Longitudinal analysis of circulating tumor cell numbers improves tracking metastatic breast cancer progression.

Hormone-responsive breast cancer represents the most common type and has the best prognosis, but still approximately 40% of patients with this type can develop distant metastases, dramatically worsening the patient's survival. Monitoring metastatic breast cancer (mBC) for signs of progression is an important part of disease management. Circulating tumor cell (CTC) detection and molecular characteristics gain importance as a diagnostic tool, but do not represent a clinical standard and its value as a predictor of progression is not yet established. The main objective of this study was to estimate the prognostic value of not only the CTC numbers, but also the dynamics of the CTC numbers in the same patient during the continuous evaluation of CTCs in patients with advanced breast cancer. The other objective was to assess the molecular changes in CTCs compared to primary tumor samples by genetic analysis of the seven genes associated with estrogen signaling pathway, mutations in which are often responsible for the resistance to endocrine therapy, and subsequent progression. This approach was taken to evaluate if genetic analysis of CTCs can be used in tracking the resistance, signaling that hormonal therapy should be replaced. Consequently, this report presents the results of a longitudinal CTC study based on three subsequent blood collections from 135 patients with metastatic breast cancer, followed by molecular analysis of the isolated single CTCs. CTCs were detected and isolated using an image-based, EpCAM-independent system CytoTrack; this approach allowed evaluation of EpCAM expression in detected CTCs. Isolated CTCs were subjected to NGS analysis to assess mutational changes. The results confirm the importance of the status of the CTC for progression-free survival and overall survival and provide new data on the dynamics of the CTC during a long monitoring period and in relation to clinical progression, highlighting the advantage of constant monitoring over the single count of CTC. Furthermore, high genetic and phenotypic inter- and intrapatient heterogeneity observed in CTCs suggest that metastatic lesions are divergent. High genetic heterogeneity in the matching CTC/primary tumor samples may indicate early dissemination. The tendency towards the accumulation of activating/oncogenic mutation in CTCs, leading to anti-estrogen resistant disease, was not confirmed in this study.

A Multi-Faceted Analysis Showing CRNDE Transcripts and a Recently Confirmed Micropeptide as Important Players in Ovarian Carcinogenesis.

CRNDE is considered an oncogene expressed as long non-coding RNA. Our previous paper is the only one reporting CRNDE as a micropeptide-coding gene. The amino acid sequence of this micropeptide (CRNDEP) has recently been confirmed by other researchers. This study aimed at providing a mass spectrometry (MS)-based validation of the CRNDEP sequence and an investigation of how the differential expression of CRNDE(P) influences the metabolism and chemoresistance of ovarian cancer (OvCa) cells. We also assessed cellular localization changes of CRNDEP, looked for its protein partners, and bioinformatically evaluated its RNA-binding capacities. Herein, we detected most of the CRNDEP sequence by MS. Moreover, our results corroborated the oncogenic role of CRNDE, portraying it as the gene impacting carcinogenesis at the stages of DNA transcription and replication, affecting the RNA metabolism, and stimulating the cell cycle progression and proliferation, with CRNDEP being detected in the centrosomes of dividing cells. We also showed that CRNDEP is located in nucleoli and revealed interactions of this micropeptide with p54, an RNA helicase. Additionally, we proved that high CRNDE(P) expression increases the resistance of OvCa cells to treatment with microtubule-targeted cytostatics. Furthermore, altered CRNDE(P) expression affected the activity of the microtubular cytoskeleton and the formation of focal adhesion plaques. Finally, according to our in silico analyses, CRNDEP is likely capable of RNA binding. All these results contribute to a better understanding of the CRNDE(P) role in OvCa biology, which may potentially improve the screening, diagnosis, and treatment of this disease.

Endometrial Cancer-Adjacent Tissues Express Higher Levels of Cancer-Promoting Genes than the Matched Tumors.

Molecular alterations in tumor-adjacent tissues have recently been recognized in some types of cancer. This phenomenon has not been studied in endometrial cancer. We aimed to analyze the expression of genes associated with cancer progression and metabolism in primary endometrial cancer samples and the matched tumor-adjacent tissues and in the samples of endometria from cancer-free patients with uterine leiomyomas. Paired samples of tumor-adjacent tissues and primary tumors from 49 patients with endometrial cancer (EC), samples of endometrium from 25 patients with leiomyomas of the uterus, and 4 endometrial cancer cell lines were examined by the RT-qPCR, for MYC, NR5A2, CXCR2, HMGA2, LIN28A, OCT4A, OCT4B, OCT4B1, TWIST1, STK11, SNAI1, and miR-205-5p expression. The expression levels of MYC, NR5A2, SNAI1, TWIST1, and STK11 were significantly higher in tumor-adjacent tissues than in the matched EC samples, and this difference was not influenced by the content of cancer cells in cancer-adjacent tissues. The expression of MYC, NR5A2, and SNAI1 was also higher in EC-adjacent tissues than in samples from cancer-free patients. In addition, the expression of MYC and CXCR2 in the tumor related to non-endometrioid adenocarcinoma and reduced the risk of recurrence, respectively, and higher NR5A2 expression in tumor-adjacent tissue increased the risk of death. In conclusion, tissues proximal to EC present higher levels of some cancer-promoting genes than the matched tumors. Malignant tumor-adjacent tissues carry a diagnostic potential and emerge as new promising target of anticancer therapy.

PROM1, CXCL8, RUNX1, NAV1 and TP73 genes as independent markers predictive of prognosis or response to treatment in two cohorts of high-grade serous ovarian cancer patients.

Considering the vast biological diversity and high mortality rate in high-grade ovarian cancers, identification of novel biomarkers, enabling precise diagnosis and effective, less aggravating treatment, is of paramount importance. Based on scientific literature data, we selected 80 cancer-related genes and evaluated their mRNA expression in 70 high-grade serous ovarian cancer (HGSOC) samples by Real-Time qPCR. The results were validated in an independent Northern American cohort of 85 HGSOC patients with publicly available NGS RNA-seq data. Detailed statistical analyses of our cohort with multivariate Cox and logistic regression models considering clinico-pathological data and different TP53 mutation statuses, revealed an altered expression of 49 genes to affect the prognosis and/or treatment response. Next, these genes were investigated in the validation cohort, to confirm the clinical significance of their expression alterations, and to identify genetic variants with an expected high or moderate impact on their products. The expression changes of five genes, PROM1, CXCL8, RUNX1, NAV1, TP73, were found to predict prognosis or response to treatment in both cohorts, depending on the TP53 mutation status. In addition, we revealed novel and confirmed known SNPs in these genes, and showed that SNPs in the PROM1 gene correlated with its elevated expression.

Cryptic MYC insertions in Burkitt lymphoma: New data and a review of the literature.

The occurrence of MYC-negative Burkitt lymphoma (BL) has been discussed for many years. The real frequency of the MYC insertion in MYC-negative BL is still unknown. Fine-needle aspiration biopsies of 108 consecutive patients with clinicopathologically suspected BL (suspBL) were evaluated by flow cytometry, classical cytogenetics, and fluorescence in situ hybridization (FISH). We found 12 cases (11%) without the MYC rearrangement by FISH with a MYC breakapart probe: two patients (1.9%) with cryptic MYC/IGH fusion (finally diagnosed as BL) and 10 patients (9.3%) with 11q gain/loss (finally diagnosed as Burkitt-like lymphoma with 11q aberration). The exact breakpoints of the cryptic MYC/IGH were investigated by next-generation sequencing. The MYC insertions' breakpoints were identified in PVT1 in the first case, and 42 kb upstream of 5'MYC in the second case. To date, a molecular characterization of the MYC insertion in BL has only been reported in one case. Detailed descriptions of our MYC insertions in a routinely and consecutively diagnosed suspBL cohort will contribute to resolving the issue of MYC negativity in BL. In our opinion, the presence of the MYC insertions in BL and other lymphomas might be underestimated, because routine genetic diagnostics are usually based on FISH only, without karyotyping.

A Set of 17 microRNAs Common for Brain and Cerebrospinal Fluid Differentiates Primary Central Nervous System Lymphoma from Non-Malignant Brain Tumors.

The diagnosis of primary central nervous system (CNS) lymphoma, which is predominantly of the diffuse large B-cell lymphoma type (CNS DLBCL), is challenging. MicroRNAs (miRs) are gene expression-regulating non-coding RNAs that are potential biomarkers. We aimed to distinguish miR expression patterns differentiating CNS DLBCL and non-malignant CNS diseases with tumor presentation (n-ML). Next generation sequencing-based miR profiling of cerebrospinal fluids (CSFs) and brain tumors was performed. Sample source-specific (CSF vs. brain tumor) miR patterns were revealed. Even so, a set of 17 miRs differentiating CNS DLBCL from n-ML, no matter if assessed in CSF or in a tumor, was identified. Along with the results of pathway analyses, this suggests their pathogenic role in CNS DLBCL. A combination of just four of those miRs (miR-16-5p, miR-21-5p, miR-92a-3p, and miR-423-5p), assessed in CSFs, discriminated CNS DLBCL from n-ML samples with 100% specificity and 67.0% sensitivity. Analyses of paired CSF-tumor samples from patients with CNS DLBCL showed significantly lower CSF levels of miR-26a, and higher CSF levels of miR-15a-5p, miR-15b-5p, miR-19a-3p, miR-106b-3p, miR-221-3p, and miR-423-5p. Noteworthy, the same miRs belonged to the abovementioned set differentiating CNS DLBCL from non-malignant CNS diseases. Our results not only add to the basic knowledge, but also hold significant translational potential.

Pleiotropy-guided transcriptome imputation from normal and tumor tissues identifies candidate susceptibility genes for breast and ovarian cancer.

Familial, sequencing, and genome-wide association studies (GWASs) and genetic correlation analyses have progressively unraveled the shared or pleiotropic germline genetics of breast and ovarian cancer. In this study, we aimed to leverage this shared germline genetics to improve the power of transcriptome-wide association studies (TWASs) to identify candidate breast cancer and ovarian cancer susceptibility genes. We built gene expression prediction models using the PrediXcan method in 681 breast and 295 ovarian tumors from The Cancer Genome Atlas and 211 breast and 99 ovarian normal tissue samples from the Genotype-Tissue Expression project and integrated these with GWAS meta-analysis data from the Breast Cancer Association Consortium (122,977 cases/105,974 controls) and the Ovarian Cancer Association Consortium (22,406 cases/40,941 controls). The integration was achieved through application of a pleiotropy-guided conditional/conjunction false discovery rate (FDR) approach in the setting of a TWASs. This identified 14 candidate breast cancer susceptibility genes spanning 11 genomic regions and 8 candidate ovarian cancer susceptibility genes spanning 5 genomic regions at conjunction FDR < 0.05 that were >1 Mb away from known breast and/or ovarian cancer susceptibility loci. We also identified 38 candidate breast cancer susceptibility genes and 17 candidate ovarian cancer susceptibility genes at conjunction FDR < 0.05 at known breast and/or ovarian susceptibility loci. The 22 genes identified by our cross-cancer analysis represent promising candidates that further elucidate the role of the transcriptome in mediating germline breast and ovarian cancer risk.

Cytoplasmic HAX1 Is an Independent Risk Factor for Breast Cancer Metastasis.

HAX1 is an antiapoptotic factor involved in the regulation of cell migration and calcium homeostasis, overexpressed in several cancers, including breast cancer. It has been suggested that HAX1 is also implicated in metastasis. Herein we report the results of meta-analysis of HAX1 expression, based on publicly available data, which confirms its significant overexpression in breast cancer and demonstrates copy number gain and prognostic value of HAX1 overexpression for metastatic relapse in ER+ tumors. IHC analysis reported here also reveals its significant overexpression in breast cancer samples from primary tumors, indicating significantly higher HAX1 protein levels in a group of patients who developed distant metastases in a disease course. Moreover, we demonstrate that HAX1 localization is important for the prediction of metastatic relapse and that cytoplasmic but not nuclear HAX1 is an independent risk factor for breast cancer metastasis.

Clinical importance of FANCD2, BRIP1, BRCA1, BRCA2 and FANCF expression in ovarian carcinomas.

OBJECTIVE: DNA repair pathways are potential targets of molecular therapy in cancer patients. The FANCD2, BRIP1, BRCA1/2, and FANCF genes are involved in homologous recombination DNA repair, which implicates their possible role in cell response to DNA-damaging agents. We evaluated a clinical significance of pre-treatment expression of these genes at mRNA level in 99 primary, advanced-stage ovarian carcinomas from patients, who later received taxane-platinum (TP) or platinum-cyclophosphamide (PC) treatment. METHODS: Gene expression was determined with the use of Real-Time PCR. The BRCA2 and BRIP1 gene sequence was investigated with the use of SSCP, dHPLC, and PCR-sequencing. RESULTS: Increased FANCD2 expression occurred to be a negative prognostic factor for all patients (PC+TP:HR 3.85, p = 0.0003 for the risk of recurrence; HR 1.96, p = 0.02 for the risk of death), and this association was even stronger in the TP-treated group (HR 6.7, p = 0.0002 and HR 2.33, p = 0.01, respectively). Elevated BRIP1 expression was the only unfavorable molecular factor in the PC-treated patients (HR 8.37, p = 0.02 for the risk of recurrence). Additionally, an increased FANCD2 and BRCA1/2 expression levels were associated with poor ovarian cancer outcome in either TP53-positive or -negative subgroups of the TP-treated patients, however these groups were small. Sequence analysis identified one protein truncating variant (1/99) in BRCA2 and no mutations (0/56) in BRIP1. CONCLUSIONS: Our study shows for the first time that FANCD2 overexpression is a strong negative prognostic factor in ovarian cancer, particularly in patients treated with TP regimen. Moreover, increased mRNA level of the BRIP1 is a negative prognostic factor in the PC-treated patients. Next, changes in the BRCA2 and BRIP1 genes are rare and together with other analyzed FA genes considered as homologous recombination deficiency may not affect the expression level of analyzed genes.

Clinical importance of the EMSY gene expression and polymorphisms in ovarian cancer.

EMSY, a BRCA2-associated protein, is amplified and overexpressed in various sporadic cancers. This is the first study assessing the clinical impact of its expression and polymorphisms on ovarian cancer (OvCa) outcome in the context of the chemotherapy regimen used. In 134 frozen OvCa samples, we assessed EMSY mRNA expression with Reverse Transcription-quantitative PCR, and also investigated the EMSY gene sequence using SSCP and/or PCR-sequencing. Clinical relevance of changes in EMSY mRNA expression and DNA sequence was evaluated in two subgroups treated with either taxane/platinum (TP, n=102) or platinum/cyclophosphamide (PC, n=32). High EMSY expression negatively affected overall survival (OS), disease-free survival (DFS) and sensitivity to treatment (PS) in the TP-treated subgroup (p-values: 0.001, 0.002 and 0.010, respectively). Accordingly, our OvCa cell line studies showed that the EMSY gene knockdown sensitized A2780 and IGROV1 cells to paclitaxel. Interestingly, EMSY mRNA expression in surviving cells was similar as in the control cells. Additionally, we identified 24 sequence alterations in the EMSY gene, including the previously undescribed: c.720G>C, p.(Lys240Asn); c.1860G>A, p.(Lys620Lys); c.246-76A>G; c.421+68A>C. In the PC-treated subgroup, a heterozygous genotype comprising five SNPs (rs4300410, rs3814711, rs4245443, rs2508740, rs2513523) negatively correlated with OS (p-value=0.009). The same SNPs exhibited adverse borderline associations with PS in the TP-treated subgroup. This is the first study providing evidence that high EMSY mRNA expression is a negative prognostic and predictive factor in OvCa patients treated with TP, and that the clinical outcome may hinge on certain SNPs in the EMSY gene as well.

Changes in plasma miR-9, miR-16, miR-205 and miR-486 levels after non-small cell lung cancer resection.

PURPOSE: The majority of non-small cell lung cancer (NSCLC) patients presents with an advanced-stage disease and, consequently, exhibits a poor overall survival rate. We aimed to assess changes in plasma miR-9, miR-16, miR-205 and miR-486 levels and their potential as biomarkers for the diagnosis and monitoring of NSCLC patients. METHODS: Plasma was collected from 50 healthy donors and from NSCLC patients before surgery (n = 61), 1 month after surgery (n = 37) and 1 year after surgery (n = 14). microRNA levels were quantified using qRT-PCR. RESULTS: We found in NSCLC patients before treatment, both with squamous cell carcinoma (SQCC) and adenocarcinoma (ADC), significantly higher plasma miR-16 and miR-486 levels than in healthy individuals. Pre-treatment miR-205 concentrations were found to be significantly higher in SQCC than in ADC patients, and only SQCC patients presented significantly higher circulating miR-205 levels than healthy donors. SQCC plasma miR-9 levels were not different from normal control levels, but in ADC they were found to be significantly decreased. A combination of plasma miR-16, miR-205 and miR-486 measurements was found to discriminate NSCLC patients from healthy persons, with a specificity of 95% and a sensitivity of 80%. Following tumor resection, we found that the miR-9 and miR-205 levels significantly decreased, even below the normal level, whereas the increased miR-486 level persisted up to one year after surgery, and the miR-16 level decreased to normal. After tumor resection, none of the miR levels tested was found to relate to recurrence. CONCLUSIONS: Our data indicate that miR-9, miR-16, miR-205 and miR-486 may serve as NSCLC biomarkers. The observed cancer-related pre- and post-operative changes in their plasma levels may not only reflect the presence of a primary cancer, but also of a systemic response to cancer.

Prognosis of patients with BRCA1-associated ovarian carcinomas depends on TP53 accumulation status in tumor cells.

OBJECTIVE: TP53 mutation is the most frequent molecular event in BRCA1-associated ovarian carcinomas. TP53 status may be a confounding factor in the evaluation of clinical importance of other proteins. We aimed to evaluate the clinical significance of BRCA1 mutations with respect to the TP53 accumulation status in 159 high-grade ovarian carcinomas. METHODS: Statistical analyses were done with the Kaplan-Meier method, log-rank test, the Cox's and logistic regression models for all patients, and in subgroups with and without TP53 accumulation (TP53+ and TP53-, respectively). RESULTS: Forty of 159 ovarian carcinomas (25.2%) were diagnosed in patients with BRCA1 germline mutations; 102 tumors (64.2%) were TP53+ and 57 (37.8%) were TP53-. Among patients with TP53+ carcinomas, BRCA1 carriers had increased odds of recurrence compared with sporadic cases (HR 2.25, P=0.003; median disease-free survival time 7.7 vs. 18.4months, respectively). In the smaller TP53- subgroup, BRCA1 mutation reduced the risk of death by 46% (HR 0.54, P=0.099, median overall survival time 42.7 vs. 28.1months), but beyond the border of significance. When the TP53 status was not taken into account, BRCA1 mutations did not show any significance, however, there was a trend toward increased odds of complete remission for women with BRCA1 mutations compared to non-carriers (OR 2.47, P=0.064). Taxane-platinum therapy showed advantage over the platinum-cyclophosphamide one in the entire group of patients and in the TP53+ subgroup. CONCLUSIONS: Our results suggest that the TP53 accumulation status determines the prognosis of BRCA1 mutation carriers with high-grade ovarian carcinomas.

The significance of c.690G>T polymorphism (rs34529039) and expression of the CEBPA gene in ovarian cancer outcome.

The CEBPA gene is known to be mutated or abnormally expressed in several cancers. This is the first study assessing the clinical impact of CEBPA gene status and expression on the ovarian cancer outcome. The CEBPA gene sequence was analyzed in 118 ovarian cancer patients (44 platinum/cyclophosphamide (PC)-treated and 74 taxane/platinum (TP)-treated), both in tumors and blood samples, and in blood from 236 healthy women, using PCR-Sanger sequencing and Real-Time quantitative PCR (qPCR)-based genotyping methods, respectively. The CEBPA mRNA level was examined with Reverse Transcription quantitative PCR (RT-qPCR). The results were correlated to different clinicopathological parameters. Thirty of 118 (25.4%) tumors harbored the CEBPA synonymous c.690G>T polymorphism (rs34529039), that we showed to be related to up-regulation of CEBPA mRNA levels (p=0.0059). The presence of the polymorphism was significantly associated with poor prognosis (p=0.005) and poor response to the PC chemotherapy regimen (p=0.024). In accordance, elevated CEBPA mRNA levels negatively affected patient survival (p<0.001) and tumor response to the PC therapy (p=0.014). The rs34529039 SNP did not affect the risk of developing ovarian cancer. This is the first study providing evidence that the c.690G>T, p.(Thr230Thr) (rs34529039) polymorphism of the CEBPA gene, together with up-regulation of its mRNA expression, are negative factors worsening ovarian cancer outcome. Their adverse clinical effect depends on a therapeutic regimen used, which might make them potential prognostic and predictive biomarkers for response to DNA-damaging chemotherapy.

The putative oncogene, CRNDE, is a negative prognostic factor in ovarian cancer patients.

The CRNDE gene seems to play an oncogenic role in cancers, though its exact function remains unknown. Here, we tried to assess its usefulness as a molecular prognostic marker in ovarian cancer. Based on results of our microarray studies, CRNDE transcripts were further analyzed by Real-Time qPCR-based profiling of their expression. The qPCR study was conducted with the use of personally designed TaqMan assays on 135 frozen tissue sections of ovarian carcinomas from patients treated with platinum compounds and either cyclophosphamide (PC, N = 32) or taxanes (TP, N = 103). Elevated levels of two different CRNDE transcripts were a negative prognostic factor; they increased the risk of death and recurrence in the group of patients treated with TP, but not PC (DNA-damaging agents only). Higher associations were found for overexpression of the short CRNDE splice variant (FJ466686): HR 6.072, 95% CI 1.814-20.32, p = 0.003 (the risk of death); HR 15.53, 95% CI 3.812-63.28, p < 0.001 (the risk of recurrence). Additionally, accumulation of the TP53 protein correlated with decreased expression of both CRNDE transcripts in tumor cells. Our results depict CRNDE as a potential marker of poor prognosis in women with ovarian carcinomas, and suggest that its significance depends on the therapeutic regimen used.

The Novel Gene CRNDE Encodes a Nuclear Peptide (CRNDEP) Which Is Overexpressed in Highly Proliferating Tissues.

CRNDE, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the CRNDE gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two CRNDE transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of CRNDE. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our in silico results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the CRNDE gene.

Germline SMARCA4 mutations in patients with ovarian small cell carcinoma of hypercalcemic type.

BACKGROUND: SMARCA4 mutations have recently been identified as driving lesions of the ovarian small cell carcinoma of hypercalcemic type (SCCHT). Familial occurrence of this neoplasm was described previously. METHODS: We looked for germline SMARCA4 alterations in eight patients with the SCCHT. DNA was extracted from probands' and their relatives' blood. The SMARCA4 coding sequence, previously found altered in all the tumors, was PCR amplified and sequenced in the germline DNA. RESULTS: Two patients carried a heterozygous germline SMARCA4 alteration: c.3760G > T and c.2352insG, respectively. The analysis of the probands' next of kins revealed that the c.3760G > T mutation was inherited by the proband and her sister from their father, and the sisters' four children also carried the mutation. The proband's sister was diagnosed with a carcinoma of the parotid gland at age 2. A brother of the other proband was tested negative. CONCLUSIONS: Our study suggests that some women develop the ovarian SCCHT due to the inherited or possibly de novo-occurring germline alterations in the SMARCA4 gene, however, its penetrance appears limited. Nevertheless, because of high aggressiveness of the SCCHT, a molecular diagnostics of the SMARCA4 gene and careful follow-up should be offered to patients with this cancer and their families.

p19(INK4d) mRNA and protein expression as new prognostic factors in ovarian cancer patients.

p19(INK4d) (CDKN2D) is a negative regulator of the cell cycle. Little is known of its role in cancer development and prognosis. We aimed to evaluate the clinical significance of p19(INK4d) expression in ovarian carcinomas with respect to the TP53 accumulation status, as well as the frequency of CDKN2D mutations. p19(INK4d) and TP53 expression was evaluated immunohistochemically in 445 ovarian carcinomas: 246 patients were treated with platinum-cyclophosphamide (PC/PAC), while 199 were treated with taxane-platinum agents (TP). CDKN2D gene expression (mRNA) was examined in 106 carcinomas, while CDKN2D mutations in 68 tumors. Uni- and multivariate statistical analyses (logistic regression and the Cox proportional hazards model) were performed for patient groups divided according to the chemotherapeutic regimen administered, and in subgroups with and without TP53 accumulation. High p19(INK4d) expression increased the risk of death, but only in patients with the TP53-negative carcinomas (HR 1.61, P = 0.049 for PC/PAC-treated patients, HR 2.00, P = 0.015 for TP-treated patients). This result was confirmed by the mRNA analysis (HR 4.24, P = 0.001 for TP-treated group). High p19(INK4d) protein expression associated with adverse clinicopathological factors. We found no alterations in the CDKN2D gene; the c.90C>G (p.R30R; rs1968445) polymorphism was detected in 10% of tumors. Our results suggest that p19(INK4d) expression is a poor prognostic factor in ovarian cancer patients. Analyses of tumor groups according to the TP53 accumulation status facilitate the identification of cancer biomarkers.

High frequency of allelic loss at the BRCA1 locus in ovarian cancers: clinicopathologic and molecular associations.

BRCA1 dysfunction may occur by different mechanisms that are rarely evaluated concomitantly. We aimed to analyze BRCA1 germline mutations, loss of heterozygosity (LOH) and promoter methylation in unselected ovarian carcinomas in the context of their clinicopathologic characteristics and other molecular changes. BRCA1 mutations were analyzed in 257 carcinomas using single-strand conformation polymorphism (SSCP), heteroduplex, and sequencing methods. LOH at the BRCA1 locus was screened for in 180 cancers. Methylation analysis was performed for 241 tumors using quantitative methylation specific PCR (qMSP). BRCA1 alterations, comprising germline mutations, allelic loss, and/or aberrant promoter methylation, were found in 77.6% (125/161) of ovarian carcinomas. Patients with germline mutations were younger than non-carriers (P < 0.0001). Germline mutations and LOH were associated with advanced stages (P=0.009, P < 0.0001), high tumor grade (P=0.005, P < 0.0001), and TP53 mutations (P=0.003, P < 0.0001, for mutations and LOH, respectively). LOH was also associated with the serous histological type (P=0.004) and PIK3CA amplification (P=0.003). Aberrant promoter methylation was associated with LOH (P=0.017) and absence of germline mutations (P=0.037). The high frequency of LOH at the BRCA1 locus suggests that LOH may be an important mechanism of BRCA1 deficiency in ovarian carcinomas. Tumors with various BRCA1 alterations have a similar phenotype of high-grade, high-stage carcinomas with frequent TP53 mutations.

Nuclear survivin expression is a positive prognostic factor in taxane-platinum-treated ovarian cancer patients.

BACKGROUND: Survivin is an inhibitor of apoptosis and a regulator of mitotic progression. TP53 protein is a negative transcriptional regulator of survivin. The aim of our study was to evaluate the clinical significance of survivin expression in advanced stages ovarian cancer with respect to the TP53 status. METHODS: Survivin and TP53 expression was evaluated immunohistochemically in 435 archival samples of ovarian carcinomas (244 patients were treated with platinum/cyclophosphamide-PC/PAC; 191-with taxane-platinum (TP) agents). Univariate and multivariate statistical analyses were performed in patients groups divided according to the administered chemotherapeutic regimen, and in subgroups with and without TP53 accumulation (TP53+ and TP53-, respectively). RESULTS: Nuclear and cytoplasmic survivin expression was observed in 92% and 74% of the carcinomas, respectively. In patients treated with TP, high nuclear survivin expression decreased the risk of disease recurrence and death, and increased the probability of high platinum sensitivity (p < 0.01), but only in the TP53(+) group, and not in the TP53(-) group. CONCLUSIONS: It appears that TP53 status determines the clinical importance of nuclear survivin expression in taxane-platinum treated ovarian cancer patients.