Jolanta Kupryjanczyk

A novel germline PALB2 deletion in Polish breast and ovarian cancer patients

BackgroundPALB2 protein was recently identified as a partner of BRCA1 and BRCA2 which determines their proper function in DNA repair.MethodsInitially, the entire coding sequence of the PALB2 gene with exon/intron boundaries was evaluated by the PCR-SSCP and direct sequencing methods on 70 ovarian carcinomas. Sequence variants of interest were further studied on enlarged groups of ovarian carcinomas (total 339 non-consecutive ovarian carcinomas), blood samples from 334 consecutive sporadic and 648 consecutive familial breast cancer patients, and 1310 healthy controls from central Poland.ResultsTen types of sequence variants were detected, and among them four novel polymorphisms: c.2996+58T>C in intron 9; c.505C>A (p.L169I), c.618T>G (p.L206L), both in exon 4; and c.2135C>T (A712V) in exon 5 of the PALB2 gene. Another two polymorphisms, c.212-58A>C and c.2014G>C (E672Q) were always detected together, both in cancer (7.5% of patients) and control samples (4.9% of controls, p = 0.2). A novel germline truncating mutation, c.509_510delGA (p.R170fs) was found in exon 4: in 2 of 339 (0.6%) unrelated ovarian cancer patients, in 4 of 648 (0.6%) unrelated familial breast cancer patients, and in 1 of 1310 controls (0.08%, p = 0.1, p = 0.044, respectively). One ovarian cancer patient with the PALB2 mutation had also a germline nonsense mutation of the BRCA2 gene.ConclusionsThe c.509_510delGA is a novel PALB2 mutation that increases the risk of familial breast cancer. Occurrence of the same PALB2 alteration in seven unrelated women suggests that c.509_510delGA (p.R170fs) is a recurrent mutation for Polish population.

PIK3CA amplification associates with resistance to chemotherapy in ovarian cancer patients.

PI3K/AKT signalling pathway controls important cellular processes such as the cell proliferation and apoptosis. PIK3CA gene encoding a catalytic subunit of the PI3K is mutated and/or amplified in various neoplasms, including ovarian cancer. We aimed to evaluate PIK3CA alterations and their clinical importance in ovarian cancer patients. Molecular analysis was performed on 117 ovarian carcinomas with the use of qPCR, SSCP and sequencing. In a group of 98 patients with complete clinical data, 62 patients were treated with standard taxane-platinum regimens and 36 patients with platinum-cyclophosphamide regimens. A multivariate analysis was performed by the Cox’s and logistic regression models. PIK3CA mutations occurred in 5/117 (4.3%) carcinomas, exclusively in the endometrioid and clear cell types (P=0.0002); they were also associated with low FIGO stage (P=0.0003), low tumor grade (P=0.045) and early patient’s age at diagnosis (P=0.0005). The PIK3CA amplification (predominantly a low-level) was found in 28/117 (24%) ovarian carcinomas. It was more frequent in TP53 mutant tumors (P=0.012) and tended to associate with high pAKT expression (P=0.061). The PIK3CA amplification strongly diminished odds of complete remission (OR=0.25, P=0.033) and platinum sensitive response (PS, OR=0.12, P=0.004) in the taxane-platinum treated patients. The odds of PS were also much lower in all patients with the PIK3CA amplification evaluated together, regardless of the treatment applied (OR=0.18, P=0.001). Our results suggest that PIK3CA amplification may be a marker predicting ovarian cancer response to chemotherapy.

HAX-1 overexpression, splicing and cellular localization in tumors

BackgroundHAX-1 has been described as a protein potentially involved in carcinogenesis and especially metastasis. Its involvement in regulation of apoptosis and cell migration along with some data indicating its overexpression in cancer cell lines and tumors suggests that HAX-1 may play a role in neoplastic transformation. Here we present the first systematic analysis of HAX-1 expression in several solid tumors.MethodsUsing quantitative RT-PCR, we have determined the mRNA levels of HAX1 splice variant I in several solid tumors. We have also analyzed by semiquantitative and quantitative RT-PCR the expression of five HAX-1 splice variants in breast cancer samples and in normal tissue from the same individuals. Quantitative PCR was also employed to analyze the effect of estrogen on HAX1 expression in breast cancer cell line. Immunohistochemical analysis of HAX-1 was performed on normal and breast cancer samples.ResultsThe results reveal statistically important HAX1 up-regulation in breast cancer, lung cancer and melanoma, along with some minor variations in the splicing pattern. HAX-1 up-regulation in breast cancer samples was confirmed by immunohistochemical analysis, which also revealed an intriguing HAX-1 localization in the nuclei of the tumor cells, associated with strong ER status.ConclusionHAX-1 elevated levels in cancer tissues point to its involvement in neoplastic transformation, especially in breast cancer. The connection between HAX-1 nuclear location and ER status in breast cancer samples remains to be clarified.

Androgen, progesterone, and FSH receptor polymorphisms in ovarian cancer risk and outcome

Genes encoding hormone receptors are among candidate genes modulating the risk of ovarian cancer. We aimed to assess a frequency of PGRG+331A, FSHRAla307Thr, and FSHRSer680Asn polymorphic variants, and the length of (CAG)n and (GGN)n repeat tracts in the androgen receptor gene (AR) with respect to ovarian cancer risk and outcome. We genotyped 215 ovarian cancer patients and 352 unaffected control subjects. Statistical analysis was performed with the logistic regression model with adjustment for age. Clinical importance of the polymorphic variants was evaluated in multivariate models on 69 patients treated with taxane-platinum chemotherapy, with respect to TP53 status. Longer AR (GGN)n and (CAG)n repeat tracts decreased the risk of ovarian cancer. For (GGN)n, each additional repeat decreased the risk by 17% (P=0.011) or 27% (P=0.002), while the presence of at least 23 repeats decreased the risk by 41% (P=0.032) or 68% (P=0.008), for the shorter or longer allele respectively. The risk of disease was also decreased by 11% with each additional (CAG)n repeat (P=0.006 for the longer allele). FSHRAla307Ala or FSHRSer680Ser polymorphisms increased ovarian cancer risk by 1.8 times (P=0.042). In all 69 patients, longer AR (CAG)n repeats decreased the risk of recurrence (P=0.031). In the group with TP53 accumulation, longer AR (CAG)n repeats decreased the risk of recurrence (P=0.003) and death (P=0.03), while the FSHRSer680Ser polymorphism increased the risk of recurrence (P=0.037). Progesterone receptor polymorphisms analyzed did not show any associations. Our results support both the androgen and gonadotropin hypotheses of ovarian cancer development.

NANOS1 and PUMILIO2 bind microRNA biogenesis factor GEMIN3, within chromatoid body in human germ cells

Nanos and pumilio bind each other to regulate translation of specific mRNAs in germ cells of model organisms, such as D. melanogaster or C. elegans. Recently described human homologues NANOS1 and PUMILIO2 form a complex similar to their ancestors. This study was aimed to identify the proteins interacting with NANOS1-PUMILIO2 complex in the human spermatogenic cells. Here, using the yeast two-hybrid system we found that NANOS1 and PUMILIO2 proteins interact with RNA DEAD-box helicase GEMIN3, a microRNA biogenesis factor. Moreover, GEMIN3 coimmunoprecipitates with NANOS1 and PUMILIO2 in transfected mammalian cells. By double immunofluorescence staining, we observed that complexes built of NANOS1, PUMILIO2 and GEMIN3 are located within cytoplasmic region of germ cells. These proteins condense to form a compact aggregate in the round spermatids of the human and mouse germ cells. This aggregate was reminiscent of the chromatoid body (CB), a perinuclear structure present in the mammalian male germ line. This structure is considered evolutionary remnant of germ plasm, a hallmark structure of germ cells in lower metazoan. Using a CB marker VASA protein, we demonstrated that CBs are present in the human round spermatids, as they are in the mouse. Moreover, NANOS1, PUMILIO2 and GEMIN3 colocalize with VASA protein. We demonstrated for the first time that a mammalian Nanos-Pumilio complex functions within CB, a center of RNA storing and processing, involving microRNAs. NANOS1-PUMILIO2 complex, together with GEMIN3 and small noncoding RNAs, possibly regulate mRNA translation within CB of the human germ cells.

TP53 status and taxane-platinum versus platinum-based therapy in ovarian cancer patients: A non-randomized retrospective study

BackgroundTaxane-platinum therapy (TP) has replaced platinum-based therapy (PC or PAC, DNA damaging chemotherapy) in the postoperative treatment of ovarian cancer patients; however, it is not always effective. TP53 protein plays a differential role in response to DNA-damaging agents and taxanes. We sought to define profiles of patients who benefit the most from TP and also of those who can be treated with PC.MethodsWe compared the effectiveness of PC/PAC (n = 253) and TP (n = 199) with respect to tumor TP53 accumulation in ovarian cancer patients with FIGO stage IIB-IV disease; this was a non-randomized retrospective study. Immunohistochemical analysis was performed on 452 archival tumors; univariate and multivariate analysis by the Coxs and logistic regression models was performed in all patients and in subgroups with [TP53(+)] and without TP53 accumulation [TP53(-)].ResultsThe advantage of taxane-platinum therapy over platinum-based therapy was seen in the TP53(+), and not in the TP53(-) group. In the TP53(+) group taxane-platinum therapy enhanced the probability of complete remission (p = .018), platinum sensitivity (p = .014), platinum highly sensitive response (p = .038) and longer survival (OS, p = .008). Poor tumor differentiation diminished the advantage from taxane-platinum therapy in the TP53(+) group. In the TP53(-) group PC/PAC was at least equally efficient as taxane-platinum therapy and it enhanced the chance of platinum highly sensitive response (p = .010). However, in the TP53(-) group taxane-platinum therapy possibly diminished the risk of death in patients over 53 yrs (p = .077). Among factors that positively interacted with taxane-platinum therapy in some analyses were endometrioid and clear cell type, FIGO III stage, bulky residual tumor, more advanced age of patient and moderate tumor differentiation.ConclusionOur results suggest that taxane-platinum therapy is particularly justified in patients with TP53(+) tumors or older than 53 years. In the group of patients ≤53 yrs and with TP53(-) tumors platinum-based therapy is possibly equally efficient. We provide hints for planning randomized trials to verify these observations.

Gene Expression Analysis in Ovarian Cancer – Faults and Hints from DNA Microarray Study

The introduction of microarray techniques to cancer research brought great expectations for finding biomarkers that would improve patients’ treatment; however, the results of such studies are poorly reproducible and critical analyses of these methods are rare. In this study, we examined global gene expression in 97 ovarian cancer samples. Also, validation of results by quantitative RT-PCR was performed on 30 additional ovarian cancer samples. We carried out a number of systematic analyses in relation to several defined clinicopathological features. The main goal of our study was to delineate the molecular background of ovarian cancer chemoresistance and find biomarkers suitable for prediction of patients’ prognosis. We found that histological tumor type was the major source of variability in genes expression, except for serous and undifferentiated tumors that showed nearly identical profiles. Analysis of clinical endpoints [tumor response to chemotherapy, overall survival, disease-free survival (DFS)] brought results that were not confirmed by validation either on the same group or on the independent group of patients. CLASP1 was the only gene that was found to be important for DFS in the independent group, whereas in the preceding experiments it showed associations with other clinical endpoints and with BRCA1 gene mutation; thus, it may be worthy of further testing. Our results confirm that histological tumor type may be a strong confounding factor and we conclude that gene expression studies of ovarian carcinomas should be performed on histologically homogeneous groups. Among the reasons of poor reproducibility of statistical results may be the fact that despite relatively large patients’ group, in some analyses one has to compare small and unequal classes of samples. In addition, arbitrarily performed division of samples into classes compared may not always reflect their true biological diversity. And finally, we think that clinical endpoints of the tumor probably depend on subtle changes in many and, possibly, alternative molecular pathways, and such changes may be difficult to demonstrate.

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

BackgroundSurvivin 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.MethodsSurvivin 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).ResultsNuclear 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.ConclusionsIt appears that TP53 status determines the clinical importance of nuclear survivin expression in taxane-platinum treated ovarian cancer patients.

TP53, BCL-2 and BAX analysis in 199 ovarian cancer patients treated with taxane-platinum regimens.

OBJECTIVE In cell line studies, BCL-2 and BAX proteins interfere with cancer response to taxanes. This issue has not received much attention with regard to taxane-platinum (TP)-treated ovarian cancer patients. METHODS We evaluated prognostic/predictive significance of BCL-2 and BAX with regard to TP53 status. Immunohistochemical analysis was performed on 199 ovarian carcinomas FIGO stage IIB-IV treated with TP; the results were analyzed by the Cox and logistic regression models. RESULTS Clinicopathological parameters (residual tumor size, FIGO stage and/or tumor grade, but not patients age) were the only or the strongest predictors of patients outcome. Platinum highly sensitive response showed a positive association with TP53 accumulation (p=0.045). As in our previously published analysis on platinum-cyclophosphamide-treated group, complete remission showed a borderline negative (paradoxic) association with high BAX expression in the whole group (p=0.058) and with BCL-2 expression in the TP53(-) group (p=0.058). CONCLUSION Our results suggest that TP53, BCL-2 and BAX proteins carry some predictive potential in taxane-platinum-treated ovarian cancer patients, auxiliary to clinicopathological factors. We have confirmed on another patient group that clinical importance of BCL-2 may depend on TP53 status.

Analysis of Over 10,000 Cases Finds No Association between Previously Reported Candidate Polymorphisms and Ovarian Cancer Outcome

Background: Ovarian cancer is a leading cause of cancer-related death among women. In an effort to understand contributors to disease outcome, we evaluated single-nucleotide polymorphisms (SNP) previously associated with ovarian cancer recurrence or survival, specifically in angiogenesis, inflammation, mitosis, and drug disposition genes. Methods: Twenty-seven SNPs in VHL, HGF, IL18, PRKACB, ABCB1, CYP2C8, ERCC2, and ERCC1 previously associated with ovarian cancer outcome were genotyped in 10,084 invasive cases from 28 studies from the Ovarian Cancer Association Consortium with over 37,000-observed person-years and 4,478 deaths. Cox proportional hazards models were used to examine the association between candidate SNPs and ovarian cancer recurrence or survival with and without adjustment for key covariates. Results: We observed no association between genotype and ovarian cancer recurrence or survival for any of the SNPs examined. Conclusions: These results refute prior associations between these SNPs and ovarian cancer outcome and underscore the importance of maximally powered genetic association studies. Impact: These variants should not be used in prognostic models. Alternate approaches to uncovering inherited prognostic factors, if they exist, are needed. Cancer Epidemiol Biomarkers Prev; 22(5); 987–. ©2013 AACR.