical findings highlight the relevance of E2F3a in the biology of ovarian cancer. Moreover, identification of EGFR-independent mechanisms in E2F3a control can be helpful in explaining the non-responsiveness of therapeutic EGFR targeting in ovarian cancer.

Intratumoral interferon regulatory factor (IRF)-1 but not IRF-2 is of relevance in predicting patient outcome in ovarian cancer

IRF‐1 and IRF‐2 expression was determined by real‐time PCR in 138 ovarian cancer samples and 30 healthy ovarian biopsies and was correlated with the expression of other relevant immunologic parameters and common clinicopathologic variables. Regulation of IRF‐1 and IRF‐2 was evaluated by cytokine treatment of various ovarian cancer cell lines, human peritoneal mesothelial cells and ovarian surface epithelium. IRF‐1 but not IRF‐2 was constitutively over‐expressed in 5 of 7 ovarian cancer cell lines. Both IRFs were inducible with IFN‐γ and to a lesser extent with IL‐1 or TNF‐α, but not with IL‐6. Epidermal growth factor (EGF) treatment down‐regulated both IRFs. In ovarian cancer samples only IRF‐1, but not IRF‐2 mRNA, was up‐regulated when compared with healthy ovarian tissue. IRF‐1 but not IRF‐2 expression was significantly associated with interferon (IFN)‐γ and forkhead box P3 (FoxP3). In univariate survival analysis, strong expression of IRF‐1 and IRF‐2 predicted improved disease‐free survival (DFS) and overall survival (OS). In Cox regression analyses, IRF‐1 retained independent prognostic significance for DFS and OS and IFN‐γ for OS. In contrast to other solid tumors, IRF‐2 expression cannot be regarded as a classic oncoprotein associated with poor prognosis in ovarian cancer. Of the immunologic parameters investigated, intratumoral IRF‐1 expression is the most powerful independent predictor of a favorable clinical outcome.

The N-terminally truncated p53 isoform Δ40p53 influences prognosis in mucinous ovarian cancer.

Objective The tumor suppressor p53 generates the N-terminally truncated isoforms &Dgr;40p53 and &Dgr;133p53 that possess the ability to modulate p53 function in vitro. The aim of the present study was to evaluate the clinical relevance of p53 isoforms in the main histological subtypes of ovarian cancer. Methods &Dgr;40p53, &Dgr;133p53, and full-length p53 (FLp53) expression was determined in 45 mucinous, 30 endometrioid, and 91 serous ovarian cancer specimens as well as 42 normal ovarian tissues using reverse transcriptase–quantitative polymerase chain reaction. In a subgroup of mucinous ovarian cancer cases, &Dgr;40p53 expression was examined using Western blot analysis. A functional yeast-based assay and subsequent sequencing were performed to analyze the p53 mutational status. Results In endometrioid cancer specimens, &Dgr;133p53 expression was significantly lower than in mucinous and serous cases (P = 0.016) or in normal tissues (P = 0.004). Mucinous cancer samples showed elevated &Dgr;40p53 expression as compared with normal ovarian tissues (P = 0.003). In addition, high &Dgr;40p53 expression constituted an independent prognostic marker for recurrence-free but not for overall survival in patients with mucinous ovarian cancer (hazard ratio, 0.267; 95% confidence interval, 0.094–0.756 [P = 0.013]; hazard ratio, 0.453, 95% confidence interval, 0.193–1.064 [P = 0.069]). Western blot analysis confirmed the presence of p53β and &Dgr;40p53&agr; in a subset of patients with mucinous ovarian cancer. Expression of p53 isoforms was not associated with p53 mutational status or clinicopathologic parameters. Conclusions We show that expression of p53 isoforms differs in histological subtypes, thus supporting the hypothesis that histological subtypes represent distinct disease entities. In addition, we provide first evidence for a favorable role of &Dgr;40p53 in patients with mucinous ovarian cancer.

High expression of 90K (Mac-2 BP) is associated with poor survival in node-negative breast cancer patients not receiving adjuvant systemic therapies

90K (Mac‐2 BP) expression was evaluated by immunohistochemistry in paraffin‐embedded tissue from a consecutive series of lymph‐node negative breast cancer patients who did not receive adjuvant systemic treatment. An independent series of patients served as validation set. The association of 90K expression with risk of recurrence and death was examined in survival analyses together with known prognostic factors. High levels of 90K expression (IHC score > 8) were observed in 43 (25.3%) of 170 tumors examined. We found elevated risks of distant recurrence and overall mortality in patients with high 90K expression compared with patients with low 90K expression in their tumors. This increase persisted after adjusting for other prognostic factors in multivariate analysis (hazard ratio = 4.084; p < 0.001 for recurrence; hazard ratio = 4.298; p < 0.001 for death). These findings were confirmed in the validation set. Therefore, evaluation of 90K expression may be beneficial to identify lymph‐node negative breast cancer patients at lower risk of disease recurrence and death.