Eleonor Olsson

High-resolution genomic profiles of breast cancer cell lines assessed by tiling BAC array comparative genomic hybridization.

A BAC‐array platform for comparative genomic hybridization was constructed from a library of 32,433 clones providing complete genome coverage, and evaluated by screening for DNA copy number changes in 10 breast cancer cell lines (BT474, MCF7, HCC1937, SK‐BR‐3, L56Br‐C1, ZR‐75‐1, JIMT1, MDA‐MB‐231, MDA‐MB‐361, and HCC2218) and one cell line derived from fibrocystic disease of the breast (MCF10A). These were also characterized by gene expression analysis and found to represent all five recently described breast cancer subtypes using the “intrinsic gene set” and centroid correlation. Three cell lines, HCC1937 and L56BrC1 derived from BRCA1 mutation carriers and MDA‐MB‐231, were of basal‐like subtype and characterized by a high frequency of low‐level gains and losses of typical pattern, including limited deletions on 5q. Four estrogen receptor positive cell lines were of luminal A subtype and characterized by a different pattern of aberrations and high‐level amplifications, including ERBB2 and other 17q amplicons in BT474 and MDA‐MB‐361. SK‐BR‐3 cells, characterized by a complex genome including ERBB2 amplification, massive high‐level amplifications on 8q and a homozygous deletion of CDH1 at 16q22, had an expression signature closest to luminal B subtype. The effects of gene amplifications were verified by gene expression analysis to distinguish targeted genes from silent amplicon passengers. JIMT1, derived from an ERBB2 amplified trastuzumab resistant tumor, was of the ERBB2 subtype. Homozygous deletions included other known targets such as PTEN (HCC1937) and CDKN2A (MDA‐MB‐231, MCF10A), but also new candidate suppressor genes such as FUSSEL18 (HCC1937) and WDR11 (L56Br‐C1) as well as regions without known genes. The tiling BAC‐arrays constitute a powerful tool for high‐resolution genomic profiling suitable for cancer research and clinical diagnostics.

Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease.

Metastatic breast cancer is usually diagnosed after becoming symptomatic, at which point it is rarely curable. Cell‐free circulating tumor DNA (ctDNA) contains tumor‐specific chromosomal rearrangements that may be interrogated in blood plasma. We evaluated serial monitoring of ctDNA for earlier detection of metastasis in a retrospective study of 20 patients diagnosed with primary breast cancer and long follow‐up. Using an approach combining low‐coverage whole‐genome sequencing of primary tumors and quantification of tumor‐specific rearrangements in plasma by droplet digital PCR, we identify for the first time that ctDNA monitoring is highly accurate for postsurgical discrimination between patients with (93%) and without (100%) eventual clinically detected recurrence. ctDNA‐based detection preceded clinical detection of metastasis in 86% of patients with an average lead time of 11 months (range 0–37 months), whereas patients with long‐term disease‐free survival had undetectable ctDNA postoperatively. ctDNA quantity was predictive of poor survival. These findings establish the rationale for larger validation studies in early breast cancer to evaluate ctDNA as a monitoring tool for early metastasis detection, therapy modification, and to aid in avoidance of overtreatment.

CXCL14 is an autocrine growth factor for fibroblasts and acts as a multi-modal stimulator of prostate tumor growth

This study explored the role of secreted fibroblast-derived factors in prostate cancer growth. Analyses of matched normal and tumor tissue revealed up-regulation of CXCL14 in cancer-associated fibroblasts of a majority of prostate cancer. Fibroblasts over-expressing CXCL14 promoted the growth of prostate cancer xenografts, and increased tumor angiogenesis and macrophage infiltration. Mechanistic studies demonstrated that autocrine CXCL14-stimulation of fibroblasts stimulate migration and ERK-dependent proliferation of fibroblasts. CXCL14-stimulation of monocyte migration was also demonstrated. Furthermore, CXCL14-producing fibroblasts, but not recombinant CXCL14, enhanced in vitro proliferation and migration of prostate cancer cells and in vivo angiogenesis. These studies thus identify CXCL14 as a novel autocrine stimulator of fibroblast growth and migration, with multi-modal tumor-stimulatory activities. In more general terms, our findings suggest autocrine stimulation of fibroblasts as a previously unrecognized mechanism for chemokine-mediated stimulation of tumor growth, and suggest a novel mechanism whereby cancer-associated fibroblasts achieve their pro-tumorigenic phenotype.

CD44 isoforms are heterogeneously expressed in breast cancer and correlate with tumor subtypes and cancer stem cell markers

BackgroundThe CD44 cell adhesion molecule is aberrantly expressed in many breast tumors and has been implicated in the metastatic process as well as in the putative cancer stem cell (CSC) compartment. We aimed to investigate potential associations between alternatively spliced isoforms of CD44 and CSCs as well as to various breast cancer biomarkers and molecular subtypes.MethodsWe used q-RT-PCR and exon-exon spanning assays to analyze the expression of four alternatively spliced CD44 isoforms as well as the total expression of CD44 in 187 breast tumors and 13 cell lines. ALDH1 protein expression was determined by IHC on TMA.ResultsBreast cancer cell lines showed a heterogeneous expression pattern of the CD44 isoforms, which shifted considerably when cells were grown as mammospheres. Tumors characterized as positive for the CD44+/CD24- phenotype by immunohistochemistry were associated to all isoforms except the CD44 standard (CD44S) isoform, which lacks all variant exons. Conversely, tumors with strong expression of the CSC marker ALDH1 had elevated expression of CD44S. A high expression of the CD44v2-v10 isoform, which retain all variant exons, was correlated to positive steroid receptor status, low proliferation and luminal A subtype. The CD44v3-v10 isoform showed similar correlations, while high expression of CD44v8-v10 was correlated to positive EGFR, negative/low HER2 status and basal-like subtype. High expression of CD44S was associated with strong HER2 staining and also a subgroup of basal-like tumors. Unsupervised hierarchical cluster analysis of CD44 isoform expression data divided tumors into four main clusters, which showed significant correlations to molecular subtypes and differences in 10-year overall survival.ConclusionsWe demonstrate that individual CD44 isoforms can be associated to different breast cancer subtypes and clinical markers such as HER2, ER and PgR, which suggests involvement of CD44 splice variants in specific oncogenic signaling pathways. Efforts to link CD44 to CSCs and tumor progression should consider the expression of various CD44 isoforms.

STC1 Expression By Cancer-Associated Fibroblasts Drives Metastasis of Colorectal Cancer

Platelet-derived growth factor (PDGF) receptor signaling is a major functional determinant of cancer-associated fibroblasts (CAF). Elevated expression of PDGF receptors on stromal CAFs is associated with metastasis and poor prognosis, but mechanism(s) that underlie these connections are not understood. Here, we report the identification of the secreted glycoprotein stanniocalcin-1 (STC1) as a mediator of metastasis by PDGF receptor function in the setting of colorectal cancer. PDGF-stimulated fibroblasts increased migration and invasion of cocultured colorectal cancer cells in an STC1-dependent manner. Analyses of human colorectal cancers revealed significant associations between stromal PDGF receptor and STC1 expression. In an orthotopic mouse model of colorectal cancer, tumors formed in the presence of STC1-deficient fibroblasts displayed reduced intravasation of tumor cells along with fewer and smaller distant metastases formed. Our results reveal a mechanistic basis for understanding the contribution of PDGF-activated CAFs to cancer metastasis.

Characterization of bone marrow-derived mesenchymal stromal cells (MSC) based on gene expression profiling of functionally defined MSC subsets.

BACKGROUND AIMS Human mesenchymal stromal cells (MSC) are promising candidates for cell therapy because of their intriguing properties (high proliferation and differentiation capacity, microenvironmental function and immune modulation). However, MSC are heterogeneous and a better understanding of the heterogeneity of the cells that form the MSC cultures is critical. METHODS Human MSC were generated in standard cultures and stained with carboxyfluorescein succinimidyl ester (CFSE) for cell division tracking. Gene expression profiling of MSC that were sorted based on functional parameters (i.e. proliferation characteristics) was utilized to characterize potential MSC subpopulations (progenitor content and differentiation capacity) and identify potential MSC subpopulation markers. RESULTS The majority of MSC had undergone more than two cell divisions (79.7+/-2.0%) after 10 days of culture, whereas 3.5+/-0.9% of MSC had not divided. MSC were then sorted into rapidly dividing cells (RDC) and slowly/non-dividing cells (SDC/NDC). Colony-forming unit-fibroblast (CFU-F) frequencies were lowest in NDC and highest in RDC with low forward-/side-scatter properties (RDC(lolo)). Comparative microarray analysis of NDC versus RDC identified 102 differentially expressed genes. Two of these genes (FMOD and VCAM1) corresponded to cell-surface molecules that enabled the prospective identification of a VCAM1(+)/FMOD(+) MSC subpopulation, which increased with passage and showed very low progenitor activity and limited differentiation potential. CONCLUSIONS These data clearly demonstrate functional differences within MSC cultures. Furthermore, this study shows that cell sorting based on proliferation characteristics and gene expression profiling can be utilized to identify surface markers for the characterization of MSC subpopulations.

Cancer-associated fibroblasts expressing CXCL14 rely upon NOS1-derived nitric oxide signaling for their tumor-supporting properties.

Cancer-associated fibroblasts (CAF) stimulate tumor growth and metastasis. Signals supporting CAF function are thus emerging as candidate therapeutic targets in the tumor microenvironment. The chemokine CXCL14 is a potent inducer of CAF protumorigenic functions. This study is aimed at learning how the protumoral functions of CXCL14-expressing CAF are maintained. We found that the nitric oxide synthase NOS1 is upregulated in CXCL14-expressing CAF and in fibroblasts stimulated with CXCL14. Induction of Nos1 was associated with oxidative stress and occurred together with activation of NRF2 and HIF1α signaling in CXCL14-expressing CAF. Genetic or pharmacologic inhibition of NOS1 reduced the growth of CXCL14-expressing fibroblasts along with their ability to promote tumor formation following coinjection with prostate or breast cancer cells. Tumor analysis revealed reduced macrophage infiltration, with NOS1 downregulation in CXCL14-expressing CAF and lymphangiogenesis as a novel component of CXCL14-promoted tumor growth. Collectively, our findings defined key components of a signaling network that maintains the protumoral functions of CXCL14-stimulated CAF, and they identified NOS1 as intervention target for CAF-directed cancer therapy.

Prognostic Significance in Breast Cancer of a Gene Signature Capturing Stromal PDGF Signaling

In this study, we describe a novel gene expression signature of platelet-derived growth factor (PDGF)-activated fibroblasts, which is able to identify breast cancers with a PDGF-stimulated fibroblast stroma and displays an independent and strong prognostic significance. Global gene expression was compared between PDGF-stimulated human fibroblasts and cultured resting fibroblasts. The most differentially expressed genes were reduced to a gene expression signature of 113 genes. The biological significance and prognostic capacity of this signature were investigated using four independent clinical breast cancer data sets. Concomitant high expression of PDGFβ receptor and its cognate ligands is associated with a high PDGF signature score. This supports the notion that the signature detects tumors with PDGF-activated stroma. Subsequent analyses indicated significant associations between high PDGF signature score and clinical characteristics, including human epidermal growth factor receptor 2 positivity, estrogen receptor negativity, high tumor grade, and large tumor size. A high PDGF signature score is associated with shorter survival in univariate analysis. Furthermore, the high PDGF signature score acts as a significant marker of poor prognosis in multivariate survival analyses, including classic prognostic markers, Ki-67 status, a proliferation gene signature, or other recently described stroma-derived gene expression signatures.

Targeted sequencing of BRCA1 and BRCA2 across a large unselected breast cancer cohort suggests that one-third of mutations are somatic

We carried out targeted sequencing of BRCA1/2 in an unselected cohort of patients diagnosed with primary breast cancer within a population without strong founder mutations. Eleven percent of cases harbored a germline or somatic BRCA1/2 mutation, and the ratio of germline versus somatic mutation was 2 : 1. This has implications for treatment, genetic counseling, and interpretation of tumor-only testing.

Contralateral breast cancer can represent a metastatic spread of the first primary tumor: determination of clonal relationship between contralateral breast cancers using next-generation whole genome sequencing

IntroductionBy convention, a contralateral breast cancer (CBC) is treated as a new primary tumor, independent of the first cancer (BC1). Although there have been indications that the second tumor (BC2) sometimes may represent a metastatic spread of BC1, this has never been conclusively shown. We sought to apply next-generation sequencing to determine a “genetic barcode” for each tumor and reveal the clonal relationship of CBCs.MethodsTen CBC patients with detailed clinical information and available fresh frozen tumor tissue were studied. Using low-coverage whole genome DNA-sequencing data for each tumor, chromosomal rearrangements were enumerated and copy number profiles were generated. Comparisons between tumors provided an estimate of clonal relatedness for tumor pairs within individual patients.ResultsBetween 15–256 rearrangements were detected in each tumor (median 87). For one patient, 76 % (68 out of 90) of the rearrangements were shared between BC1 and BC2, highly consistent with what has been seen for true primary-metastasis pairs (>50 %) and thus confirming a common clonal origin of the two tumors. For most of the remaining cases, BC1 and BC2 had similarly low overlap as unmatched randomized pairs of tumors from different individuals, suggesting the CBC to represent a new independent primary tumor.ConclusionUsing rearrangement fingerprinting, we show for the first time with certainty that a contralateral BC2 can represent a metastatic spread of BC1. Given the poor prognosis of a generalized disease compared to a new primary tumor, these women need to be identified at diagnosis of CBC for appropriate determination of treatment. Our approach generates a promising new method to assess clonal relationship between tumors. Additional studies are required to confirm the frequency of CBCs representing metastatic events.