Low expression levels of ATM may substitute for CHEK2 /TP53 mutations predicting resistance towards anthracycline and mitomycin chemotherapy in breast cancer.

INTRODUCTION: Mutations affecting p53 or its upstream activator Chk2 are associated with resistance to DNA-damaging chemotherapy in breast cancer. ATM (Ataxia Telangiectasia Mutated protein) is the key activator of p53 and Chk2 in response to genotoxic stress. Here, we sought to evaluate ATM's potential role in resistance to chemotherapy. METHODS: We sequenced ATM and assessed gene expression levels in pre-treatment biopsies from 71 locally advanced breast cancers treated in the neoadjuvant setting with doxorubicin monotherapy or mitomycin combined with 5-fluorouracil. Findings were confirmed in a separate patient cohort treated with epirubicin monotherapy. Each tumor was previously analyzed for CHEK2 and TP53 mutation status. RESULTS: While ATM mutations were not associated with chemo-resistance, low ATM expression levels predicted chemo-resistance among patients with tumors wild-type for TP53 and CHEK2 (P = 0.028). Analyzing the ATM-chk2-p53 cascade, low ATM levels (defined as the lower 5 to 50% percentiles) or mutations inactivating TP53 or CHEK2 robustly predicted anthracycline resistance (P-values varying between 0.001 and 0.027 depending on the percentile used to define "low" ATM levels). These results were confirmed in an independent cohort of 109 patients treated with epirubicin monotherapy. In contrast, ATM-levels were not suppressed in resistant tumors harboring TP53 or CHEK2 mutations (P > 0.5). CONCLUSIONS: Our data indicate loss of function of the ATM-Chk2-p53 cascade to be strongly associated with resistance to anthracycline/mitomycin-containing chemotherapy in breast cancer.

Persistence of disseminated tumor cells after neoadjuvant treatment for locally advanced breast cancer predicts poor survival.

INTRODUCTION: Presence of disseminated tumor cells (DTCs) in bone marrow (BM) and circulating tumor cells (CTC) in peripheral blood (PB) predicts reduced survival in early breast cancer. The aim of this study was to determine the presence of and alterations in DTC- and CTC-status in locally advanced breast cancer patients undergoing neoadjuvant chemotherapy (NACT) and to evaluate their prognostic impact. METHODS: Bone marrow and peripheral blood were collected before NACT (BM1: n = 231/PB1: n = 219), at surgery (BM2: n = 69/PB2: n = 71), and after 12 months from start of NACT (BM3: n = 162/PB3: n = 141). Patients were included from 1997 to 2003 and followed until 2009 (or ten years follow-up). DTC- and CTC-status were determined by morphological evaluation of immunocytochemically detected cytokeratin-positive cells. The prognostic significance of DTCs/CTCs was assessed by univariate and multivariate Cox-regression analyses. RESULTS: Before NACT, DTCs and CTCs were detected in 21.2% and 4.9% of the patients, respectively. At surgery, 15.9% and 1.4% had DTC- and CTC-presence, compared to 26.5% and 4.3% at 12 months from start of NACT. Of patients for whom DTC results both before NACT and at 12 months were available, concordant results were observed in 68%, and 14 out of 65 had positive DTC-status at both time points. Presence of ≥ 1 DTC 12 months from start of NACT, but not at other time points, predicted reduced disease-free survival (DFS; HR 2.3, p = 0.003), breast cancer-specific survival (BCSS; HR 3.0, p < 0.001) and overall survival (OS; HR 2.8, p < 0.001). Before NACT, presence of ≥ 3 DTCs was also associated with unfavorable outcome, and reduced BCSS was observed for CTC-positive patients (HR 2.2, p = 0.046). In multivariate analysis, DTC status (

Psychosocial interventions as part of breast cancer rehabilitation programs? Results from a systematic review.

OBJECTIVE: This systematic review aimed to determine the effectiveness of psychoeducation, cognitive behavioural therapy (CBT) and social support interventions used in the rehabilitation of breast cancer (BC) patients. METHODS: We conducted a systematic literature search to identify randomised controlled trials of female BC patients who underwent different psychosocial interventions during or after primary cancer treatment. The methodological quality of all studies was independently assessed by two reviewers. Studies with low quality, less than 20 participants in each group, patients with metastatic cancer, data not presented separately for BC and studies that included other cancer types were excluded. RESULTS: Among 9617 identified studies, only 18 RCTs published between 1999 and 2008, including 3272 patients were finally included in this systematic evaluation. Outcome measures were categorised into quality of life (QoL), fatigue, mood, health behaviour and social function. Six trials examined psychoeducation had inconsistent results, both during and after the primary treatment. Seven trials examined the effect of CBT, four of which given after primary treatment (range 6-12 weeks) demonstrated improvements in QoL; the other three CBT studies given during primary treatment (range 9-20 weeks) had inconsistencies. Five studies addressed social support and showed no conclusive impacts of this intervention. CONCLUSIONS: Limited documentation exists on the efficacy of psychosocial rehabilitation interventions among BC patients. However, we found that patients might have QoL benefits from CBT given after primary BC treatment. More documentation is needed regarding the effects of CBT during primary treatment and the effects of psychoeducation and social support.

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.

Gene expression profiles do not consistently predict the clinical treatment response in locally advanced breast cancer.

Neoadjuvant treatment offers an opportunity to correlate molecular variables to treatment response and to explore mechanisms of drug resistance in vivo. Here, we present a statistical analysis of large-scale gene expression patterns and their relationship to response following neoadjuvant chemotherapy in locally advanced breast cancers. We analyzed cDNA expression data from 81 tumors from two patient series, one treated with doxorubicin alone (51) and the other treated with 5-fluorouracil and mitomycin (30), and both were previously studied for correlations between TP53 status and response to therapy. We observed a low frequency of progressive disease within the luminal A subtype from both series (2 of 36 versus 13 of 45 patients; P = 0.0089) and a high frequency of progressive disease among patients with luminal B type tumors treated with doxorubicin (5 of 8 patients; P = 0.0078); however, aside from these two observations, no other consistent associations between response to chemotherapy and tumor subtype were observed. These specific associations could possibly be explained by covariance with TP53 mutation status, which also correlated with tumor subtype. Using supervised analysis, we could not uncover a gene profile that could reliably (>70% accuracy and specificity) predict response to either treatment regimen.

Influence of letrozole and anastrozole on total body aromatization and plasma estrogen levels in postmenopausal breast cancer patients evaluated in a randomized, cross-over study.

PURPOSE: To compare the effects of the two novel, potent, nonsteroidal aromatase inhibitors anastrozole and letrozole on total-body aromatization and plasma estrogen levels. PATIENTS AND METHODS: Twelve postmenopausal women with estrogen receptor-positive, metastatic breast cancer were treated with anastrozole 1 mg orally (PO) and letrozole 2.5 mg PO once daily, each given for a time interval of 6 weeks in a randomized sequence. Total-body aromatization was determined before treatment and at the end of each treatment period using a dual-label isotopic technique involving isolation of the metabolites with high-performance liquid chromatography. Plasma levels of estrone (E(1)), estradiol (E(2)), and estrone sulfate (E(1)S) were determined in samples obtained before each injection using highly sensitive radioimmunoassays. RESULTS: Pretreatment aromatase levels ranged from 1.68% to 4.27%. On-treatment levels of aromatase were detectable in 11 of 12 patients during treatment with anastrozole (mean percentage inhibition in the whole group, 97.3%) but in none of the 12 patients during treatment with letrozole (> 99.1% suppression in all patients; Wilcoxon, P =.0022, comparing the two drug regimens). Treatment with anastrozole suppressed plasma levels of E(1), E(2), and E(1)S by a mean of 81.0%, 84.9%, and 93.5%, respectively, whereas treatment with letrozole caused a corresponding decrease of 84.3%, 87.8% and 98.0%, respectively. The suppression of E(1) and E(1)S was found to be significantly better during treatment with letrozole compared with anastrozole (P =.019 and.0037, respectively). CONCLUSION: This study revealed letrozole (2.5 mg once daily) to be a more potent suppressor of total-body aromatization and plasma estrogen levels compared with anastrozole (1 mg once daily) in postmenopausal women with metastatic breast cancer.

Treatment with high-dose estrogen (diethylstilbestrol) significantly decreases plasma estrogen and androgen levels but does not influence in vivo aromatization in postmenopausal breast cancer patients.

While growth factors and hormones are known to influence aromatase expression in experimental systems, little is know about potential factors influencing peripheral aromatization in postmenopausal women. The fact that peripheral aromatase activity is higher in old compared to young women and the finding of relatively high tissue estradiol (E2) concentrations after the menopause suggests peripheral aromatization could be influenced by estrogen concentration. To test this hypothesis, we determined plasma hormone levels (n=9) and in vivo aromatization (n=3) in postmenopausal women suffering from advanced breast cancer before and during treatment (4 weeks) with diethylstilbestrol (DES) 5mg three times daily. Plasma levels of cortisol (C), corticosteroid-binding globulin (CBG), and sex hormone binding globulin (SHBG) were significantly increased in all patients (P<0.05 for all). While we found no change in total body aromatization and plasma estrone (E(1)) levels, estradiol (E(2)) and estrone sulfate (E(1)S) were suppressed by a mean of 48.8 and 68.2%, respectively (P=0.043 and 0.008). Surprisingly, plasma levels of androstenedione (A), testosterone (T), dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) were also suppressed by a mean in the range of 32.1 to 52.6% (P<0.05 for all androgens). In contrast, no change in plasma progesterone or 17alpha-hydroxyprogesterone was found. Thus, one possible explanation to our findings could be that DES administered in high doses reduces 17,20-lyase activity in the adrenal gland.

TP53 gene mutations predict the response to neoadjuvant treatment with 5-fluorouracil and mitomycin in locally advanced breast cancer.

PURPOSE: Recent studies have found an association between certain TP53 mutations and resistance to anthracycline-based primary medical therapy in breast cancer. The purpose of this study was to investigate whether TP53 mutational status also might influence the response to a non-anthracycline-containing regimen in primary breast cancer. EXPERIMENTAL DESIGN: Thirty-five patients with locally advanced breast cancer were investigated for TP53 mutations before receiving combination chemotherapy with 5-fluorouracil (1000 mg/m(2) on days 1 and 2) and mitomycin (6 mg/m(2) on day 2), administered every 3 weeks for 2-10 cycles in the neoadjuvant setting. RESULTS: Mutations in the TP53 gene, in particular those affecting loop domains L2 or L3 of the p53 protein, were associated with lack of response to chemotherapy (i.e., increase in the diameter product of tumor lesion by >/=25%; P = 0.177 for all mutations and P = 0.006 for those affecting L2/L3 domains, respectively). No statistically significant correlation between TP53 LOH and response to therapy was seen. CONCLUSION: This study revealed a significant association between lack of response to 5-fluorouracil and mitomycin and mutations affecting the L2/L3 domains of the p53 protein. Together with our previous finding that such mutations predict resistance to weekly doxorubicin, our data suggest that mutations affecting this particular domain of the p53 protein may cause resistance to several different cytotoxic compounds applied in breast cancer treatment.

Cardiac and pulmonary doses and complication probabilities in standard and conformal tangential irradiation in conservative management of breast cancer.

BACKGROUND AND PURPOSE: The clinical benefit of irradiating the intact breast after lumpectomy must be weighted against the risk of severe toxicity. We present a study on cardiac and pulmonary dose-volume data and the related complication probabilities of tangential breast irradiation having the following objectives: (1) to quantify the sparing of the organs at risk (ORs), the heart and the lung, achieved by three-dimensional (3-D) conformal tangential irradiation (CTI) as compared to standard tangential irradiation (STI); (2) to elucidate the uncertainty in radiation tolerance data; and (3) to analyse the relation between the amount of OR irradiated and the resulting morbidity risk. MATERIAL AND METHODS: Computed tomography (CT)-based 3-D treatment plans of 26 patients prescribed to CTI of the intact breast were applied. Contour-based STI has been our routine treatment, and was reconstructed for all patients. Dose-volume data and normal tissue complication probability (NTCP) predictions from the probit and relative seriality models with several cardiac and pulmonary tolerance parameterizations were analysed and compared. RESULTS AND CONCLUSIONS: A significant amount of normal tissues can be spared from radiation by using CT-based CTI, resulting in a 50% reduction of the average excess cardiac mortality risk in the left-sided cases. The risks for pericarditis and pneumonitis were too low to reveal any clinically significant difference between the treatments. For the STI set-up, a regression analysis showed that the excess cardiac mortality risk increased when larger parts of the heart were inside the fields. However, the different excess cardiac mortality and pneumonitis tolerance parameters resulted in statistically significant different NTCPs, which precluded the ability to accurately predict absolute NTCPs after tangential breast irradiation. Despite this uncertainty the different series of cardiac and pulmonary risk predictions were in relatively good agreement when small volumes of the ORs were irradiated. From the present data and without consideration of patient or organ motion, it therefore appears that tangential breast irradiation with less than 1 cm of the heart and 2-2.5 cm of the lung included inside the treatment fields will cause at most 1 per thousand risk for cardiac mortality and pulmonary morbidity. CT-based CTI should be considered, in particular for the left-sided cases, if these requirements cannot be met.

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/m(2) (n = 109) or paclitaxel 200 mg/m(2) (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/m(2) 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 mutations 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.

Monitoring of disseminated tumor cells in bone marrow in high-risk breast cancer patients treated with high-dose chemotherapy.

The present study aimed to investigate the clinical relevance of disseminated tumor cells (DTC) in breast cancer patients before and after high-dose adjuvant chemotherapy with or without progenitor stem-cell support. One hundred and eighteen high-risk stage II breast cancer patients entering the Scandinavian Study Group multicenter trial were randomized to 9 cycles of tailored and dose-escalated FEC (5-fluorouracil, epirubicin, cyclophosphamide) or 3 cycles of standard FEC followed by high-dose chemotherapy. Bone marrow (BM) samples at diagnosis and 6 months after completion of chemotherapy were assessed for the presence of cytokeratin positive (CK+) cells. Before treatment, 29% of the patients were CK+ (21% in the dose-escalated group and 36% in the high-dose-group). Six months after treatment, 17% of the patients were CK+ (17 and 16% respectively). Of the 95 patients who were evaluated 6 months after treatment, 60% were consistently CK-. CK+ cells in BM was evaluated as a prognostic and predictive marker and compared to other defined prognostic factors of the primary tumor. Monitoring BM changes at the time of diagnosis and 6 months posttreatment is an independent predictive factor for breast-cancer-specific survival (BCS) (p = 0.001). Those who have consistent CK negative (-) BM findings constitute a group of patients with good prognosis. Our results suggest that changes in CK+ cells in BM before and after chemotherapy can be used clinically as a surrogate maker to predict outcome in breast cancer patients.