Ranjan Chrisanthar

Predictive and prognostic impact of TP53 mutations and MDM2 promoter genotype in primary breast cancer patients treated with epirubicin or paclitaxel.

Background TP53 mutations have been associated with resistance to anthracyclines but not to taxanes in breast cancer patients. The MDM2 promoter single nucleotide polymorphism (SNP) T309G increases MDM2 activity and may reduce wild-type p53 protein activity. Here, we explored the predictive and prognostic value of TP53 and CHEK2 mutation status together with MDM2 SNP309 genotype in stage III breast cancer patients receiving paclitaxel or epirubicin monotherapy. Experimental Design Each patient was randomly assigned to treatment with epirubicin 90 mg/m2 (n = 109) or paclitaxel 200 mg/m2 (n = 114) every 3rd week as monotherapy for 4–6 cycles. Patients obtaining a suboptimal response on first-line treatment requiring further chemotherapy received the opposite regimen. Time from last patient inclusion to follow-up censoring was 69 months. Each patient had snap-frozen tumor tissue specimens collected prior to commencing chemotherapy. Principal Findings While TP53 and CHEK2 mutations predicted resistance to epirubicin, MDM2 status did not. Neither TP53/CHEK2 mutations nor MDM2 status was associated with paclitaxel response. Remarkably, TP53 mutations (p = 0.007) but also MDM2 309TG/GG genotype status (p = 0.012) were associated with a poor disease-specific survival among patients having paclitaxel but not patients having epirubicin first-line. The effect of MDM2 status was observed among individuals harbouring wild-type TP53 (p = 0.039) but not among individuals with TP53 mutated tumors (p>0.5). Conclusion TP53 and CHEK2 mutations were associated with lack of response to epirubicin monotherapy. In contrast, TP53 mutations and MDM2 309G allele status conferred poor disease-specific survival among patients treated with primary paclitaxel but not epirubicin monotherapy.

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.

Mutations and polymorphisms of the p21B transcript in breast cancer

p21WAF1/CIP1, transcribed from the CDKN1A locus, plays a key role executing p53‐induced growth arrest. The recent discovery that an alternative transcript, p21B, induces apoptosis, suggests an additional important function of this gene. Here, we report p21 and p21B mutation status in large cohorts of breast cancers and compare distributions of p21B polymorphisms in cancer patients to healthy controls. In 521 breast tumor samples analyzed, only one point mutation affecting the p21B protein was observed. No mutations were found when screening a panel of 20 established cell lines. A novel polymorphism, p21BG128T was identified. Haplotype analysis revealed no association between this variant and the previously identified p21B polymorphism p21BT35C or any of the known p21WAF1/CIP1 polymorphisms. As previously reported for p21BT35C, distribution of p21BG128T was similar among breast cancer patients and healthy controls (n = 691 and 1,015; incidence 6.1 vs. 4.8%; p = 0.273, respectively). No association between p21BG128T or p21BT35C and response to chemotherapy with an anthracycline‐containing regimen or paclitaxel was recorded. Our findings do not suggest mutations or polymorphisms of p21B to play a major role with respect to either breast cancer risk or sensitivity towards chemotherapy.

The Novel p21 Polymorphism p21G251A Is Associated with Locally Advanced Breast Cancer

Purpose: p21 is a main effector of growth arrest induced by p53. In addition, a second transcript from the same gene (p21B) has been linked to apoptosis. We previously analyzed p21 status in breast cancer and reported two novel polymorphisms of the p21 gene. In the present study, we present a larger study designed to explore a possible association between these novel polymorphisms and breast cancer. Experimental Design: The p21/p21B polymorphisms were analyzed in 507 breast cancer patients and 1,017 healthy individuals using cDNA or genomic DNA from tumor and/or blood samples. Results: We detected five polymorphisms of the p21 gene. Three of these polymorphisms are earlier reported by others, whereas two were reported for the first time in a recent study by us. The presence of the A allele of the p21G251A polymorphism was observed more frequently among patients with primary stage III breast cancer (4.5%) compared with stage I and II tumors (1.5%) and healthy female controls (1.4%; P = 0.007, comparing the three groups; P = 0.0049 and P = 0.0057, comparing locally advanced to stage I/II and healthy controls, or to healthy controls alone, respectively). The allele frequencies of the remaining four polymorphisms were evenly distributed among patients and healthy individuals. Discussion: The finding of an association between locally advanced breast cancer and one particular polymorphism of the p21 gene suggests this polymorphism to be related to tumor behavior, including enhanced growth rate. If confirmed in other studies, this may add significant information to our understanding of the biology as well as of the clinical behaviour of locally advanced breast cancers.

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

P21/WAF1 mutation and drug resistance to paclitaxel in locally advanced breast cancer.

We read with great interest the recent article by Galmarini and colleagues in the journal. The authors reported a point mutation in exon 2 of the p21 gene (WAF1, CIP1, CDKN1A), leading to a premature STOP in codon 127 and paclitaxel resistance in noncancerous epithelial breast cells. The cyclin-dependent kinase inhibitor p21 is a main effector of growth arrest induced by p53. The role of p21 in breast cancer is still unclear, particularly with respect to drug resistance. Thus, if corroborated by clinical findings, identification of p21 as a key element for response to paclitaxel would be a major breakthrough to our understanding of drug resistance.

Adjuvant treatment: the contribution of expression microarrays

Although gene expression microarrays provide novel tools and hold great promise in cancer research, achievements thus far in terms of improved prognostication and, in particular, prediction of drug sensitivity have been moderate. To improve clinical therapy, we believe that it is imperative to integrate gene expression arrays with other laboratory methods based on functional concepts [1,2].

Effect of WBC BRCA1 promoter methylation on ovarian cancer risk.

5029 Background: Recently, some studies have reported BRCA1 WBC hypermethylation to be associated with increased risk of breast cancer. We assessed WBC BRCA1 promoter methylations in ovarian (OC) and breast cancer (BC) patients and healthy controls. In an OC subgroup harboring WBC BRCA1 methylation, we confirmed promoter methylation status in normal tissue. METHODS WBC DNA from 899 OC patients, 425 BC patients and 719 healthy controls were analyzed for BRCA1 promoter methylation by methylation specific PCR. In addition, we analyzed WBC DNA from 256 OC and 393 BC patients in blood samples drawn prior to diagnosis in a population-based study. We also analyzed 24 BC patients with a family history (BRCAPRO scores > 80%; Manchester score >40) without BRCA1/2 mutations. Paraffin-embedded normal tissue from 5 OC patients harboring WBC BRCA1 methylation was analyzed for BRCA1 methylation status. Finally, to determine potential risk factors in cis, we investigated BRCA1 haplotype status in a sub-cohort of 10 individuals with WBC methylated BRCA1and 13 controls. RESULTS We detected WBC BRCA1 promoter hypermethylation in 2.4% of healthy controls and 3.1% of BC patients (all BC individuals). No difference in BRCA1 methylation incidence was recorded between BC patients with blood samples drawn before (4.1%) or after (2.1%) diagnosis. In contrast, we detected WBC BRCA1 methylation in 10.3% of OC patients having blood samples drawn at diagnosis. Among OC patients with blood sampling 0-13 years (median 4.6 y) prior to diagnosis, BRCA1 promoter methylation was detected in 6.6%. BRCA1 promoter methylation was associated with a non-significant elevated risk of BC (HR 1.302; 95% CI 0.697-2.431) but a significantly increased risk of OC in the cohort of patients with blood samples drawn at time of (OR 4.765; CI 2.814-8.069) or prior to (OR 2.937; CI 1.476-5.845) diagnosis. We confirmed BRCA1 promoter methylation in normal tissue from all 5 individuals analyzed, excluding WBC promoter methylation being due to circulating DNA contamination. No association between BRCA1methylation and promoter haplotype was found. CONCLUSIONS WBC BRCA1 promoter methylation is associated with increased risk of ovarian cancer.This finding has clinical as well as biological implications..