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 wildtype 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

Olaparib monotherapy as primary treatment in unselected triple negative breast cancer

Background - The antitumor efficacy of PARP inhibitors (PARPi) for breast cancer patients harboring germline BRCA1/2 (gBRCA1/2) mutations is well established. While PARPi monotherapy was ineffective in patients with metastatic triple negative breast cancer (TNBC) wild type for BRCA1/2, we hypothesized that PARPi may be effective in primary TNBCs without previous chemotherapy exposure. Patients and methods - In the phase II PETREMAC trial, patients with primary TNBC >2 cm received olaparib for up to 10 weeks before chemotherapy. Tumor biopsies collected before and after olaparib underwent targeted DNA sequencing (360 genes) and BRCA1 methylation analyses. In addition, BRCAness (multiplex ligation-dependent probe amplification), PAM50 gene expression, RAD51 foci, tumor-infiltrating lymphocytes (TILs) and PD-L1 analyses were performed on pretreatment samples. Results - The median pretreatment tumor diameter was 60 mm (range 25-112 mm). Eighteen out of 32 patients obtained an objective response (OR) to olaparib (56.3%). Somatic or germline mutations affecting homologous recombination (HR) were observed in 10/18 responders [OR 55.6%, 95% confidence interval (CI) 33.7-75.4] contrasting 1/14 non-responders (OR 7.1%; CI 1.3-31.5, P = 0.008). Among tumors without HR mutations, 6/8 responders versus 3/13 non-responders revealed BRCA1 hypermethylation (P = 0.03). Thus, 16/18 responders (88.9%, CI 67.2-96.9), in contrast to 4/14 non-responders (28.6%, CI 11.7-54.7, P = 0.0008), carried HR mutations and/or BRCA1 methylation. Excluding one gPALB2 and four gBRCA1/2 mutation carriers, 12/14 responders (85.7%, CI 60.1-96.0) versus 3/13 non-responders (23.1%, CI 8.2-50.3, P = 0.002) carried somatic HR mutations and/or BRCA1 methylation. In contrast to BRCAness signature or basal-like subtype, low RAD51 scores, high TIL or high PD-L1 expression all correlated to olaparib response. Conclusion - Olaparib yielded a high clinical response rate in treatment-naïve TNBCs revealing HR deficiency, beyond germline HR mutations

Homologous Recombination Deficiency Across Subtypes of Primary Breast Cancer

Purpose - Homologous recombination deficiency (HRD) is highly prevalent in triple-negative breast cancer (TNBC) and associated with response to PARP inhibition (PARPi). Here, we studied the prevalence of HRD in non-TNBC to assess the potential for PARPi in a wider group of patients with breast cancer. Methods - HRD status was established using targeted gene panel sequencing (360 genes) and BRCA1 methylation analysis of pretreatment biopsies from 201 patients with primary breast cancer in the phase II PETREMAC trial (ClinicalTrials.gov identifier: NCT02624973). HRD was defined as mutations in BRCA1, BRCA2, BRIP1, BARD1, or PALB2 and/or promoter methylation of BRCA1 (strict definition; HRD-S). In secondary analyses, a wider definition (HRD-W) was used, examining mutations in 20 additional genes. Furthermore, tumor BRCAness (multiplex ligation-dependent probe amplification), PAM50 subtyping, RAD51 nuclear foci to test functional HRD, tumor-infiltrating lymphocyte (TIL), and PD-L1 analyses were performed. Results - HRD-S was present in 5% of non-TNBC cases (n = 9 of 169), contrasting 47% of the TNBC tumors (n = 15 of 32). HRD-W was observed in 23% of non-TNBC (n = 39 of 169) and 59% of TNBC cases (n = 19 of 32). Of 58 non-TNBC and 30 TNBC biopsies examined for RAD51 foci, 4 of 4 (100%) non-TNBC and 13 of 14 (93%) TNBC cases classified as HRD-S had RAD51 low scores. In contrast, 4 of 17 (24%) non-TNBC and 15 of 19 (79%) TNBC biopsies classified as HRD-W exhibited RAD51 low scores. Of nine non-TNBC tumors with HRD-S status, only one had a basal-like PAM50 signature. There was a high concordance between HRD-S and either BRCAness, high TIL density, or high PD-L1 expression (each P Conclusion - The prevalence of HRD in non-TNBC suggests that therapy targeting HRD should be evaluated in a wider breast cancer patient population. Strict HRD criteria should be implemented to increase diagnostic precision with respect to functional HRD

New clinical and biological insights from the international TARGIT-A randomised trial of targeted intraoperative radiotherapy during lumpectomy for breast cancer

BACKGROUND: The TARGIT-A trial reported risk-adapted targeted intraoperative radiotherapy (TARGIT-IORT) during lumpectomy for breast cancer to be as effective as whole-breast external beam radiotherapy (EBRT). Here, we present further detailed analyses. METHODS: In total, 2298 women (≥45 years, invasive ductal carcinoma ≤3.5 cm, cN0-N1) were randomised. We investigated the impact of tumour size, grade, ER, PgR, HER2 and lymph node status on local recurrence-free survival, and of local recurrence on distant relapse and mortality. We analysed the predictive factors for recommending supplemental EBRT after TARGIT-IORT as part of the risk-adapted approach, using regression modelling. Non-breast cancer mortality was compared between TARGIT-IORT plus EBRT vs. EBRT. RESULTS: Local recurrence-free survival was no different between TARGIT-IORT and EBRT, in every tumour subgroup. Unlike in the EBRT arm, local recurrence in the TARGIT-IORT arm was not a predictor of a higher risk of distant relapse or death. Our new predictive tool for recommending supplemental EBRT after TARGIT-IORT is at https://targit.org.uk/addrt . Non-breast cancer mortality was significantly lower in the TARGIT-IORT arm, even when patients received supplemental EBRT, HR 0.38 (95% CI 0.17-0.88) P = 0.0091. CONCLUSION: TARGIT-IORT is as effective as EBRT in all subgroups. Local recurrence after TARGIT-IORT, unlike after EBRT, has a good prognosis. TARGIT-IORT might have a beneficial abscopal effect. TRIAL REGISTRATION: ISRCTN34086741 (21/7/2004), NCT00983684 (24/9/2009)

Metabolic profiling of human brain metastases using in vivo proton MR spectroscopy at 3T

<p>Abstract</p> <p>Background</p> <p>Metastases to the central nervous system from different primary cancers are an oncologic challenge as the overall prognosis for these patients is generally poor. The incidence of brain metastases varies with type of primary cancer and is probably increasing due to improved therapies of extracranial metastases prolonging patient's overall survival and thereby time for brain metastases to develop. In addition, the greater access to improved neuroimaging techniques can provide earlier diagnosis. The aim of this study was to investigate the feasibility of using proton magnetic resonance spectroscopy (MRS) and multivariate analyses to characterize brain metastases originating from different primary cancers, to assess changes in spectra during radiation treatment and to correlate the spectra to clinical outcome after treatment.</p> <p>Methods</p> <p>Patients (n = 26) with brain metastases were examined using single voxel MRS at a 3T clinical MR system. Five patients were excluded due to poor spectral quality. The spectra were obtained before start (n = 21 patients), immediately after (n = 6 patients) and two months after end of treatment (n = 4 patients). Principal component analysis (PCA) and partial least square regression analysis (PLS) were applied in order to identify clustering of spectra due to origin of metastases and to relate clinical outcome (survival) of the patients to spectral data from the first MR examination.</p> <p>Results</p> <p>The PCA results indicated that brain metastases from primary lung and breast cancer were separated into two clusters, while the metastases from malignant melanomas showed no uniformity. The PLS analysis showed a significant correlation between MR spectral data and survival five months after MRS before start of treatment.</p> <p>Conclusion</p> <p>MRS determined metabolic profiles analysed by PCA and PLS might give valuable clinical information when planning and evaluating the treatment of brain metastases, and also when deciding to terminate further therapies.</p

Merging transcriptomics and metabolomics - advances in breast cancer profiling

Background Combining gene expression microarrays and high resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS) of the same tissue samples enables comparison of the transcriptional and metabolic profiles of breast cancer. The aim of this study was to explore the potential of combining these two different types of information. Methods Breast cancer tissue from 46 patients was analyzed by HR MAS MRS followed by gene expression microarrays. Two strategies were used to combine the gene expression and metabolic data; first using multivariate analyses to identify different groups based on gene expression and metabolic data; second correlating levels of specific metabolites to transcripts to suggest new hypotheses of connections between metabolite levels and the underlying biological processes. A parallel study was designed to address experimental issues of combining microarrays and HR MAS MRS. Results In the first strategy, using the microarray data and previously reported molecular classification methods, the majority of samples were classified as luminal A. Three subgroups of luminal A tumors were identified based on hierarchical clustering of the HR MAS MR spectra. The samples in one of the subgroups, designated A2, showed significantly lower glucose and higher alanine levels than the other luminal A samples, suggesting a higher glycolytic activity in these tumors. This group was also enriched for genes annotated with Gene Ontology (GO) terms related to cell cycle and DNA repair. In the second strategy, the correlations between concentrations of myo-inositol, glycine, taurine, glycerophosphocholine, phosphocholine, choline and creatine and all transcripts in the filtered microarray data were investigated. GO-terms related to the extracellular matrix were enriched among the genes that correlated the most to myo-inositol and taurine, while cell cycle related GO-terms were enriched for the genes that correlated the most to choline. Additionally, a subset of transcripts was identified to have slightly altered expression after HR MAS MRS and was therefore removed from all other analyses. Conclusions Combining transcriptional and metabolic data from the same breast carcinoma sample is feasible and may contribute to a more refined subclassification of breast cancers as well as reveal relations between metabolic and transcriptional levels. See Commentary: http://www.biomedcentral.com/1741-7015/8/7

Associations between tamoxifen, estrogens, and FSH serum levels during steady state tamoxifen treatment of postmenopausal women with breast cancer

<p>Abstract</p> <p>Background</p> <p>The cytochrome P450 (CYP) enzymes 2C19, 2D6, and 3A5 are responsible for converting the selective estrogen receptor modulator (SERM), tamoxifen to its active metabolites 4-hydroxy-tamoxifen (4OHtam) and 4-hydroxy-<it>N</it>-demethyltamoxifen (4OHNDtam, endoxifen). Inter-individual variations of the activity of these enzymes due to polymorphisms may be predictors of outcome of breast cancer patients during tamoxifen treatment. Since tamoxifen and estrogens are both partly metabolized by these enzymes we hypothesize that a correlation between serum tamoxifen and estrogen levels exists, which in turn may interact with tamoxifen on treatment outcome. Here we examined relationships between the serum levels of tamoxifen, estrogens, follicle-stimulating hormone (FSH), and also determined the genotypes of CYP2C19, 2D6, 3A5, and SULT1A1 in 90 postmenopausal breast cancer patients.</p> <p>Methods</p> <p>Tamoxifen and its metabolites were measured by liquid chromatography-tandem mass spectrometry. Estrogen and FSH levels were determined using a sensitive radio- and chemiluminescent immunoassay, respectively.</p> <p>Results</p> <p>We observed significant correlations between the serum concentrations of tamoxifen, <it>N</it>-dedimethyltamoxifen, and tamoxifen-<it>N</it>-oxide and estrogens (p < 0.05). The genotype predicted CYP2C19 activity influenced the levels of both tamoxifen metabolites and E1.</p> <p>Conclusions</p> <p>We have shown an association between tamoxifen and its metabolites and estrogen serum levels. An impact of CYP2C19 predicted activity on tamoxifen, as well as estrogen kinetics may partly explain the observed association between tamoxifen and its metabolites and estrogen serum levels. Since the role of estrogen levels during tamoxifen therapy is still a matter of debate further prospective studies to examine the effect of tamoxifen and estrogen kinetics on treatment outcome are warranted.</p

Prognostic value of metabolic response in breast cancer patients receiving neoadjuvant chemotherapy

<p>Abstract</p> <p>Background</p> <p>Today's clinical diagnostic tools are insufficient for giving accurate prognosis to breast cancer patients. The aim of our study was to examine the tumor metabolic changes in patients with locally advanced breast cancer caused by neoadjuvant chemotherapy (NAC), relating these changes to clinical treatment response and long-term survival.</p> <p>Methods</p> <p>Patients (n = 89) participating in a randomized open-label multicenter study were allocated to receive either NAC as epirubicin or paclitaxel monotherapy. Biopsies were excised pre- and post-treatment, and analyzed by high resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS). The metabolite profiles were examined by paired and unpaired multivariate methods and findings of important metabolites were confirmed by spectral integration of the metabolite peaks.</p> <p>Results</p> <p>All patients had a significant metabolic response to NAC, and pre- and post-treatment spectra could be discriminated with 87.9%/68.9% classification accuracy by paired/unpaired partial least squares discriminant analysis (PLS-DA) (<it>p </it>< 0.001). Similar metabolic responses were observed for the two chemotherapeutic agents. The metabolic responses were related to patient outcome. Non-survivors (< 5 years) had increased tumor levels of lactate (<it>p </it>= 0.004) after treatment, while survivors (≥ 5 years) experienced a decrease in the levels of glycine (<it>p </it>= 0.047) and choline-containing compounds (<it>p </it>≤ 0.013) and an increase in glucose (<it>p </it>= 0.002) levels. The metabolic responses were not related to clinical treatment response.</p> <p>Conclusions</p> <p>The differences in tumor metabolic response to NAC were associated with breast cancer survival, but not to clinical response. Monitoring metabolic responses to NAC by HR MAS MRS may provide information about tumor biology related to individual prognosis.</p

Genome-Wide Profile of Pleural Mesothelioma versus Parietal and Visceral Pleura: The Emerging Gene Portrait of the Mesothelioma Phenotype

Malignant pleural mesothelioma is considered an almost incurable tumour with increasing incidence worldwide. It usually develops in the parietal pleura, from mesothelial lining or submesothelial cells, subsequently invading the visceral pleura. Chromosomal and genomic aberrations of mesothelioma are diverse and heterogenous. Genome-wide profiling of mesothelioma versus parietal and visceral normal pleural tissue could thus reveal novel genes and pathways explaining its aggressive phenotype.Well-characterised tissue from five mesothelioma patients and normal parietal and visceral pleural samples from six non-cancer patients were profiled by Affymetrix oligoarray of 38 500 genes. The lists of differentially expressed genes tested for overrepresentation in KEGG PATHWAYS (Kyoto Encyclopedia of Genes and Genomes) and GO (gene ontology) terms revealed large differences of expression between visceral and parietal pleura, and both tissues differed from mesothelioma. Cell growth and intrinsic resistance in tumour versus parietal pleura was reflected in highly overexpressed cell cycle, mitosis, replication, DNA repair and anti-apoptosis genes. Several genes of the “salvage pathway” that recycle nucleobases were overexpressed, among them TYMS, encoding thymidylate synthase, the main target of the antifolate drug pemetrexed that is active in mesothelioma. Circadian rhythm genes were expressed in favour of tumour growth. The local invasive, non-metastatic phenotype of mesothelioma, could partly be due to overexpression of the known metastasis suppressors NME1 and NME2. Down-regulation of several tumour suppressor genes could contribute to mesothelioma progression. Genes involved in cell communication were down-regulated, indicating that mesothelioma may shield itself from the immune system. Similarly, in non-cancer parietal versus visceral pleura signal transduction, soluble transporter and adhesion genes were down-regulated. This could represent a genetical platform of the parietal pleura propensity to develop mesothelioma.Genome-wide microarray approach using complex human tissue samples revealed novel expression patterns, reflecting some important features of mesothelioma biology that should be further explored