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p53 Mutations in epithelial ovarian cancers: Possible role in predicting chemoresistance
Abstract
The investigators undertook a retrospective study to determine (1) whether p53 mutations are predictors of survival in patients with advanced epithelial ovarian cancer, (2) whether p53 status by sequencing is associated with established prognostic indicators, and (3) the agreement of results between direct sequencing of p53 mutations and immunohistochemistry.
This study was retrospective review of 43 patients with advanced epithelial ovarian cancer treated with surgery and by paclitaxel-based chemotherapy. Clinical data were extracted from the charts. By use of paraffin-embedded blocks, p53 analysis was carried out by (1) direct sequencing and (2) immunohistochemistry. Kaplan-Meier estimates were used for overall and disease-free survivals. To determine whether p53 mutation (sequencing) was related to prognosis and to determine the agreement between p53 abnormalities by sequencing and immunohistochemistry, risk ratios were calculated.
Mean age at diagnosis was 57.4 years. Surgical stages were as follows: 5% were stage IIC, 79% were stage III, and 16% were stage IV. Seventy-seven percent of tumors were serous, and 56% of tumors were grade 3. All patients received paclitaxel-based chemotherapy. Mean disease-free and overall survivals were 16.4 and 22.6 months, respectively. p53 abnormalities were detected by sequencing in 53% of cases and by immunohistochemistry in 70%. Agreement between both techniques was 68%. Patients with stages IIC/IV had a risk of 1.7 of having a p53 mutation by sequencing; grade, histology, disease-free survival and overall survival were not predictive of p53 mutation status.
The 54% mutation rate may be underestimated by limiting our analysis to exons 5 to 9. p53 mutation status was not predictive of survival (disease free and overall) or of chemoresistance; this suggests that paclitaxel-based apoptosis is independent of the p53 gene. The concomitant use of cisplatin, an inducer of apoptosis that is dependent on the normal function of p53, makes the interpretation of these results difficult. Histopathologic factors were not statistically associated with p53 status, but more advanced surgical stage and tumor grade were suggestive of higher rates of p53 mutations, implying more aggressive behavior. Finally, the lack of agreement between results obtained by sequencing and immunohistochemistry highlights the limitation of the latter technique and the possibility of underestimating abnormal p53 function. In conclusion, because discrepancies exist between the two techniques, we recommend direct sequencing of exons 4 through 10 to determine the true prevalence of p53 mutation. Furthermore, larger randomized studies are required to elucidate the role of p53 in predicting chemoresponse in advanced epithelial ovarian cancer.
... The results from 41 studies were included in the pooled analysis of p53 mutations in epithelial ovarian tumors. 17,26,36,42,48,50,53,58,59,78,81,82,84,89,92,93,95,103,107, Carcinomas were examined in all the studies, tumors of LMP were examined in 19 studies, 17,26,36, 42,48,50,53,59,103,115,116,118 -121,129,132-134 and benign tumors were examined in 10 studies. 26,36,103,115,119,120,123,124,132,133 Frozen tissue 42,50,59,84,92,107,115,116,119,120,[126][127][128]133 and fixed, paraffin-embedded tissue 36,48,58,78,81,89,93,95,103,117,121,124,129 were studied the most often, followed by a combination of tissue types 26,53,82,118,130,134,135 and fresh tissue. ...
... 17,26,36,42,48,50,53,58,59,78,81,82,84,89,92,93,95,103,107, Carcinomas were examined in all the studies, tumors of LMP were examined in 19 studies, 17,26,36, 42,48,50,53,59,103,115,116,118 -121,129,132-134 and benign tumors were examined in 10 studies. 26,36,103,115,119,120,123,124,132,133 Frozen tissue 42,50,59,84,92,107,115,116,119,120,[126][127][128]133 and fixed, paraffin-embedded tissue 36,48,58,78,81,89,93,95,103,117,121,124,129 were studied the most often, followed by a combination of tissue types 26,53,82,118,130,134,135 and fresh tissue. 17,123,125,136 Tissue type was not documented in three studies. ...
... The exons screened varied across studies (exons 5-8 in 14 studies; 26,36,42,48,58,59,93,103,116,119,127,131,132,134 exons 5-9 in 9 studies; 78,81,82,84,92,120,121,123,136 135 ). Among those studies in which exons 5-8 or exons 5-9 were evaluated, the p53 mutation prevalence estimates were similar: 42% (95% CI, 38 -46%) and 43% (95% CI, 47-59%), respectively. ...
BACKGROUND
In the current study, the authors present pooled data from studies that investigated p53 protein expression and/or mutation in human epithelial ovarian tumors.METHODS
The English literature in the MEDLINE, PubMed, and Ingenta databases was searched to the end of the year 2000 to identify relevant studies. Data were pooled across eligible studies, and the prevalence of p53 expression and mutation among benign, low malignant potential (LMP), and invasive tumors was determined. Prevalence estimates by tumor histology, International Federation of Gynecology and Obstetrics (FIGO) stage, and grade also were calculated.RESULTSThe pooled prevalence estimate for p53 overexpression among epithelial ovarian carcinomas was 51% (95% confidence intervals [95% CI], 50–53%) compared with 17% (95% CI, 15–20%) among LMP tumors and 7% (95% CI, 5–10%) among benign tumors. p53 mutation prevalence estimates were 45% (95% CI, 42–47%), 5% (95% CI, 2–9%), and 1% (95% CI, 0–5%), respectively, for invasive, LMP, and benign tumors. The prevalence of these p53 abnormalities was found to be associated positively with increasing tumor grade and stage. Differences based on histologic subtype also were found.CONCLUSIONS
Although these pooled estimates might appear to offer support for various hypotheses regarding the role of p53 in ovarian carcinoma, the limitations inherent in these data hamper the interpretation of the significance of any of the findings. Future studies will require innovative methods to address the limitations of many previous investigations and more comprehensive investigation into defective tumor suppression mechanisms. Cancer 2003;97:389–404. © 2003 American Cancer Society.DOI 10.1002/cncr.11064
... P53 gene inactivation has been found to confer resistance to cisplatin and other DNAdamaging agents (11). Conversely, recent clinical studies reported that patients with mutant p53 tumors were responsive to paclitaxel-plus platinum-based chemotherapy (18,19,(21)(22)(23). The mechanism of action of taxanes consists of alterations in microtubule function and the presence of a functional p53 gene does not seem to be required for apoptotic cell death induction by antimicrotubule agents (22). ...
... Moreover, p53 status was found to be prognostically irrelevant for the patients treated with paclitaxel-based regimens. In a retrospective investigation on 43 patients with advanced ovarian cancer treated with paclitaxel-based chemotherapy, Laframbroise et al. (23) showed that p53 status was neither predictive of chemoresistance nor prognostic of disease-free and overall survival. A multicentric Italian study (22) assessed p53 status by genetic analysis of exons 5 through 8 in tumor specimens collected at the time of initial surgery from 48 advanced ovarian cancer patients who subsequently received paclitaxel-plus platinum-based chemotherapy. ...
... Patients with mutant p53 gene showed a lower response rate and worse clinical outcome when compared to patients with either wild-type or homozygous polymorphic p53 gene, thus confirming the negative predictive and prognostic role of a mutated p53 gene in human malignancies. The addition of paclitaxel to carboplatin does not seem as effective in the treatment of p53-mutated ovarian carcinomas as reported in previous studies (19,22,23). Discrepancies with the literature may be ascribed, in part, to the different histological features of the tumors analyzed in different series. ...
The aim of this retrospective study was to assess whether p53 gene status has any predictive or prognostic relevance in patients with advanced, poorly-differentiated serous epithelial ovarian cancer treated with paclitaxel- plus platinum-based chemotherapy.
The study was conducted on 46 patients who underwent surgery followed by paclitaxel- plus carboplatin-based chemotherapy. The tumor tissue samples were analyzed for p53 gene mutations. The median follow-up of survivors was 50.3 months.
Twenty-three patients (50%) showed p53 mutations at exons 5 to 9. Sixteen (34.8%) patients had a polymorphism at codon 72 in exon 4 (SNP codon 72): 10 were Pro/Pro homozygous and 6 Pro/Arg heterozygous. Four polymorphic patients had a second mutation at exons 5 to 9. An inverse correlation was evidenced between the SNP codon 72 and mutations at exons 5 to 9, with the latter more frequently found in wild-type (Arg/Arg) codon 72 (19/30 versus 4/16, 63.3% versus 25.0%; p=0.03) cases. A clear trend for a higher response rate and longer progression-free and overall survival was observed in wild-type p53 and Pro/Pro polymorphic patients as compared to patients with mutant p53.
The addition of paclitaxel to carboplatin does not appear to overcome the negative predictive and prognostic significance of p53 gene mutations in serous ovarian cancer. Nevertheless, the comprehensive analysis of p53 genotype, including the SNP codon 72, warrants further investigation in order to envisage individual responsiveness to cancer therapy.
... In turn, this could be the reason for the good prognosis of some patients with cancer, even though wild-type p53 over-expression is not always related with a favourable outcome (Taylor et al., 2000;Ryan et al., 2001). The prognostic significance of p53 mutation has been observed in many tumors, (including gastric cancer (Lam, 1999;Matturri et al., 1998), astrocytoma (Nokazi et al., 1999), non-small cell lung cancer (Bennett et al., 1998), breast carcinoma (Powell et al., 2000;Hurlimann et al., 1995), ovarian cancer (Laframboise et al., 2000). These studies have found a reduction of apoptotic phenomena associated to p53 mutation and poor prognosis. ...
... Furthermore, it helps in identifying patients that need additional peri-operative therapies (Matturri et al., 1998). Moreover, a more advanced surgical stage or tumor grade were suggestive of higher rates of p53 mutations (Laframboise et al., 2000), determining a more aggressive surgery. ...
The transcription factor p53 and the cytokine receptor FasL are two of the most famous regulators of cell life, and their alterations can cause a large number of pathologies, including cancer. In this review, we focused on how they can determine defective apoptosis, one of the causes of tumorigenesis and tumor progression. The importance of this knowledge lies in the new perspectives that gene therapy can offer to cure cancer.
... We found neither ERBB2 expression nor its mutation status can predict OvCa prognosis (Fig. 3A). Furthermore, we found FBXL20 outperformed other potential OvCa prognostic molecular markers, including mutation status of BRAC1/2 [19] and TP53 [20], and expression levels of WTAP (Wilms' tumor 1-associating protein) [21] and EGFR [22] (Supplementary 4). We also found that FBXL20 expression levels and cellular protein levels do not correlate with OvCa staging, indicating FBXL20 can be an independent OvCa prognostic biomarker (Supplementary 5). ...
About 70% of ovarian cancer (OvCa) cases are diagnosed at advanced stages (stage III/IV) with only 20–40% of them survive over 5 years after diagnosis. A reliably screening marker could enable a paradigm shift in OvCa early diagnosis and risk stratification. Age is one of the most significant risk factors for OvCa. Older women have much higher rates of OvCa diagnosis and poorer clinical outcomes. In this article, we studied the correlation between aging and genetic alterations in The Cancer Genome Atlas Ovarian Cancer dataset. We demonstrated that copy number variations (CNVs) and expression levels of the F-Box and Leucine-Rich Repeat Protein 20 (FBXL20), a substrate recognizing protein in the SKP1-Cullin1-F-box-protein E3 ligase, can predict OvCa overall survival, disease-free survival and progression-free survival. More importantly, FBXL20 copy number loss predicts the diagnosis of OvCa at a younger age, with over 60% of patients in that subgroup have OvCa diagnosed at age less than 60 years. Clinicopathological studies further demonstrated malignant histological and radiographical features associated with elevated FBXL20 expression levels. This study has thus identified a potential biomarker for OvCa prognosis.
... The tumor suppressor p53 plays a major role in cell apoptosis, cell cycle arrest, DNA repair, and senescence. p53 is typically mutated in serous ovarian cancer and also associated with ovarian cancer chemoresistance [25][26][27] . Studies have shown that STAT3 and p53 regulate each other's activities [28][29][30] . ...
Chemoresistance has been the biggest obstacle in ovarian cancer treatment, and STAT3 may play an important role in chemoresistance of multiple cancers, but the underlying mechanism of STAT3 in ovarian cancer chemoresistance has long been truly illusive, particularly in association with p53 and RAS signaling. In this study, by using wild type, constitutive active, and dominant negative STAT3 constructs, wild-type p53, and RAS-mutant V12, we performed a series of in vitro and in vivo experiments by gene overexpression, drug treatment, and animal assays. We found that phosphorylation of STAT3 Y705 but not S727 promoted cancer cell EMT and metastasis through the Slug-mediated regulation of E-cadherin and Vimentin. The phosphorylation of STAT3 at Y705 also activated the MAPK and PI3K/AKT signaling to inhibit the ERS-mediated autophagy through down-regulation of pPERK, pelf2α, ATF6α, and IRE1α, which led to increased cisplatin resistance. Induction of wild type p53 in STAT3-DN-transfected cells further diminished the chemoresistance and tumor growth through the upregulation of the MAPK- and PI3K/AKT-mediated ERS and autophagy. Introduction of STAT3-DN deprived the RASV12-induced ERS, autophagy, oncogenicity, and cisplatin resistance, whereas introduction of p53 in STAT3-DN/RASV12 expressing cells induced additional tumor retardation and cisplatin sensitivity. Thus, our data provide strong evidence that the crosstalk between STAT3 and p53/RAS signaling controls ovarian cancer cell metastasis and cisplatin resistance via the Slug/MAPK/PI3K/AKT-mediated regulation of EMT and autophagy.
... The tumor suppressor p53, which is a dominant force in promoting cell apoptosis, cell cycle arrest, DNA repair, and senescence in multiple cancer types, is typically mutated in serous ovarian cancer [2], and is associated with ovarian cancer chemoresistance [3]. Reles et al. reported that p53 alterations were significantly correlated with platinum-based chemoresistance [4]. ...
The interplay between p53 and RAS signaling regulates cancer chemoresistance, but the detailed mechanism is unclear. In this study, we investigated the interactive effects of p53 and RAS on ovarian cancer cisplatin resistance to explore the potential therapeutic targets.
Methods: An inducible p53 and RAS mutants active in either MAPK/ERK (S35 and E38) or PI3K/AKT (C40) or both (V12) were sequentially introduced into a p53-null ovarian cancer cell line-SKOV3. Comparative microarray analysis was performed using Gene Chip Prime View Human Gene Expression arrays (Affymetrix). In vitro assays of autophagy and apoptosis and in vivo animal experiments were performed by p53 induction and/or cisplatin treatment using the established cell lines. The correlation between HDAC4 and HIF-1α or CREBZF and the association of HDAC4, HIF-1α, CREBZF, ERK, AKT, and p53 mRNA levels with patient survival in 523 serous ovarian cancer cases from TCGA was assessed.
Results: We show that p53 and RAS mutants differentially control cellular apoptosis and autophagy to inhibit or to promote chemoresistance through dysregulation of Bax, Bcl2, ATG3, and ATG12. ERK and AKT active RAS mutants are mutually suppressive to confer or to deprive cisplatin resistance. Further studies demonstrate that p53 induces HIF-1α degradation and HDAC4 cytoplasmic translocation and phosphorylation. S35, E38, and V12 but not C40 promote HDAC4 phosphorylation and its cytoplasmic translocation along with HIF-1α. Wild-type p53 expression in RAS mutant cells enhances HIF-1α turnover in ovarian and lung cancer cells. Autophagy and anti-apoptotic processes can be promoted by the overexpression and cytoplasmic translocation of HDAC4 and HIF1-α. Moreover, the phosphorylation and cytoplasmic translocation of HDAC4 activate the transcription factor CREBZF to promote ATG3 transcription. High HDAC4 or CREBZF expression predicted poor overall survival (OS) and/or progression-free survival (PFS) in ovarian cancer patients, whereas high HIF-1α expression was statistically correlated with poor or good OS depending on p53 status.
Conclusion: HIF-1α and HDAC4 may mediate the interaction between p53 and RAS signaling to actively control ovarian cancer cisplatin resistance through dysregulation of apoptosis and autophagy. Targeting HDAC4, HIF-1α and CREBZF may be considered in treatment of ovarian cancer with p53 and RAS mutations.
... As a famous tumor suppressor, p53 plays an important role in chemoresistance by regulating the proliferation and apoptosis of cancer cells (34,35). p53 status can predict chemotherapy response and prognosis in various types of cancers (11,(36)(37)(38). The interaction of S100B and p53 has been demonstrated. ...
Chemoresistance is one of the most important causes of ovarian cancer‑related deaths. Recently, cancer stem cells (CSCs) have been recognized as the source of chemoresistance in ovarian cancer. However, the underlying mechanisms that regulate the chemoresistance of ovarian CSCs (OCSCs) remain unclear. The aim of the present study was to investigate the roles of S100B in the regulation of OCSC chemoresistance, which provides a novel therapeutic target. We observed high expression of S100B in CD133+ OCSCs derived from ovarian cancer cell lines and primary tumors and in cisplatin‑resistant patient samples. Then, we determined that S100B knockdown promoted the apoptosis of OCSCs after treatment with different concentrations of cisplatin. The underlying mechanism of S100B‑mediated chemoresistance in OCSCs may be through p53 inhibition. Furthermore, drug‑resistance genes, including MDR1 and MRP1, were involved in the process of S100B‑mediated OCSC chemoresistance. In conclusion, our results elucidated the importance of S100B in the maintenance of OCSC chemoresistance, which may provide a promising therapeutic target for ovarian cancer.
... The incidence increases with age, reaching its peak in the eighth decade [4]. Furthermore, the later occurrence of ovarian cancer in sporadic cancer patient if compared to patients with hereditary cancer was explained as that, in women with positive family history of ovarian cancer the disease occurs at a younger age than sporadic cancer [25]. ...
... It has recently been suggested that 100% of HGSOC are in fact TP53 mutant 20 . These figures are higher than first thought from reports that used less sensitive methods such as Sanger sequencing and/or only focussed on mutational 'hot-spot' regions of this gene [21][22][23][24] . TP53 is in fact the only gene that is frequently mutated at the somatic level in HGSOC, this malignancy otherwise being characterised by low prevalence (2-6%) of recurrently mutated genes such as BRCA1, BRCA2, RB1 and NF1, and a long tail of infrequently mutated genes 18 . ...
The tumour suppressor p53 is mutated in cancer, including over 96% of high-grade serous ovarian cancer (HGSOC). Mutations cause loss of wild-type p53 function due to either gain of abnormal function of mutant p53 (mutp53), or absent to low mutp53. Massively parallel sequencing (MPS) enables increased accuracy of detection of somatic variants in heterogeneous tumours. We used MPS and immunohistochemistry (IHC) to characterise HGSOCs for TP53 mutation and p53 expression. TP53 mutation was identified in 94% (68/72) of HGSOCs, 62% of which were missense. Missense mutations demonstrated high p53 by IHC, as did 35% (9/26) of non-missense mutations. Low p53 was seen by IHC in 62% of HGSOC associated with non-missense mutations. Most wild-type TP53 tumours (75%, 6/8) displayed intermediate p53 levels. The overall sensitivity of detecting a TP53 mutation based on classification as ‘Low’, ‘Intermediate’ or ‘High’ for p53 IHC was 99%, with a specificity of 75%. We suggest p53 IHC can be used as a surrogate marker of TP53 mutation in HGSOC; however, this will result in misclassification of a proportion of TP53 wild-type and mutant tumours. Therapeutic targeting of mutp53 will require knowledge of both TP53 mutations and mutp53 expression.
... TP53 proteini, overde moleküler hedef olarak kullanılma potansiyeli olan değerli bir prognostik belirteçtir (13). Bazı araştırmacılar, over karsinomlarında TP53 mutasyonları ile sağkalım ve kemoterapi yanıtı arasında ilişki saptarken (14)(15)(16)(17), diğer bazı araştırmacılar anlamlı bir ilişki saptamamışlardır (18)(19)(20)(21)(22). ...
Objective: To investigate TP53 and Ki-67 expressions in malignant, borderline and benign epithelial ovarian tumors; correlate results with histopathological parameters; compare TP53 and Ki-67 expressions and discuss ovarian carcinogenesis. Material and Methods: Standard immunohistochemistry was performed in 100 malignant, 27 borderline and 31 benign tumors and 42 non-tumoral ovarian epithelia. Results: TP53 overexpression was higher in malignant (61.0%) than in borderline tumors (40.7%); in malignant cases above 50 years-of-age, with high grade and in undifferentiated and serous subtypes. Carcinomas with solid architecture in serous, high mitosis in non-serous and high pleomorphism in both subtypes; lymph node involvement, and advanced stage also showed higher expressions of TP53 (p<0.05). The mean Ki-67 index was 41.3% in malignant and 17.2% in borderline tumors. Ki-67 index tended to increase with increasing grade, and decrease with lymph node involvement. The coexpression of proteins according to grade, lymph node involvement and stage in different subtypes, showed significant differences and correlations. Conclusion: Our findings support the role of TP53 overexpression in development of "borderline" and malignant epithelial ovarian tumors. The degree of TP53 expression differed in terms of histopathological parameters; and was correlated with Ki-67 index. Especially in early stages, these biological factors could provide additional information to stratify patients for case specific therapies.
... It is well established that more than 50% of advanced ovarian cancers contain TP53 mutations that are mostly missense [15]. Although TP53 mutation status was not predictive of survival (disease free and overall) or of chemoresistance [16][17][18][19][20], the TP53 missense mutation-mediated cisplatin resistance may be associated with reduced transactivation and expression of BAX, an apoptosis-inducing gene, in ovarian carcinoma cell systems [19]. These studies reported that TP53 mutations in ovarian cancers were mostly involved in exons of DNA binding domains (17) but not involved in the tetramerization domain of TP53 that is responsible for its nuclear export signal (NES) and translocation to mitochondria ( Supplementary Fig. 1). ...
TP53 K351N mutation is associated with acquired cisplatin resistance in ovarian cancer cells following exposure to cisplatin. We investigated the effect of TP53 K351N mutation on outcome in patients with epithelial ovarian cancer (EOC) that received platinum-based chemotherapy.
We assessed TP53 K351N mutations by allele specific real-time PCR (AS-PCR) and DNA sequencing in tumor samples of 153 patients with stage IIIC/IV EOC. Clinicopathologic and follow-up data were collected by a retrospective chart review.
TP53 K351N mutations were detected in 8 (11.27%) of 71 patients underwent neoadjuvant chemotherapy with interval debulking surgery (NACT-IDS) but not in 82 patients underwent primary debulking surgery (PDS) (P<0.01). In patients with relapse within 6 months, the relapse rate was 14 (19.72%) of 71 patients for NACT-IDS compared to 15 (18.29%) of 82 patients for PDS (P=0.49), and TP53 K351N mutation was observed in 8 of NACT-IDS 14 patients (57.14% P<0.01). In the patients retreated at first recurrence within 6 months, 7 with TP53 K351N mutation of 14 NACT-IDS patients exhibited progression of disease, compared to 2 of PDS 15 patients (50.00% vs. 13.33%, P=0.04). The median disease-free survival (DFS) for NACT-IDS was 13.0months compared to 15.0months for PDS (P=0.02). In multivariate analysis, TP53 K351N mutation is an independent factor for shorter DFS in the patients underwent NACT-IDS (HR=19.05; P=0.01).
TP53 K351N mutation may be associated with induction of platinum resistance after NACT in advanced EOC.
... Many clinical studies have attempted to correlate the presence of a TP53 mutation with patient survival or the development of chemoresistance [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. However, the conclusions of these studies are conflicting, due in large part to insufficient analysis and inadequate methods. ...
Ovarian cancer is the most lethal gynecological malignancy, with an alarmingly poor prognosis attributed to late detection and chemoresistance. Initially, most tumors respond to chemotherapy but eventually relapse due to the development of drug resistance. Currently, there are no biological markers that can be used to predict patient response to chemotherapy. However, it is clear that mutations in the tumor suppressor gene TP53, which occur in 96% of serous ovarian tumors, alter the core molecular pathways involved in drug response. One subtype of TP53 mutations, widely termed gain-of-function (GOF) mutations, surprisingly converts this protein from a tumor suppressor to an oncogene. We term the resulting change an oncomorphism. In this review, we discuss particular TP53 mutations, including known oncomorphic properties of the resulting mutant p53 proteins. For example, several different oncomorphic mutations have been reported, but each mutation acts in a distinct manner and has a different effect on tumor progression and chemoresistance. An understanding of the pathological pathways altered by each mutation is necessary in order to design appropriate drug interventions for patients suffering from this deadly disease.
... This is supported by the differences in disease-free and overall survival between these two TP53 mutation-positive groups. Studies that have evaluated the correlation of p53-immunoreactivity with clinical outcome have produced conflicting results, and this may have been due to analyses that included different histological subtypes of ovarian cancer as well as incomplete assessment of TP53 mutation status, where mutation status was inferred by istry or where mutation screening was limited to specific exons [6,454647484950. Indeed our results are consistent with a recent report associating p53 protein null HGSCs, which were also assessed for TP53 mutation, with poorer overall outcome [6]. ...
High-grade ovarian serous carcinomas (HGSC) are characterized by TP53 mutations and non-random patterns of chromosomal anomalies, where the nature of the TP53 mutation may correlate with clinical outcome. However, the frequency of common somatic genomic events occurring in HGSCs from demographically defined populations has not been explored. Whole genome SNP array, and TP53 mutation, gene and protein expression analyses were assessed in 87 confirmed HGSC samples with clinical correlates from French Canadians, a population exhibiting strong founder effects, and results were compared with independent reports describing similar analyses from unselected populations. TP53 mutations were identified in 91% of HGSCs. Anomalies observed in more than 50% of TP53 mutation-positive HGSCs involved gains of 3q, 8q and 20q, and losses of 4q, 5q, 6q, 8p, 13q, 16q, 17p, 17q, 22q and Xp. Nearly 400 regions of non-overlapping amplification or deletion were identified, where 178 amplifications and 98 deletions involved known genes. The subgroup expressing mutant p53 protein exhibited significantly prolonged overall and disease-free survival as compared with the p53 protein null subgroup. Interestingly, a comparative analysis of genomic landscapes revealed a significant enrichment of gains involving 1q, 8q, and 12p intervals in the subgroup expressing mutant p53 protein as compared with the p53 protein null subgroup. Although the findings show that the frequency of TP53 mutations and the genomic landscapes observed in French Canadian samples were similar to those reported for samples from unselected populations, there were differences in the magnitude of global gains/losses of specific chromosomal arms and in the spectrum of amplifications and deletions involving focal regions in individual samples. The findings from our comparative genomic analyses also support the notion that there may be biological differences between HGSCs that could be related to the nature of the TP53 mutation.
... Accordingly, p53 overexpression (a specific feature of mutant p53 protein in cancer cells), has been associated with poor prognosis, poor overall survival and altered sensitivity to chemotherapy in patients with ovarian cancer (Fujita et al., 1994; Ferrandina et al., 1999; Sengupta et al., 2000; Reles et al., 2001; Hashiguchi et al., 2001; Tachibana et al., 2003; Bali et al., 2004; Bartel et al., 2008; Bernardini et al., 2010; Lee et al., 2011). However, others studies showed that overexpression of p53 is not associated with patient outcome (Havrilesky et al., 2003), has no prognostic value (Laframboise et al., 2000; Fallows et al., 2001) and is not predictive for responsiveness to platinum-based chemotherapy (Bauerschlag et al., 2010). Recently, a meta-analysis of studies on the prognostic value of p53 expression, showed that aberrant p53 status is associated only with poor overall survival (de Graeffet al., 2009), although there was ample heterogeneity among studies. ...
... Moreover, Goodell and colleagues evaluated the relationship between antibody immunity to the p53 oncogenic protein with EOC outcome, and they demonstrated the presence of p53-Ab was an independent prognostic indicator of overall survival in advanced-stage patients. 147 Other studies have associated overexpression of mutant p53 with worse survival, FIGO stages, [148][149][150][151] poorer histological grade of differentiation, and high proliferative fraction. Shahin et al observed an association between worse prognosis and p53 mutation status in patients with EOC. ...
Epithelial ovarian cancer (EOC) remains the most lethal gynecological malignancy despite several decades of progress in diagnosis and treatment. Taking advantage of the robust development of discovery and utility of prognostic biomarkers, clinicians and researchers are developing personalized and targeted treatment strategies. This review encompasses recently discovered biomarkers of ovarian cancer, the utility of published prognostic biomarkers for EOC (especially biomarkers related to angiogenesis and key signaling pathways), and their integration into clinical practice.
... Other studies have suggested that alterations in p53 expression in ovarian cancer affect sensitivity to chemotherapy (35,38). In contrast, there are a number of studies that suggest that p53 expression has no prognostic value in EOC (8,25). ...
Epithelial ovarian carcinoma is worldwide the sixth most common female cancer, and this malignancy carries the highest mortality among all gynecological cancers. The high mortality is due mostly to the fact that the tumor is frequently diagnosed late, in advanced stage, as the early disease is often asymptomatic and no effective screening methods are available. The most important prognostic factors in ovarian carcinoma are the stage, size of residual tumor following surgery, presence of ascites, age and the general condition of the patient, tumor histology, and, in patients with early disease, also the grade of the tumor. Large number of studies on prognostic and predictive factors in epithelial ovarian carcinoma has been published, often with contradictory results. The most intensely studied prognostic factors are those for expression of hormonal receptors, for tumor proliferation activity (mainly by antigen Ki-67 and topoisomerase IIalpha), the markers of apoptosis (p53, p21, mdm2, bcl-2 and other proteins), or other oncoproteins (particularly HER-2/neu).
... Overabundance of p53 by immunohistochemistry has been commonly used as a surrogate marker for TP53 mutation in a wide range of cancers and although the method is cost effective, it does not have the required sensitivity to predict TP53 mutations [12,22]. Dideoxy sequencing remains the gold standard method to detect these mutations. ...
p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples.
We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status.
One cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons.
HRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53.
... Another mechanism seems to be inducible or acquired resistance of the tumor to apoptosis. In this way, the role of apoptosis related genes, NFκB, p16, p21, Bcl-xL and p53 might be important (22)(23)(24)(25). Here, our experiment investigated the inhibition of NFκB activity on the growth of paclitaxel-treated NCI-H460 tumors and expression of p53. ...
Nuclear factor kappaB (NFkappaB) is a transcription factor which is importantly implicated in cancer cell growth. In a previous report, we confirmed that lung cancer cell growth was suppressed significantly by the blockade of NFkappaB function. In this study the combination effect of chemotherapy and inhibition of NFkappaB on the human lung cancer cell line, NCI-H460, in vitro and in vivo was investigated.
In the in vitro experiment, 50% of cell growth inhibitory concentrations (IC50) of chemotherapy agents were determined alone or when combined with adenovirus mediated IkappaBalpha gene transfer. Annexin-V/PI stain and caspase 3 activity measurement were used to detect the apoptosis caused by treatment. In the in vivo experiment, the tumor growth suppressive effect of combination treatment was evaluated for tumor-bearing mice. NFkappaB, p53 and VEGF expression in the tumors were also analyzed immunohistologically.
Several chemotherapy agents, including paclitaxel, showed lower IC50s when combined with AdIkappaBalpha infection in vitro. Apoptosis through activation of the caspase 3 pathway was enhanced by the combination treatment. For established NCI-H460 tumors, combined treatment significantly inhibited tumor growth. Immunohistochemical staining showed increased expression of p65 after paclitaxel treatment, while paclitaxel in combination with AdIkappaBalpha intratumoral injection eliminated this expression accompanied by the slightly reduced expression of VEGF, with stable p53 status.
A combination of chemotherapy and IkappaBalpha could inhibit tumor growth effectively by blocking the expression of NFkappaB and inducing apoptosis. Moreover, it might allow reduction of the dose of chemotherapy agents and provide benefit for clinical application.
Selected patients with peritoneal metastases are treated with a curative intent, the backbone of which is cytoreductive surgery (CRS) with or without hyperthermic intraperitoneal chemotherapy (HIPEC). Despite this aggressive and morbid treatment, a large majority of the patients develop recurrence and/or progressive disease. In the era of molecular oncology, molecular markers have been gaining increasing focus as they not only have prognostic value and can improve patient selection, but can also serve as therapeutic targets for developing systemic therapies. Whole genome sequencing has accelerated the process of biomarker discovery. As the field of peritoneal oncology is evolving, many studies have been done and are ongoing, to assess the utility of biomarkers in various aspects of management of peritoneal metastases. This chapter provides a review of the known and emerging biomarkers related to some common peritoneal tumors.
Introduction: Paclitaxel and platinum analogs are standard drugs implemented as first-line therapy in advanced ovarian cancer, after previous surgery. One of causes of resistance to therapy is, among others, disruption of mechanisms leading to cell apoptosis. Aim of the paper is to determine the significance of expression of TP53, BAX and BCL-2 in response to first-line chemotherapy using paclitaxel in patients with advanced ovarian cancer, as well as to assess the role of TP53 as a prognostic and predictive factor in ovarian cancer. Material and method: The study included 164 patients with advanced ovarian cancer. Immunohistochemical staining was performed on histological specimens obtained from paraffin blocks. Monoclonal antibodies anti-BAX, anti-BCL-2 and anti-p53 were used. Results: Accumulation of TP53 was noticed in 60% of tumors. BCL-2 was absent in half of tumors studied. Significant expression of BCL-2 was noticed in 24% of tumors, while that of BAX was seen in 34% of tumors. There was a correlation of histological grade, volume of residual tumor left after surgery and BAX expression with overall survival. Enhanced expression of BAX correlated directly with increased death risk. Accumulation of none of proteins studied had any influence on disease-free survival. Complete remission was obtained in 106 patients. Size of residual tumor has a significant impact on disease-free survival. Conclusions: 1) In ovarian cancer patients, clinical factors clearly have far greater prognostic value than molecular factors. 2) There was no correlation between TP53 expression and clinical response to first-line chemotherapy using paclitaxel, cisplatin or platinum analogs. 3) TP53 expression is not a prognostic or predictive factor in ovarian cancer.
The analysis of dihydroptychantol (DHA) and its chemically synthesized macrocyclic bisbibenzyl derivatives (DHA1, 2 and 3) led to the selection of DHA2 as a potential drug candidate for ovarian cancer. The exposure of ovarian cancer SKOV3 cells to DHA2 resulted in the downregulation of the anti-apoptotic X-linked inhibitor of apoptosis protein (XIAP) and Bcl-2 and led to caspaseindependent cell death. The overexpression of XIAP reversed DHA2-induced cell death, and the depletion of XIAP had the opposite effect. DHA2 could induce autophagy, as evidenced by increases in the formation of acidic vesicular organelles and the processing of LC3B-I to LC3B-II. Pretreatment with autophagy inhibitors potentiated DHA2-mediated cell death. We showed that typical PI3K/Akt signaling was involved in DHA2-mediated cell death. The overexpression of Akt almost completely reversed DHA2-induced cell death, and the inactivation of Akt significantly facilitated DHA2-induced cell death. The administration of DHA2 to xenograft mice reduced tumor growth by causing Akt inactivation, the conversion of LC3B and proliferation inhibition. Our study demonstrates that the inhibition of XIAP and the inactivation of Akt/mTOR facilitate the efficacy of DHA2 and curcumin in ovarian cancer cells.
Epithelial ovarian cancer is the most lethal gynecological malignancy. Due to its lack of symptoms, this disease is diagnosed at an advanced stage when the cancer has already spread to secondary sites. While initial rates of response to first treatment is >80%, the overall survival rate of patients is extremely low, mainly due to development of drug resistance. To date, there are no reliable clinical factors that can properly stratify patients for suitable chemotherapy strategies. Clinical parameters such as disease stage, tumor grade and residual disease, although helpful in the management of patients after their initial surgery to establish the first line of treatment, are not efficient enough. Accordingly, reliable markers that are independent and complementary to clinical parameters are needed for a better management of these patients. For several years, efforts to identify prognostic factors have focused on molecular markers, with a large number having been investigated. This review aims to present a summary of the recent advances in the identification of molecular biomarkers in ovarian cancer patient tissues, as well as an overview of the need and importance of molecular markers for personalized medicine in ovarian cancer.
More than 50% of the women diagnosed with epithelial ovarian cancer will succumb to their disease. This high death rate is because of two substantial clinical problems:
1.
A typically late detection of the disease.
2.
The common resistance of ovarian tumors to current therapeutic strategies.
In ovarian cancer, translational research on the prognostic impact of molecular biological factors has until now not led to clinical implementation of any of these factors. This is partly due to the often conflicting results of different prognostic factor studies on the same molecular biological factor. We have performed meta-analyses on studies in ovarian cancer on four putative prognostic molecular biological factors, epidermal growth factor-receptor (EGFR), HER-2/neu, glutathione-S-transferase (GST)-pi and p53. Odds ratios were estimated for the increase in death at 1 and 5 years for patients with ovarian cancer, harbouring aberrant EGFR, HER-2/neu, GST-pi and p53, respectively. Patients with aberrant Her2/neu or p53 in their tumours had significantly worse odds of surviving 1 and 5 years, respectively. Patients with aberrant EGFR in their tumours only had a significantly greater risk of mortality at 5 years, while there seemed to be a trend for a decreased probability of 5-year survival for patients with aberrant GST-pi in their tumours. Despite inevitable flaws (such as small individual study sizes, publication bias, etc.) our meta-analysis confirms that therapeutic drugs targeted at EGFR, HER-2/neu, GSTpi and p53 may have therapeutic potential. Since ovarian cancer is a relatively rare disease, international collaboration to increase the number of patients to be analysed is critical for progress in translational research on the prognostic impact of molecular biological factors and on innovative treatment in ovarian cancer. In addition it is important to reach a consensus about guidelines for the design, conduct and analysis of translational studies in ovarian cancer.
Flavopiridol that inhibits cyclin-dependent kinase, can cause cell cycle arrest, induce apoptosis in human tumor cell lines. In the present study, we investigated apoptotic effects of flavopiridol and the underlying molecular mechanisms in human ovarian cancer cell lines.
During the last three years, we have demonstrated frequent activation (39%) of PI3K/AKT1 pathway in human primary ovarian cancer. The activation of this pathway is associated with late stage and high grade tumors, indicating that activation of PI3K/AKT1 plays a important role in ovarian tumor progression rather than initiation. Activation of - PI3K/AKT1 pathway induces malignant transformation and chemoresistance. Inhibition of this pathway by PI3K inhibitor or dominant negative AKT1 results in apoptosis and cell growth arrest as well as sensitizes ovarian cancer cells to cisplatin-induced cell death. We have also demonstrated that AKT targets ASK1/JNK/p38 pathway to promote cell survival. Further, we documented that farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase I Inhibitors (GGTI) inhibit PI3K/AKT pathway to overcome chemoresistance in human ovarian cancer cells.
Immunohistochemical staining for p53 is used as a surrogate for mutational analysis in the diagnostic workup of carcinomas of multiple sites including ovarian cancers. Strong and diffuse immunoexpression of p53 is generally interpreted as likely indicating a TP53 gene mutation. The immunoprofile that correlates with wild-type TP53, however, is not as clear. In particular, the significance of completely negative immunostaining is controversial. The aim of this study was to clarify the relationship of the immunohistochemical expression of p53 with the mutational status of the TP53 gene in ovarian cancer. A total of 57 ovarian carcinomas (43 high-grade serous ovarian/peritoneal carcinomas, 2 malignant mesodermal mixed tumors (carcinosarcomas), 2 low-grade serous carcinomas, 4 clear cell carcinomas, 1 well-differentiated endometrioid carcinoma, and 5 carcinomas with mixed epithelial differentiation) were analyzed for TP53 mutations by nucleotide sequencing (exons 4-9), and subjected to immunohistochemical analysis of p53 expression. Thirty six tumors contained functional mutations and 13 had wild type TP53. Five tumors were found to harbor known TP53 polymorphism and changes in the intron region were detected in three. Tumors with wild-type TP53 displayed a wide range of immunolabeling patterns, with the most common pattern showing ≤10% of positive cells in 6 cases (46%). Mutant TP53 was associated with 60-100% positive cells in 23 cases (64% of cases). This pattern of staining was also seen in three cases with wild-type TP53. Tumors that were completely negative (0% cells staining) had a mutation of TP53 in 65% of cases and wild-type TP53 in 11%. Combining two immunohistochemical labeling patterns associated with TP53 mutations (0% and 60-100% positive cells), correctly identified a mutation in 94% of cases (P<0.001). Immunohistochemical analysis can be used as a robust method for inferring the presence of a TP53 mutation in ovarian carcinomas. In addition to a strong and diffuse pattern of p53 expression (in greater than 60% of cells), complete absence of p53 immunoexpression is commonly associated with a TP53 mutation. Accordingly, this latter pattern, unlike low-level expression (10-50% cells), should not be construed as indicative of wild-type TP53.
P53, EGFR and HER-2/neu are the most frequently studied molecular biological parameters in epithelial ovarian cancer, but their prognostic impact is still unequivocal. We performed a meta-analysis to more precisely estimate their prognostic significance.
Published studies that investigated the association between p53, EGFR and HER-2/neu status and survival were identified. Meta-analysis was performed using a DerSimonian-Laird model. Publication bias was investigated using funnel plots and sources of heterogeneity were identified using meta-regression analysis.
A total of 62 studies were included for p53, 15 for EGFR and 20 for HER-2/neu. P53, EGFR and HER-2/neu status had a modest effect on overall survival (pooled HR 1.47, 95% CI 1.33-1.61 for p53; HR 1.65, 95% CI 1.25-2.19 for EGFR and HR 1.67, 95% CI 1.34-2.08 for HER-2/neu). Meta-regression analysis for p53 showed that FIGO stage distribution influenced study outcome. For EGFR and HER-2/neu, considerable publication bias was present.
Although p53, EGFR and HER-2/neu status modestly influences survival, these markers are, by themselves, unlikely to be useful as prognostic markers in clinical practice. Our study highlights the need for well-defined, prospective clinical trials and more complete reporting of results of prognostic factor studies.
Bortezomib, an approved drug for the treatment of certain haematological neoplasms, is currently being tested in clinical trials as a potential therapeutic agent against several types of solid cancer, including ovarian cancer. We have analyzed the effect of bortezomib on ovarian cancer cells and tissue explants either as a single agent or in combination with carboplatin, taxol, or TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). Bortezomib alone efficiently induced apoptosis in ovarian cancer cells. Apoptosis was preceded by an upregulation of the endoplasmic reticulum stress sensor ATF3, and increased the expression of cytoplasmic heat shock proteins. Bortezomib enhanced the sensitivity of ovarian cancer cells and tissue explants to an apoptosis-inducing TRAIL receptor antibody by upregulating the TRAIL receptor DR5. In contrast to the synergistic effect observed for TRAIL, the efficacy of the taxol treatment was reduced by bortezomib, and bortezomib inhibited the G2/M phase accumulation of ovarian cancer cells treated with taxol. Bortezomib alone or in combination with taxol induced a cell cycle arrest within the S phase, and downregulation of cdk1, a cyclin-dependent kinase that is necessary for the entry into the M phase. Thus, bortezomib can be regarded as a promising agent for the treatment of ovarian cancer and could either be administered as a single agent or in combination with TRAIL. However, a combination treatment with taxanes may not be beneficial and may even be less effective.
The correlation between inactivation of the TP53 gene through mutation or the presence of high-risk human papillomavirus (HPV) DNA and intrinsic paclitaxel sensitivity was studied in 27 gynaecological cancer cell lines. IC(50) values, as a measure of drug sensitivity, were determined using a 96-well clonogenic assay. TP53 mutations were investigated with polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and direct DNA sequencing. HPV status was studied with PCR using HPV consensus primers. TP53 mutations were found in 7/11 vulvar SCC cell lines. Only 2/9 endometrial and 1/7 ovarian cancer cell lines carried TP53 mutations. One vulvar and one endometrial cancer cell line were HPV-positive; both carrying HPV type-16 DNA. Thus, TP53 was functionally normal in 3/11 vulvar, 6/9 endometrial and 6/7 ovarian cancer cell lines. The IC(50) values for paclitaxel were 0.60-2.9, 0.49-2.3 and 0.40-3.4 nM in the vulvar, endometrial and ovarian cancer cell lines, respectively. No correlation could be demonstrated between inactivation of the TP53 gene and paclitaxel sensitivity in vitro; the cell lines were evaluated as one group or according to their anatomical origin or histology. Previous reports have given inconclusive results, partly due to the cell types used, i.e. normal, cancerous or transformed cells. Our results support the view that paclitaxel sensitivity of tumour-derived cancer cell lines is not related to the TP53 status.
Although cisplatin derivatives are first-line chemotherapeutic agents for the treatment of epithelial ovarian cancer, chemoresistance remains a major hurdle to successful therapy and the molecular mechanisms involved are poorly understood. Apoptosis is the cellular underpinning of cisplatin-induced cell death, which is associated with expression of specific "death" genes and down-regulation of "survival" counterparts. The X-linked inhibitor of apoptosis proteins (Xiap), an intracellular anti-apoptotic protein, plays a key role in cell survival by modulating death signaling pathways and is a determinant of cisplatin resistance in ovarian cancer cells in vitro. This review focuses on the role of Xiap and its interactions with the phosphoinositide-3 kinase (PI3K)/Akt cell survival pathway in conferring resistance of ovarian cancer cells to chemotherapeutic agents and discusses potential therapeutic strategies in overcoming chemoresistant ovarian cancer.
The aim of this study was to use the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (MTS) assay to determine the response of 17 endometrial and 27 cervical tumours to cytotoxic drugs. Tumour samples were taken at surgery and cultured using the explant technique. Cells were incubated with chemotherapy drugs. The MTS cytotoxicity assay was carried out to ascertain the response to the drugs and correlated retrospectively to the clinical outcome. Tumours of similar stage and grade displayed heterogeneity in their responses to the drugs. A total of 88 of 90 tumours (97.8%), including data from an earlier study of 44 ovarian tumour samples yielded chemosensitivity data. A total of 45 specimens were evaluable for in vitro-in vivo correlations. In vitro sensitivity was associated with clinical response in 26 of 30 patients and in vitro resistance with progressive disease or death in 14 of 15 patients. A randomised prospective trial should be carried out to validate chemosensitivity/resistance testing.
The glutathione S-transferases (GSTs) are a group of multifunctional enzymes that catalyze the conjugation of glutathione with a variety of electrophilic compounds, including cytotoxic agents. A significant percentage of normal individuals exhibit genetic polymorphism with a homozygous deletion (null genotype) of the genes, leading to absence of the enzyme.
In the present study we analyzed GSTM1 and GSTT1 polymorphisms in the genomic DNA isolated from peripheral blood of patients with ovarian cancer treated with chemotherapy (paclitaxel and cisplatinum) after cytoreductive surgery and assessed its correlation with the clinical outcome of these patients. The median follow-up for the patients was 30 months.
The estimated 3-year survival rate was 59.8% for all patients and 20.8% for carriers of GSTM1-wt/GSTT1-wt (wt indicates wild type) genotype combination (37.7% for GSTM1-wt alone) compared with 83.1% for non-GSTM1-wt/GSTT1-wt genotype carriers (100% for GSTM1-null). The mean survival time was significantly better in patients who are carriers of the GSTM1-null genotype (40.5 vs. 33.5; P=0.006) or carriers of non-GSTM1-wt/ GSTT1-wt genotypes (55.4 vs. 30.7; P=0.009). The progression-free interval was more favorable for GSTM1-null carriers (41.9 vs. 27.4; P=0.024).
The study suggests that characterization of the drug-metabolizing genetic individual profile can be of great interest in clinical oncology. It can define the optimal chemotherapy for each patient, improve the efficiency, and reduce the incidence of drug toxicity and poor drug responses.
Despite advances in surgery and chemotherapy, less than 20% of patients with stage III or IV ovarian cancer survive long-term. In the past, cytotoxic regimens have been developed empirically, combining active agents at maximally tolerated doses, often without a clear rationale for their interaction. Advances in understanding the biology of ovarian cancer have identified multiple molecular targets that differ in normal and malignant cells. Targets include cell cycle regulators, growth factor receptors, signal transduction pathways, molecules that confer drug resistance, and angiogenic mechanisms. A number of targeted agents have entered clinical trials. Small molecular weight inhibitors, monoclonal antibodies, and antisense and gene therapy are all being evaluated alone and in combination with cytotoxic drugs. In contrast to earlier studies, the impact of each agent on the designated target can be assessed and agents can be matched to the genotype and phenotype of malignant and normal cells. In the long run, this should facilitate individualization of more effective, less toxic therapy for women with ovarian cancer.
Much research suggests that TP53 mutations have prognostic importance and sometimes are a significant factor in clinical oncology. A considerable effort has been made to develop fast and inexpensive methods for TP53 mutations detection.
On the basis of describing the role of TP53 as tumor suppressor gene and TP53 mutation spectrum, the authors discuss conventional methods and new technologies for TP53 mutations detection. This discussion is supported by more recent publications in the field of both molecular genetics and analysis technologies.
Biosensors and gene chips are of considerable recent interest, due to their tremendous promise for obtaining sequence-specific information in a faster, simpler and cheaper manner compared to traditional methods.
New methods such as biosensors and gene chips appear promising as analytical methods of detecting mutations.
Breast and ovarian cancers, like other cancers, occur due to genetic damage. Research aimed to determine the specific genes involved in the development of breast and ovarian cancers will help to understand how normal breast and ovarian epithelial cells escape regulation of proliferation, apoptosis and senescence. It was determined that approximately 10% of ovarian cancers and 20-30% of breast cancers arise in women who have inherited mutations in cancer susceptibility genes such as BRCA1, BRCA2 and other DNA repair genes. The ability to perform genetic testing permits the identification of women at increased risk who can then be offered preventive strategies. The vast majority of ovarian and breast cancers are sporadic, presumably resulting from the accumulation of genetic damage over lifetime. Several genes involved in breast and ovarian carcinogenesis have been identified, most notably the p53 tumor suppressor. The recent availability of expression microarrays has facilitated the simultaneous screening of thousands of genes and this will extend further the understanding of molecular events involved in the dynamic development of ovarian and breast cancers. Then, all this knowledge could be translated into effective screening, surveillance, prevention, and treatment strategies in the future.
BRCA1 associated RING domain protein (BARD1) was originally identified due to its interaction with the RING domain of BRCA1. BARD1 is required for S phase progression, contact inhibition and normal nuclear division, as well as for BRCA1 independent, p53 dependent apoptosis.
To investigate whether alterations in BARD1 are involved in human breast and ovarian cancer, we used single strand conformation polymorphism analysis and sequencing on 35 breast tumours and cancer cell lines and on 21 ovarian tumours.
Along with the G2355C (S761N) missense mutation previously identified in a uterine cancer, we found two other variants in breast cancers, T2006C (C645R) and A2286G (I738V). The T2006C (C645R) mutation was also found in one ovarian tumour. A variant of uncertain consequence, G1743C (C557S), was found to be homozygous or hemizygous in an ovarian tumour. Eleven variants of BARD1 were characterised with respect to known functions of BARD1. None of the variants appears to affect localisation or interaction with BRCA1; however, putative disease associated alleles appear to affect the stability of p53. These same mutations also appear to abrogate the growth suppressive and apoptotic activities of BARD1.
These activities allowed us to identify one of the rare variants (A2286G; I738V) as a neutral polymorphism rather than a detrimental mutation, and suggested that G1743C (C557S) is not a polymorphism but may contribute to the cancer phenotype.
To sum up the knowledge about genetic aspects of ovarian cancer.
Review article.
Department of Gynecology and Obstetrics, First Faculty of Medicine, Charles University Prague and General Teaching Hospital Prague, Prague.
Compilation of data from scientific literature.
Ovarian cancer represents the most lethal malignancy among gynecological tumours. Etiology is still largely unknown. Multistep process, with accumulation of genetic alterations concerning factors with key role in cell regulation - oncogenes, tumor-supressor genes and mismatch-repair genes is supposed. Cytogenetic and molecular-genetic characteristics, in correlation with other molecular-biological parameters extend information of carcinogenesis. The final aim is to create a predictive model for clinical use.
The importance of somatic TP53 mutations and germline TP53 codon 72 genotype in the survival of women with epithelial ovarian cancer is controversial. Recent data suggest that a promoter polymorphism in the MDM2 gene may influence age of cancer onset in a gender-specific fashion. We sought to determine the relationship between somatic TP53 mutations, germline genotypes at TP53 codon 72 and MDM2 SNP309, and overall survival and response to chemotherapy in a large series of patients with ovarian and peritoneal carcinomas. Of the 188 cancers, 103 (54.8%) had a TP53 mutation, of which 71% were missense mutations and 29% were null mutations. TP53 mutation status and mutation type (null vs. missense) did not influence response to therapy or overall survival. Women with the codon 72 Pro/Pro had a decreased overall survival (median, 29 months) compared with women with one or two arginine alleles (median, 49 months; P=0.04). Somatic mutation or deletion was equally common for either codon 72 allele. Age of diagnosis was not influenced by codon 72 but showed a trend for younger age in women with somatic TP53 mutations and the MDM2 G/G genotype.
The purpose of this study was to assess the feasibility of establishing a library of ovarian cancer nucleic acids using paper matrix by: 1) confirming the stability of DNA stored on paper matrix over a prolonged period of time, 2) determining the amount of genetic material required for storage, and 3) establishing the stability of RNA. Tumor tissue from 66 patients with ovarian cancer was collected intraoperatively, frozen, and dissociated with collagenase and trypsin. A cell suspension was then prepared and spotted onto the paper. The numbers of cells that were stored on the paper were counted using a hemocytometer. The cell suspension was serially diluted and spotted on the paper matrix until the minimum cell number that can be stored and produce a PCR product was determined. PCR, STR genotyping and direct sequencing were performed on tissue stored on the paper matrix. FTA paper was used as RNA template, and RT PCR converted the RNA to cDNA. Ten to 50 mg of ovarian cancer tissue was stored on FTA paper. We stored 7 x 10(4) cells on ISOcode paper and 18 x 10(4) cells on FTA and obtained extractable DNA. PCR analysis on cards with DNA stored 18 months ago enabled us to establish the stability of DNA after storage. RNA was stable for 6 months when stored on FTA cards. Since genetic material is extractable from the paper matrices after passage of time, it could be a suitable medium for the storage of genetic material in cancer tissue banks.
The TP53 mutation frequency in ovarian serous carcinomas has been reported to range between 50% and 80%, but a stringent analysis of TP53 using purified epithelial samples has not yet been performed to accurately assess the mutation frequency and to correlate it with the histologic grade. The purpose of this study was to assess the TP53 mutational profile in a relatively large series of high-grade (53 primary and 18 recurrent) and 13 low-grade ovarian serous tumors using DNA isolated from affinity-purified tumor cells and to correlate it with in vitro drug resistance. All samples were affinity purified, and the tumor DNA was analyzed for TP53 mutations in exons 4-9. In vitro drug resistance assays to carboplatin, cisplatin, paclitaxel, and taxotere were performed on the same tumor samples and correlated with the TP53 mutation status. TP53 mutations were detected in 57 (80.3%) of 71 high-grade carcinomas and in one (7.8%) of 13 low-grade serous tumors (an invasive low-grade serous carcinoma). The mutations were predominantly missense mutations (59.6%). TP53 mutations were associated with high-grade serous carcinomas and recurrent disease (P < 0.0001). There was no statistically significant correlation between TP53 mutation status and drug resistance assays or clinical stage (P > 0.25). The frequency of TP53 mutations using purified tumor DNA from ovarian serous carcinomas was 80.3%, which is much higher than previously reported. Furthermore, we found that TP53 is not directly involved in the development of drug resistance in high-grade ovarian serous carcinomas.
The purpose of the study was to implement a uniform system for assigning tumor grade in serous ovarian cancer and evaluate its correlation with response to conventional chemotherapy.
Serous ovarian cancer tumor samples were retrospectively reviewed by 3 pathologists who were blinded to the original report. Samples were scored for architectural pattern, nuclear pleomorphism, and mitotic activity. Sum scores from these 3 indices were used to classify tumors as low grade or high grade.
A total of 21 patients were identified as low-grade tumors and 21 were identified as high-grade tumors. Of low-grade tumors, 16 (76%) were found to be platinum resistant, defined as recurrent or persistent disease, 180 days from completion of the final cycle of chemotherapy, Of 21 patients defined as high grade, 9 (43%) were platinum resistant (P = .028).
Utilization of a uniform grading system retrospectively correlates with platinum sensitivity.
Tumour stage, residual disease after initial surgery, histological type and tumour grade are the most important clinical-pathological factors related to the clinical outcome of patients with epithelial ovarian cancer. In the last years, several investigations have assessed different biological variables in sera and in tissue samples from patients with this malignancy in order to detect biomarkers able to reflect either the response to chemotherapy or survival. The present paper reviewed the literature data about the predictive or prognostic relevance of serum CA 125, soluble cytokeratin fragments, serum human kallikreins, serum cytokines, serum vascular endothelial growth factor and plasma d-dimer as well as of tissue expression of cell cycle- and apoptosis-regulatory proteins, human telomerase reverse transcriptase, membrane tyrosine kinase receptors and matrix metalloproteinases. A next future microarray technology will hopefully offer interesting perspectives of translational research for the identification of novel predictive and prognostic biomarkers for epithelial ovarian cancer.
Mutations of the p53 gene on chromosome 17p are a common genetic change in the malignant progression of many cancers. We have analyzed 38 malignant tumors of ovarian or peritoneal müllerian type for evidence of p53 variations at either the DNA or protein levels. Genetic studies were based on single-strand conformation polymorphism analysis and DNA sequencing of exons 2 through 11 of the p53 gene; mutations were detected in 79% of the tumors. These data show a statistically significant association between mutations at C.G pairs and a history of estrogen therapy. Two of 20 patients whose normal tissue could be studied carried germ-line mutations of p53. Immunohistochemical analysis of the p53 protein was carried out using monoclonal antibody PAb1801. Ninety-six percent of the missense mutations were associated with abnormal accumulation of p53 protein, but nonsense mutations, a splicing mutation, and most deletions did not result in p53 protein accumulation. A statistically significant association between p53 protein accumulation in poorly differentiated stage III serous carcinomas and small primary tumor size at diagnosis was found, perhaps suggesting that p53 protein accumulation accelerates the metastatic spread from a primary tumor. Overall, our findings indicate that alterations of p53 play a major role in ovarian cancer, including predisposition to the disease in some patients, and suggest a possible mechanism for somatic mutations leading to this cancer.
Chemotherapeutic management of ovarian cancers is a difficult task as these neoplasms show significant differences in chemosensitivity, even if they share identical clinicopathological features. The present study was undertaken to investigate the prognostic and predictive role of p53 alterations in ovarian cancer. To this end, using different technical approaches, i.e. genetic and immunohistochemical analyses, we analysed a series of 68 ovarian neoplasms including 15 low malignant potential (LMP) tumours and 53 invasive carcinomas. We never observed p53 abnormalities in LMP tumours. p53 alterations were present only in invasive ovarian carcinomas, and they were detected much more frequently in tumours characterized by high histological grade (P = 0.01) and advanced-stage disease (P = 0.006 and P = 0.05 for gene mutations and protein expression respectively). For 33 patients with invasive ovarian cancer, information was available concerning response to cisplatin-based chemotherapy. A strong correlation (P = 0.001) has emerged between p53 alterations and response to chemotherapy; only one (14%) of seven patients who had a pathological complete response to antiblastic drugs showed p53 aberrations, whereas 18 (82%) of 22 cases with partial response and all of the four non-responsive patients scored positive for p53 abnormalities. We also observed that patients with p53 mutations had a significantly shorter progression-free survival than patients with p53-negative tumours (P = 0.05). Taken together, our results strongly suggest that in epithelial ovarian malignancies tumours showing p53 aberrations are significantly less sensitive to chemotherapy and more aggressive than those with functional p53. Thus, a routine analysis of this gene could have profound implications for the treatment of ovarian cancer.
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For the past decade, cellular oncogenes have attracted the attention of biologists intent on understanding the molecular origins of cancer. As the present decade unfolds, oncogenes are yielding their place at center stage to a second group of actors, the tumor suppressor genes, which promise to teach us equally important lessons about the molecular mechanisms of cancer pathogenesis.
A new technique for characterizing somatic mutations in very small samples of cellularly heterogeneous human cancer tissue was developed and tested using mutations in the p53 gene in breast carcinomas as a model system. The technique combines touch preparation of specimens to obtain homogeneous clusters of carcinoma cells free of normal cells with a nested pair of polymerase chain reaction (PCR) amplifications of DNA to increase the amount of target gene sequence sufficiently to permit direct sequencing of the p53 gene. Touch preparations of fresh or previously frozen tissue from human adenocarcinomas derived from several organs were stained, and clusters of 10-50 malignant cells were transferred by pipette into microfuge tubes for PCR amplification. Exons 5-9 of the p53 gene, which contain the major mutational hot spots associated with most human cancers, were sequenced by the following steps: 1) two rounds of PCR amplification using DNA Taq polymerase and two sets of oligonucleotide primers, the second set being nested within the segment amplified by the first set and having attached T7 and SP6 phage promoter sequences, 2) transcription of the amplified DNA sequences with T7 and SP6 RNA polymerases, and 3) dideoxy sequencing of single-stranded RNA transcripts with reverse transcriptase and with additional oligonucleotide primers to achieve specificity for this unique region of the genome. The utility of this approach is illustrated by our success in detecting and analyzing point mutations in cell clusters from four of 11 primary adenocarcinomas of the human breast.
Epithelial carcinoma of the ovary is characterized by presentation at an advanced stage, spread primarily by an intraperitoneal (IP) route, and relative sensitivity to chemotherapy. An initial surgical approach is essential to proper staging of the disease process and to aggressive cytoreduction, which in turn improves response to chemotherapy and survival. The use of chemotherapy is the mainstay of definitive therapy after completion of the initial surgery. A large number of drugs have activity against the disease, with the most important single category of agents being the platinum compounds. Studies by the Gynecologic Oncology Group (GOG) document the superiority of cisplatin-based combination chemotherapy over single alkylating agents and combinations that do not include cisplatin. The current regimen of choice is a two-drug combination of cisplatin plus cyclophosphamide. Efforts to improve results further focus on enhancing dose intensity of the drug combination through either escalating intravenous (IV) doses or administering cisplatin via an IP route. Also offering an opportunity for further improvement of therapeutic results are three drugs identified as having activity in patients no longer candidates for cisplatin: carboplatin, ifosfamide, and taxol. Biologic agents, such as alpha-interferon, also have potential roles in future combination therapy. The management of patients with limited (stage I or II) disease is based on studies of the GOG and the Ovarian Cancer Study Group, which indicate that this population can be divided by prognostic factors into a group at low risk for recurrence and a group at high risk. Those at low risk require only surgery, whereas those at high risk should receive either IP radioactive chromic phosphate or systemic chemotherapy following surgery. The future prospects for additional improvement of results in all patients appear bright on the basis not only of studies of dose intensity and IP therapy but also of efforts directed at overcoming multidrug resistance and at devising noninvasive means of assessing disease status.
We report a rapid and sensitive method for the detection of base changes in given sequences of genomic DNA. This technique is based on the facts that specific regions of genomic sequences can be efficently labeled and amplified simultaneously by using labeled substrates in the polymerase chain reaction and that in nondenaturing polyacrylamide gels, the electrophoretic mobility of single-stranded nucleic acid depends not only on its size but also on its sequence. The process does not involve restriction enzyme digestion, blotting, or hybridization to probes. We found that most single base changes in up to 200-base fragments could be detected as mobility shifts. RAS oncogene activation was detected by this technique. We also show that the interspersed repetitive sequences of human, Alu repeats are highly polymorphic.
Chemotherapy combinations that include an alkylating agent and a platinum coordination complex have high response rates in women with advanced ovarian cancer. Such combinations provide long-term control of disease in few patients, however. We compared two combinations, cisplatin and cyclophosphamide and cisplatin and paclitaxel, in women with ovarian cancer.
We randomly assigned 410 women with advanced ovarian cancer and residual masses larger than 1 cm after initial surgery to receive cisplatin (75 mg per square meter of body-surface area) with either cyclophosphamide (750 mg per square meter) or paclitaxel (135 mg per square meter over 24 hours).
Three hundred eighty-six women met all the eligibility criteria. Known prognostic factors were similar in the two treatment groups. Alopecia, neutropenia, fever, and allergic reactions were reported more frequently in the cisplatin-paclitaxel group. Among 216 women with measurable disease, 73 percent in the cisplatin-paclitaxel group responded to therapy, as compared with 60 percent in the cisplatin-cyclophosphamide group (P = 0.01). The frequency of surgically verified complete response was similar in the two groups. Progression-free survival was significantly longer (P < 0.001) in the cisplatin-paclitaxel group than in the cisplatin-cyclophosphamide group (median, 18 vs. 13 months). Survival was also significantly longer (P < 0.001) in the cisplatin-paclitaxel group (median, 38 vs. 24 months).
Incorporating paclitaxel into first-line therapy improves the duration of progression-free survival and of overall survival in women with incompletely resected stage III and stage IV ovarian cancer.
A comparison was made between the 3 most commonly used techniques for the detection of point mutations: single-strand conformation polymorphism (SSCP), constant denaturant gel electrophoresis (CDGE), and hydroxylamine and osmium tetroxide used in amplification mismatch cleavage analysis (HOT). Using human DNA samples containing known mutations in the p53 gene, SSCP detected 90% of mutations (18/20), CDGE detected 88% (15/17) pre-decoding of the samples but 100% when the mutations were known and the CDGE conditions optimized, and the HOT technique was 100% accurate, although 1 mutation was missed through careless examination of the gel. The positive and negative aspects of each of the techniques are considered and suggestions are made regarding the particular situations in which each of them is most useful.
The perceived function of wild-type p53 is suppression of cell proliferation. An alteration in the p53 tumor suppressor gene is a common defect in human malignancies. The purpose of this study was to prospectively determine whether p53 expression, as quantified by image analysis, was related to traditional prognostic indicators as well as survival in patients epithelial ovarian cancer.
Eighty-three consecutive patients with epithelial ovarian cancer had their p53 expression studied by immunohistochemical staining and quantified by image analysis. Unless otherwise noted, p53 expression was reported as the percentage positive nuclear area staining.
The mean follow-up was 37 months (median, 30 months; range 24-55 months). In patients with serous carcinomas of the ovary, the mean p53 expression was 29.4%, whereas in patients with other histologies, the mean was 10.5% (P < 0.001). The tumors of patients with stage III or IV tumors stained significantly higher (mean 28. 7%) than the tumors of patients with stage I or II disease (mean 8. 36%) (P < 0.001). The tumors of patients with disease which could be optimally cytoreduced stained significantly lower (mean 23.0%) than the tumors of patients whose disease was unable to be optimally cytoreduced (mean 28.6%) (P = 0.041). Utilizing survival as the endpoint for multivariate analysis, FIGO stage (P = 0.006), p53 expression (P = 0.046), and the level of cytoreduction (P < 0.001) were independent prognostic indicators.
Image analysis allows quantitative measurements of p53 staining. p53 staining is significantly higher in advanced-stage, high-grade tumors which are unable to be cytoreduced than in early-stage, low-grade tumors which can be optimally cytoreduced. p53 expression is an independent prognostic indicator of survival in patients with epithelial ovarian carcinomas.