Elisabet Ognedal Berge

CHEK2 mutations affecting kinase activity together with mutations in TP53 indicate a functional pathway associated with resistance to epirubicin in primary breast cancer

Background Chemoresistance is the main obstacle to cure in most malignant diseases. Anthracyclines are among the main drugs used for breast cancer therapy and in many other malignant conditions. Single parameter analysis or global gene expression profiles have failed to identify mechanisms causing in vivo resistance to anthracyclines. While we previously found TP53 mutations in the L2/L3 domains to be associated with drug resistance, some tumors harboring wild-type TP53 were also therapy resistant. The aim of this study was; 1) To explore alterations in the TP53 gene with respect to resistance to a regular dose epirubicin regimen (90 mg/m2 every 3 week) in patients with primary, locally advanced breast cancer; 2) Identify critical mechanisms activating p53 in response to DNA damage in breast cancer; 3) Evaluate in vitro function of Chk2 and p14 proteins corresponding to identified mutations in the CHEK2 and p14(ARF) genes; and 4) Explore potential CHEK2 or p14(ARF) germline mutations with respect to family cancer incidence. Methods and Findings Snap-frozen biopsies from 109 patients collected prior to epirubicin (as preoperative therapy were investigated for TP53, CHEK2 and p14(ARF) mutations by sequencing the coding region and p14(ARF) promoter methylations. TP53 mutastions were associated with chemoresistance, defined as progressive disease on therapy (p = 0.0358; p = 0.0136 for mutations affecting p53 loop domains L2/L3). Germline CHEK2 mutations (n = 3) were associated with therapy resistance (p = 0.0226). Combined, mutations affecting either CHEK2 or TP53 strongly predicted therapy resistance (p = 0.0101; TP53 mutations restricted to the L2/L3 domains: p = 0.0032). Two patients progressing on therapy harbored the CHEK2 mutation, Arg95Ter, completely abrogating Chk2 protein dimerization and kinase activity. One patient (Epi132) revealed family cancer occurrence resembling families harboring CHEK2 mutations in general, the other patient (epi203) was non-conclusive. No mutation or promoter hypermethylation in p14(ARF) were detected. Conclusion This study is the first reporting an association between CHEK2 mutations and therapy resistance in human cancers and to document mutations in two genes acting direct up/down-stream to each other to cause therapy failure, emphasizing the need to investigate functional cascades in future studies.

Identification and characterization of retinoblastoma gene mutations disturbing apoptosis in human breast cancers

BackgroundThe tumor suppressor pRb plays a key role regulating cell cycle arrest, and disturbances in the RB1 gene have been reported in different cancer forms. However, the literature reports contradictory findings with respect to a pro - versus anti - apoptotic role of pRb, and the consequence of alterations in RB1 to chemotherapy sensitivity remains unclear. This study is part of a project investigating alterations in pivotal genes as predictive factors to chemotherapy sensitivity in breast cancer.ResultsAnalyzing 73 locally advanced (stage III) breast cancers, we identified two somatic and one germline single nucleotide changes, each leading to amino acid substitution in the pRb protein (Leu607Ile, Arg698Trp, and Arg621Cys, respectively). This is the first study reporting point mutations affecting RB1 in breast cancer tissue. In addition, MLPA analysis revealed two large multiexon deletions (exons 13 to 27 and exons 21 to 23) with the exons 21-23 deletion occurring in the tumor also harboring the Leu607Ile mutation. Interestingly, Leu607Ile and Arg621Cys point mutations both localize to the spacer region of the pRb protein, a region previously shown to harbor somatic and germline mutations. Multiple sequence alignment across species indicates the spacer to be evolutionary conserved. All three RB1 point mutations encoded nuclear proteins with impaired ability to induce apoptosis compared to wild-type pRb in vitro. Notably, three out of four tumors harboring RB1 mutations displayed primary resistance to treatment with either 5-FU/mitomycin or doxorubicin while only 14 out of 64 tumors without mutations were resistant (p = 0.046).ConclusionsAlthough rare, our findings suggest RB1 mutations to be of pathological importance potentially affecting sensitivity to mitomycin/anthracycline treatment in breast cancer.

Chk2 splice variants express a dominant-negative effect on the wild-type Chk2 kinase activity.

While the majority of RNA transcripts from protein-encoding genes in the human genome are subject to physiological splicing, pathological splicing is increasingly reported in cancer tissue. Previously, we identified >90 different splice variants of Chk2, a gene encoding a serine/threonine kinase propagating the DNA damage signal by phosphorylating and activating several downstream substrates like p53, Cdc25A, and Cdc25C involved in cell cycle arrest and apoptosis. While alternative splice forms of other genes have been reported to exert a dominant-negative effect on the wild-type molecules, the function of Chk2 splice protein variants is still unclear. Here we evaluated the function of four Chk2 splice proteins for which mRNA splice variants were identified in human breast carcinomas. These splice variants were stably expressed as nuclear proteins. Two splice forms (Chk2Delta4 and Chk2del(2-3)) expressed kinase activity while variants Chk2Delta11 and Chk2isoI were essentially kinase inactive. Independent of intrinsic kinase activity, each splice variant impaired wild-type Chk2 activity through heterodimerization. Based on our findings, we suggest alternative splicing as a possible novel mechanism for repression of the Chk2 wild-type function.

Concomitant inactivation of the p53- and pRB- functional pathways predicts resistance to DNA damaging drugs in breast cancer in vivo

Chemoresistance is the main obstacle to cancer cure. Contrasting studies focusing on single gene mutations, we hypothesize chemoresistance to be due to inactivation of key pathways affecting cellular mechanisms such as apoptosis, senescence, or DNA repair. In support of this hypothesis, we have previously shown inactivation of either TP53 or its key activators CHK2 and ATM to predict resistance to DNA damaging drugs in breast cancer better than TP53 mutations alone. Further, we hypothesized that redundant pathway(s) may compensate for loss of p53‐pathway signaling and that these are inactivated as well in resistant tumour cells. Here, we assessed genetic alterations of the retinoblastoma gene (RB1) and its key regulators: Cyclin D and E as well as their inhibitors p16 and p27. In an exploratory cohort of 69 patients selected from two prospective studies treated with either doxorubicin monotherapy or 5‐FU and mitomycin for locally advanced breast cancers, we found defects in the pRB‐pathway to be associated with therapy resistance (p‐values ranging from 0.001 to 0.094, depending on the cut‐off value applied to p27 expression levels). Although statistically weaker, we observed confirmatory associations in a validation cohort from another prospective study (n = 107 patients treated with neoadjuvant epirubicin monotherapy; p‐values ranging from 7.0 × 10−4 to 0.001 in the combined data sets). Importantly, inactivation of the p53‐and the pRB‐pathways in concert predicted resistance to therapy more strongly than each of the two pathways assessed individually (exploratory cohort: p‐values ranging from 3.9 × 10−6 to 7.5 × 10−3 depending on cut‐off values applied to ATM and p27 mRNA expression levels). Again, similar findings were confirmed in the validation cohort, with p‐values ranging from 6.0 × 10−7 to 6.5 × 10−5 in the combined data sets. Our findings strongly indicate that concomitant inactivation of the p53‐ and pRB‐ pathways predict resistance towards anthracyclines and mitomycin in breast cancer in vivo.

White Blood Cell BRCA1 Promoter Methylation Status and Ovarian Cancer Risk

Women carrying germline BRCA1 mutations are at high risk for epithelial ovarian cancer, especially high-grade serous ovarian cancer (HGSOC) (1, 2). Thus, 4% to 10% of all women with ovarian cancer may carry BRCA1 germline mutations (3, 4). Recently, germline mutations in other genes, including PALB2, BRIP1, RAD51C, and RAD51D, all acting in the same DNA repair pathway as BRCA1 and BRCA2, have been associated with familial risk for ovarian and breast cancer (58). These findings are consistent with the hypothesis that disturbances in DNA repair by homologous recombination are important for cancer development in these organs. Promoter methylation represents an alternative mechanism of gene inactivation, and promoter methylation of the MLH1 mismatch repair gene, as well as BRCA1 methylation, has been observed in normal tissues in some families with a high risk for colorectal or breast cancer who do not have germline mutations in these genes (912). For breast and ovarian cancer risk, associations with BRCA1 methylation in white blood cells (WBCs) have not been consistent (10, 1316), and most studies have been small, with limited statistical power to detect any clear differences. We hypothesized that normal tissue BRCA1 methylation may be associated with an increased risk for ovarian cancer, with a particular propensity for HGSOC, analogous to observations for germline BRCA1 mutations. Here, we determined WBC BRCA1 promoter methylation status in a large casecontrol study and then attempted to replicate the findings in a similarly designed validation study. To determine whether normal tissue BRCA1 methylation may be established early in life, we also assessed WBC BRCA1 methylation in samples of newborn girls and healthy young women. Methods Study Design Overview We compared WBC BRCA1 promoter methylation status between patients with ovarian cancer and population control participants. The initial study was followed by a similarly designed casecontrol study to validate our results. In addition, we performed extensive sensitivity analyses to test the robustness of our findings. Initial Study White blood cell DNA was available from 934 patients with epithelial ovarian cancer treated at Oslo University Hospital, Norwegian Radium Hospital, between 1993 and 2011 (Figure 1, A). All samples collected in the biobank during that period were included; however, borderline ovarian tumors were excluded. Also, all patients had been tested for pathogenic BRCA1 or BRCA2 germline mutations, and patients carrying such mutation were excluded. Samples were collected before any systemic chemotherapy, with 583 samples collected from patients after surgery and 351 collected from patients before surgery or from those who did not have surgery. As a control, we used random samples of women from CONOR (Cohort of Norway), a large collection of population studies in Norway with similar questionnaire data, clinical measurements, and blood samples (17). The CONOR participants were recruited between 1994 and 2003 (for details, see Supplement Table 1). Thus, in the initial casecontrol study, 1698 women without cancer were frequency matched by 5-year age categories (at blood sampling) to the 934 patients with ovarian cancer. Among participants with successful BRCA1 methylation analysis, the patients (age range, 15 to 90 years; median, 62 years) were somewhat older than the control participants (range, 20 to 93 years; median, 57 years); therefore, we adjusted for age at blood sampling in the casecontrol comparisons. Figure 1. Flow chart of patients with ovarian cancer and healthy control participants included and successfully analyzed in the initial (A) and validation (B) studies. HGSOC = high-grade serous ovarian cancer; LGSOC = low-grade serous ovarian cancer; qPCR = quantitative polymerase chain reaction. Supplement. Supplementary Material and Methods To assess whether the percentage of methylated alleles would display a doserisk association with ovarian cancer, we analyzed methylation-positive samples by pyrosequencing. Validation Study We estimated sample size for the validation study on the basis of the strength of association found for the HGSOC group in our initial study (Supplement). Thus, we analyzed WBC DNA from 607 patients with ovarian cancer (274 from Oslo University Hospital and 333 from Haukeland University Hospital, Bergen), including 286 with HGSOC, and 1984 population control participants randomly selected from CONOR, with frequency matching in 5-year age increments. The Oslo patients with ovarian cancer had blood collected in 2011 to 2015, and the Bergen patients in 2001 to 2015 (Figure 1, B). Among the patients with ovarian cancer, 433 had blood collected before and 174 after surgery. All the Oslo patients had been tested for pathogenic BRCA1 and BRCA2 germline mutations, with negative results, whereas the Bergen patients had not been tested. As in the initial casecontrol study, samples were collected before chemotherapy began. The control group drawn from the CONOR study (17) did not overlap with that of the initial study. We adjusted for age at blood sampling by using a procedure similar to that of the initial study. Newborns and Healthy Young Persons To assess whether normal tissue BRCA1 methylation might be established early in life, we determined WBC methylation status in umbilical cord blood from a sample of newborn girls (n= 611) from MoBa (the Norwegian Mother and Child Cohort Study) (18). In addition, WBC methylation was determined in a group of healthy women aged 20 to 25 years (n= 292) selected from the CONOR study (17). These 2 groups were not part of the control groups used in the casecontrol comparisons (Supplement). Tumor and Normal Tissue BRCA1 Methylation We hypothesized that WBC BRCA1 methylation may be a surrogate marker of BRCA1 tissue methylation in general. If correct, one would expect that many women with ovarian cancer and positive BRCA1 methylation status would have BRCA1 methylation in their tumor tissue. To address these questions, we analyzed normal and ovarian cancer tissue samples from patients with and without positive WBC methylation status (Supplement). Methylation Status Among Persons Carrying BRCA1/2 Germline Mutations To explore a potential relationship between WBC BRCA1 methylation and BRCA1/2 germline mutation, we determined BRCA1 methylation status in 251 patients with ovarian cancer and germline BRCA1 mutations, 100 with BRCA2 mutations, and 15 with familial ovarian cancer who had negative results on BRCA1/2 mutation testing. Sensitivity Analysis Previous studies suggested that cancer burden influences WBC global DNA methylation patterns (19). To evaluate whether tumor load affects BRCA1 promoter methylation in blood, we compared BRCA1 methylation frequency between samples obtained before and after surgery; we also compared methylation status among FIGO (International Federation of Gynecology and Obstetrics) stages across the pooled groups of patients with ovarian cancer and those with HGSOC. We also assessed the potential effect of previous breast cancer treatment as well as of sample storage time. Further, we measured WBC BRCA1 methylation status in a separate cohort of 658 patients with ovarian cancer, from whom blood samples were collected at various time points after chemotherapy, when most patients had limited or no detectable residual disease. Previously, global DNA methylation assessments suggested that methylation of some CpG (cytosineguanine) nucleotide sequence sites across the genome might vary among different WBC fractions (20). Thus, we examined published data sets for potential variation among WBC fractions in adults (21) as well as newborns (18) to identify any variation with respect to methylation of the BRCA1 promoter CpGs of relevance to the present study (Supplement). Laboratory Analysis In brief, DNA was isolated from the samples and bisulfite converted (>99.5% validated conversion rate) before BRCA1 promoter methylation status was determined by quantitative polymerase chain reaction (qPCR). Two researchers (E.O.B. and L.M.), who were blinded to sample identity, independently scored each sample as methylation positive or negative. Further details, including assessment of individual CpG methylation across the promoter area, methylation quantification by pyrosequencing, and haplotype analysis, are given in the Supplement. Data Analysis We compared BRCA1 methylation status between patients with ovarian cancer and population control participants by using logistic regression models adjusted for age, and we reported odds ratios (ORs) from these models. In addition, we looked for heterogeneity by using a likelihood ratio test to compare results between the initial and validation studies. Because germline BRCA1 mutation carriers seem to have a propensity for HGSOC, we analyzed this subgroup separately. In a separate analysis pooling cases and controls from the 2 studies (initial and validation), we assessed the frequency of BRCA1 methylation within 10-year age groups by using logistic regression, stratified for the 2 studies. The patient and control samples from the initial study analyzed by pyrosequencing were dichotomized according to the median value of methylation, and ORs were calculated separately for each group (Supplement). We used chi-square tests to compare the proportions of persons with BRCA1 methylation between groups. The precision of the estimates of the proportion with BRCA1 methylation was presented with 95% CIs. Potential confounding by other unknown covariates was assessed by using the method described by VanderWeele (22) (Supplement). The statistical analyses were performed in Stata, version 12.1 (StataCorp), and SPSS, version 19 (IBM). The study was conducted according to the STREGA (Strengthening the Reporting of Genetic Association Studies) and GRIPS (Strengthening the Reporting of Genetic Risk Prediction Studies) statements (23, 24). Ethical Considerations

Abstract 3139: Identification and functional studies of p53 mutants detected in breast cancers after chemotherapy treatment.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Approximately 4300 different TP53 mutations have been reported in human cancers. TP53 mutations, in particular those affecting the L2/L3 domains, are associated with resistance to anthracycline or mitomycin treatment in breast cancer patients. While many mutations have been characterised functionally, novel TP53 mutations are continuously reported. Here, we characterise 10 p53 protein variants encoded by mutated TP53 (5 within and 5 outside L2/L3) detected in locally advanced or metastatic breast cancers. Each tumour was previously characterised for response to therapy, allowing comparison between in vivo and in vitro findings. Methods: Mutated p53 variants were analysed for their ability to oligomerise with the wild-type protein and their subcellular localisation by immunoprecipitation and immunofluorescence, respectively. Their ability to induce transcription of target genes was determined by qPCR. Cellular growth rate, apoptosis and senescence were monitored by WST-1, TUNEL and beta-galactosidase assays, respectively. Results: Immunoprecipitation assays revealed each mutant protein to retain binding capacity for wild-type p53, thus potentially acting in a dominant negative manner. Even though each p53 variant located predominantly in the nucleus, the percentage of cells with only nuclear p53 localisation varied between 60-90%. None of the p53 variants were able to induce target genes to levels similar to wild-type p53, nor where they able to reduce cellular growth rate, induce apoptosis or senescence similar to wild-type p53 after anthracycline treatment in vitro. Conclusions/General Significance: Our data add further information characterising the effects of somatic TP53 mutations on p53 protein function and anthracycline resistance in breast cancer. Citation Format: Johanna Huun, Elisabet O. Berge, Johan R. Lillehaug, Per Eystein Lonning, Stian Knappskog. Identification and functional studies of p53 mutants detected in breast cancers after chemotherapy treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3139. doi:10.1158/1538-7445.AM2013-3139

Abstract 1092: Alterations of the retinoblastoma gene in stage III breast cancers

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC We report the novel finding of point mutations affecting the RB1 gene in breast cancer tissue. Analyzing 73 locally advanced (stage III) breast cancers, we identified two somatic and one germline single nucleotide changes, each leading to amino acid substitution; Leu607Ile, Arg698Trp, and Arg621Cys, respectively. In addition, MLPA analysis revealed two large multiexon deletions (exons 13 to 27 and exons 21 to 23) with the exons 21-23 deletion occurring in the tumor also harboring Leu607Ile. All three RB1 point mutations encoded stable nuclear proteins with impaired ability to induce apoptosis compared to wild-type pRb in vitro. Interestingly, Leu607Ile and Arg621Cys are both located within the spacer region of the protein. Mutations in this part of the RB1 gene have previously been identified in retinoblastoma-prone families. Multiple sequence alignment across species indicates this area to be evolutionary conserved. Notably, three out of four tumors harboring RB1 mutations displayed primary resistance to treatment with either mitomycin or doxorubicin while only 14 out of 64 tumors without mutations were resistant (p = 0.046). Although rare, our findings suggest RB1 mutations to be of biological importance in breast cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1092.