Localization of tamoxifen in human breast cancer tumors by MALDI mass spectrometry imaging

Background: Tamoxifen is used in endocrine treatment of breast cancer to inhibit estrogen signaling. A set of strati‐ ed ER‐positive and ER‐negative tumor sections was subjected to manual deposition of tamoxifen solution in order to investigate its spatial distribution upon exposure to interaction within thin tissue sections. Methods: The localization of tamoxifen in tumor sections was assessed by matrix assisted laser deposition/ioniza‐ tion mass spectrometry imaging. The images of extracted ion maps were analyzed for comparison of signal intensity distributions. Results: The precursor ion of tamoxifen (m/z 372.233) displayed heterogeneous signal intensity distributions in his‐ tological compartments of tumor tissue sections. The levels of tamoxifen in tumor cells compared with stroma were higher in ER‐positive tissues, whereas ER‐negative tissue sections showed lower signal intensities in tumor cells. Conclusions: The experimental model was successfully applied on frozen tumor samples allowing for di erentiation between ER groups based on distribution of tamoxifen

Remarkable similarities of chromosomal rearrangements between primary human breast cancers and matched distant metastases as revealed by whole-genome sequencing.

To better understand and characterize chromosomal structural variation during breast cancer progression, we enumerated chromosomal rearrangements for 11 patients by performing low-coverage whole-genome sequencing of 11 primary breast tumors and their 13 matched distant metastases. The tumor genomes harbored a median of 85 (range 18-404) rearrangements per tumor, with a median of 82 (26-310) in primaries compared to 87 (18-404) in distant metastases. Concordance between paired tumors from the same patient was high with a median of 89% of rearrangements shared (range 61-100%), whereas little overlap was found when comparing all possible pairings of tumors from different patients (median 3%). The tumors exhibited diverse genomic patterns of rearrangements: some carried events distributed throughout the genome while others had events mostly within densely clustered chromothripsis-like foci at a few chromosomal locations. Irrespectively, the patterns were highly conserved between the primary tumor and metastases from the same patient. Rearrangements occurred more frequently in genic areas than expected by chance and among the genes affected there was significant enrichment for cancer-associated genes including disruption of TP53, RB1, PTEN, and ESR1, likely contributing to tumor development. Our findings are most consistent with chromosomal rearrangements being early events in breast cancer progression that remain stable during the development from primary tumor to distant metastasis

Comprehensive molecular comparison of BRCA1 hypermethylated and BRCA1 mutated triple negative breast cancers

Funder: The Governmental Funding for Young Clinical Researchers within the National Health Service (ALF) 2017-2019Funder: Shamik Mitra is financially supported by the funding received from the European Community’s Horizon 2020 Framework Program for Research and Innovation (H2020-MSCA-ITN-2014) under Grant Agreement no. 247634Funder: Vetenskapsrådet (Swedish Research Council); doi: https://doi.org/10.13039/501100004359Funder: The Governmental Funding within the National Health Service (ALF)Funder: - The Governmental Funding of Clinical Research within the National Health Service (ALF), grant nbr 2018/40612 - The Gustav V:s Jubilee Foundation (174271 and 187041) - The research foundation at Department of Oncology in LundAbstract: Homologous recombination deficiency (HRD) is a defining characteristic in BRCA-deficient breast tumors caused by genetic or epigenetic alterations in key pathway genes. We investigated the frequency of BRCA1 promoter hypermethylation in 237 triple-negative breast cancers (TNBCs) from a population-based study using reported whole genome and RNA sequencing data, complemented with analyses of genetic, epigenetic, transcriptomic and immune infiltration phenotypes. We demonstrate that BRCA1 promoter hypermethylation is twice as frequent as BRCA1 pathogenic variants in early-stage TNBC and that hypermethylated and mutated cases have similarly improved prognosis after adjuvant chemotherapy. BRCA1 hypermethylation confers an HRD, immune cell type, genome-wide DNA methylation, and transcriptional phenotype similar to TNBC tumors with BRCA1-inactivating variants, and it can be observed in matched peripheral blood of patients with tumor hypermethylation. Hypermethylation may be an early event in tumor development that progress along a common pathway with BRCA1-mutated disease, representing a promising DNA-based biomarker for early-stage TNBC

Gene expression profiling in primary breast cancer distinguishes patients developing local recurrence after breast-conservation surgery, with or without postoperative radiotherapy

Introduction Some patients with breast cancer develop local recurrence after breast-conservation surgery despite postoperative radiotherapy, whereas others remain free of local recurrence even in the absence of radiotherapy. As clinical parameters are insufficient for identifying these two groups of patients, we investigated whether gene expression profiling would add further information. Methods We performed gene expression analysis (oligonucleotide arrays, 26,824 reporters) on 143 patients with lymph node-negative disease and tumor-free margins. A support vector machine was employed to build classifiers using leave-one-out cross-validation. Results Within the estrogen receptor-positive (ER+) subgroup, the gene expression profile clearly distinguished patients with local recurrence after radiotherapy (n = 20) from those without local recurrence (n = 80 with or without radiotherapy). The receiver operating characteristic (ROC) area was 0.91, and 5,237 of 26,824 reporters had a P value of less than 0.001 (false discovery rate = 0.005). This gene expression profile provides substantially added value to conventional clinical markers (for example, age, histological grade, and tumor size) in predicting local recurrence despite radiotherapy. Within the ER- subgroup, a weaker, but still significant, signal was found (ROC area = 0.74). The ROC area for distinguishing patients who develop local recurrence from those who remain local recurrence-free in the absence of radiotherapy was 0.66 (combined ER+/ER-). Conclusion A highly distinct gene expression profile for patients developing local recurrence after breast-conservation surgery despite radiotherapy has been identified. If verified in further studies, this profile might be a most important tool in the decision making for surgery and adjuvant therapy

Prognostic and treatment predictive factors for radio- and chemotherapy resistance in breast cancer patients- a step towards personlized medicine

Breast cancer is the most common cancer form among women in the Western world. Although treatment has improved during the last decades, there is still a significant proportion of the patients who are not cured. To further improve clinical outcome we need new treatment strategies, new prognostic markers, and new treatment predictive factors for personalized medicine.In this dissertation I have focused on local relapse and distant recurrences, where the former is a tumor recurring in the same breast and the latter is distant spread in the body. I have focused on high through-put techniques, but have also evaluated one single factor, using immunohistochemistry. The most promising result is the gene expression profile for “radioresistance” found in a patient cohort consisting of 100 lymph node negative patients operated with breast conservation surgery and either postoperative radiotherapy or not. The samples were analyzed with oligonucleotide array. A gene expression profile was found that clearly separated patients who developed local recurrences despite radiotherapy (“radioresistance”) from patients without local recurrences (either with or without radiotherapy). The clinical consequence, if these results can be confirmed, would be that patients with a“radioresistant” gene profile should be offered mastectomy instead of breast conservation surgery and radiotherapy. In another patient cohort consisting of 85 node positive breast cancer patients treated with CMF (cyclophosphamide, methotrexate, and 5-fluorouracil) we found a gene expression profile, using cDNA microarray, capable of distinguishing patients who developed distant recurrences from non-recurring patients. This profile was compared to a previously published gene list and to drug-associated genes from the literature. Our results were slightly better. However, our gene profile was not able to exceed the performance of conventional clinical markers. From the same patient cohort, we developed a protocol for protein extraction from the same samples used for RNA. We analyzed the protein expression pattern using 2-DE (two-dimensional electrophoresis) and found several differentially expressed proteins, both when comparing distant recurrences to no recurrences and estrogen receptor positive to estrogen receptor negative tumors. Similarities of regulated genes and proteins were also found when comparing the two studies. Finally, we investigated the prognostic importance of a proliferation marker, cyclin B1,in a case-control study. There were 190 lymph node negative breast cancer patients with no chemotherapy who died from breast cancer and 190 corresponding controls who were alive at the corresponding case’s time of death. Cyclin B1 was an independent prognostic proliferation marker and had a high reproducibility. The marker may be useful instead of histological grade, or as a complement, to identify patients in need of adjuvant chemotherapy. In conclusion, breast cancer is a heterogeneous disease and should be subdivided even more than it is today into different risk groups with the aid of new markers. We used different high through-put techniques to analyze hundreds to thousands of gene expressions and proteins. Our aim was to find new prognostic and predictive gene expressions and protein profiles that may improve individual treatment schemes and help provide personalized medicine. However, so far it is too early to conclude that the use of single markers can be eliminated, because we also found significant prognostic value of the proliferation marker cyclin B1

Gene expression profilers and conventional clinical markers to predict distant recurrences for premenopausal breast cancer patients after adjuvant chemotherapy.

A large proportion of breast cancer patients are treated with adjuvant chemotherapy after the primary operation, but some will recur in spite of this treatment. In order to achieve an improved and more individualised therapy, our knowledge in mechanisms for drug resistance needs to be increased. We have investigated to what extent cDNA microarray measurements could distinguish the likelihood of recurrences after adjuvant CMF (cyclophosphamide, methotrexate and 5-fluorouracil) treatment of premenopausal, lymph node positive breast cancer patients, and have also compared this with the corresponding performance when using conventional clinical variables. We tried several gene selection strategies, and built classifiers using the resulting gene lists. The best performing classifier with odds ratio (OR) = 6.5 (95% confidence interval (CI) = 1.4-62) did not outperform corresponding classifiers based on clinical variables. For the clinical variables, calibrated on the samples, either using all the clinical parameters or the Nottingham Prognostic Index (NPI) parameters, the areas under the receiver operating characteristics (ROC) curve were 0.78 and 0.79, respectively. The ORs at 90% sensitivity were 15 (95% CI = 3.1-140) and 10 (95% CI = 2.1-97), respectively. Our data have provided evidence for a comparable prediction of clinical outcome in CMF-treated breast cancer patients using conventional clinical variables and gene expression based markers. (c) 2006 Elsevier Ltd. All rights reserved

Androgen Receptor in Stage I-II Primary Breast Cancer -Prognostic Value and Distribution in Subgroups

BACKGROUND/AIM: The value of androgen receptor (AR) in breast cancer has gained renewed interest as a prognostic and treatment predictive biomarker. The aims of this work were to study the associations and the prognostic value of AR in patients from two clinical cohorts.MATERIALS AND METHODS: Cohort 1 included 208 premenopausal, node-negative patients of whom 87% had received no adjuvant medical treatment; cohort 2 consisted of 263 patients with stage II disease who had all received 2 years of adjuvant tamoxifen. A semi-quantitative assessment of nuclear AR expression divided into five groups (0-1%, 2-10%, 11-50%, 51-75%, and 76-100%) was performed. Survival analyses, stratified by cohort, were performed using both a trend-test and a cut-off of >10% for positivity.RESULTS: A total of 76% of all patients were AR+, and 89%, 48%, and 23% of the estrogen receptor-positive, negative, and triple-negative, respectively. In Cox regression, stratified by cohort, AR divided into five groups was a prognostic factor for 5-year distant disease-free survival with a hazard ratio of 0.86 per step in fraction score (p=0.018). With a predefined cut-off at 10%, moderate evidence of an effect remained (Hazard Ratio=0.67, p=0.077). In multivariable analysis, AR did not retain an independent prognostic value.CONCLUSION: AR is a weak, however, not independent prognostic factor for distant metastasis. Although the prognostic value of AR may be questionable, the study identified a subset of AR-positive triple-negative patients as being potential candidates for AR-directed therapy for which further studies are warranted

Proteomic analysis identifies candidate proteins associated with distant recurrences in breast cancer after adjuvant chemotherapy.

Breast cancer is a heterogenous disease and it is of importance to select patients with regard to different prognosis and treatment sensitivity to individualize treatment regimes. In this study we successfully adapted a protein extraction protocol from mRNA extracted tumor samples enabling two-dimensional gel electrophoresis (2-DE) analysis of samples previously analyzed by cDNA microarray. The aim was to find candidate proteins that distinguish breast cancer patients with or without recurrences after adjuvant CMF (cyclophosphamide, methotrexate and 5-FU) treatment within four years to follow-up. We identified several proteins distinguishing the recurrence group from the non-recurrence group, especially in the ER and PgR positive subgroup (n = 7). The induced proteins were involved in translation/folding, iron ion binding, and protease inhibition, whereas proteins involved in signaling, ubiquitination, and splicing were decreased in expression. These results show that it is possible to use 2-DE to separate high abundant proteins in breast cancer tissue and to find discriminating proteins to identify patients with different prognosis after adjuvant CMF treatment

Cancer associated proteins in blood plasma : Determining normal variation

Protein biomarkers have the potential to improve diagnosis, stratification of patients into treatment cohorts, follow disease progression and treatment response. One distinct group of potential biomarkers comprises proteins which have been linked to cancer, known as cancer associated proteins (CAPs). We determined the normal variation of 86 CAPs in 72 individual plasma samples collected from ten individuals using SRM mass spectrometry. Samples were collected weekly during 5 weeks from ten volunteers and over one day at nine fixed time points from three volunteers. We determined the degree of the normal variation depending on interpersonal variation, variation due to time of day, and variation over weeks and observed that the variation dependent on the time of day appeared to be the most important. Subdivision of the proteins resulted in two predominant protein groups containing 21 proteins with relatively high variation in all three factors (day, week and individual), and 22 proteins with relatively low variation in all factors. We present a strategy for prioritizing biomarker candidates for future studies based on stratification over their normal variation and have made all data publicly available. Our findings can be used to improve selection of biomarker candidates in future studies and to determine which proteins are most suitable depending on study design

Amplification of the cyclin D1 gene is associated with tumour subsite, DNA non-diploidy and high S-phase fraction in squamous cell carcinoma of the head and neck

Amplification of CCND1 (cyclin D1 gene) in squamous cell carcinoma of the head and neck (SCCHN) is correlated to poor prognosis. The purpose of this study was to investigate whether CCND1 amplification is related to different subsites and also to DNA ploidy status and S-phase fraction (SPF). Biopsies from 67 patients with SCCHN were analysed for CCND1 amplification by fluorescence in situ hybridisation (FISH) and for ploidy status and SPF by flow cytometry (FCM). Twenty-one of 67 tumours (31%) showed CCND1 amplification and the frequencies differed significantly between different subsites (p = 0.01). Tumours from hypopharynx, larynx and oropharynx showed higher rates of amplification as compared to tumours from oral cavity and epipharynx. CCND1 amplification was also associated to DNA non-diploidy and high SPF (p = 0.002 and p = 0.002, respectively). In conclusion, the rate of CCND1 amplification differed between different subsites in SCCHN and was also associated to a more aggressive tumour phenotype, as defined by DNA non-diploidy and high SPF