Marit Muri Holmen

Gene expression profiles of breast biopsies from healthy women identify a group with claudin-low features

BackgroundIncreased understanding of the variability in normal breast biology will enable us to identify mechanisms of breast cancer initiation and the origin of different subtypes, and to better predict breast cancer risk.MethodsGene expression patterns in breast biopsies from 79 healthy women referred to breast diagnostic centers in Norway were explored by unsupervised hierarchical clustering and supervised analyses, such as gene set enrichment analysis and gene ontology analysis and comparison with previously published genelists and independent datasets.ResultsUnsupervised hierarchical clustering identified two separate clusters of normal breast tissue based on gene-expression profiling, regardless of clustering algorithm and gene filtering used. Comparison of the expression profile of the two clusters with several published gene lists describing breast cells revealed that the samples in cluster 1 share characteristics with stromal cells and stem cells, and to a certain degree with mesenchymal cells and myoepithelial cells. The samples in cluster 1 also share many features with the newly identified claudin-low breast cancer intrinsic subtype, which also shows characteristics of stromal and stem cells. More women belonging to cluster 1 have a family history of breast cancer and there is a slight overrepresentation of nulliparous women in cluster 1. Similar findings were seen in a separate dataset consisting of histologically normal tissue from both breasts harboring breast cancer and from mammoplasty reductions.ConclusionThis is the first study to explore the variability of gene expression patterns in whole biopsies from normal breasts and identified distinct subtypes of normal breast tissue. Further studies are needed to determine the specific cell contribution to the variation in the biology of normal breasts, how the clusters identified relate to breast cancer risk and their possible link to the origin of the different molecular subtypes of breast cancer.

Peripheral blood cells inform on the presence of breast cancer: A population‐based case–control study

Tumor–host interactions extend beyond the local microenvironment and cancer development largely depends on the ability of malignant cells to hijack and exploit the normal physiological processes of the host. Here, we established that many genes within peripheral blood cells show differential expression when an untreated breast cancer (BC) is present, and harnessed this fact to construct a 50‐gene signature that distinguish BC patients from population‐based controls. Our results were derived from a series of large datasets within our unique population‐based Norwegian Women and Cancer cohort that allowed us to investigate the influence of medications and tumor characteristics on our blood‐based test, and were further tested in two external datasets. Our 50‐gene signature contained cytostatic signals including the specific suppression of the immune response and medications influencing transcription involved in those processes were identified as confounders. Through analysis of the biological processes differentially expressed in blood, we were able to provide a rationale as to why the systemic response of the host may be a reliable marker of BC, characterized by the underexpression of both immune‐specific pathways and “universal” cell programs driven by MYC (i.e., metabolism, growth and cell cycle). In conclusion, gene expression of peripheral blood cells is markedly perturbed by the specific presence of carcinoma in the breast and these changes simultaneously engage a number of systemic cytostatic signals emerging connections with immune escape of BC.

The longitudinal transcriptional response to neoadjuvant chemotherapy with and without bevacizumab in breast cancer

Purpose: Chemotherapy-induced alterations to gene expression are due to transcriptional reprogramming of tumor cells or subclonal adaptations to treatment. The effect on whole-transcriptome mRNA expression was investigated in a randomized phase II clinical trial to assess the effect of neoadjuvant chemotherapy with the addition of bevacizumab. Experimental Design: Tumor biopsies and whole-transcriptome mRNA profiles were obtained at three fixed time points with 66 patients in each arm. Altogether, 358 specimens from 132 patients were available, representing the transcriptional state before treatment start, at 12 weeks and after treatment (25 weeks). Pathologic complete response (pCR) in breast and axillary nodes was the primary endpoint. Results: pCR was observed in 15 patients (23%) receiving bevacizumab and chemotherapy and 8 patients (12%) receiving only chemotherapy. In the estrogen receptor–positive patients, 11 of 54 (20%) treated with bevacizumab and chemotherapy achieved pCR, while only 3 of 57 (5%) treated with chemotherapy reached pCR. In patients with estrogen receptor–positive tumors treated with combination therapy, an elevated immune activity was associated with good response. Proliferation was reduced after treatment in both treatment arms and most pronounced in the combination therapy arm, where the reduction in proliferation accelerated during treatment. Transcriptional alterations during therapy were subtype specific, and the effect of adding bevacizumab was most evident for luminal-B tumors. Conclusions: Clinical response and gene expression response differed between patients receiving combination therapy and chemotherapy alone. The results may guide identification of patients likely to benefit from antiangiogenic therapy. Clin Cancer Res; 23(16); 4662–70. ©2017 AACR.

Interactions between the tumor and the blood systemic response of breast cancer patients

Although systemic immunity is critical to the process of tumor rejection, cancer research has largely focused on immune cells in the tumor microenvironment. To understand molecular changes in the patient systemic response (SR) to the presence of BC, we profiled RNA in blood and matched tumor from 173 patients. We designed a system (MIxT, Matched Interactions Across Tissues) to systematically explore and link molecular processes expressed in each tissue. MIxT confirmed that processes active in the patient SR are especially relevant to BC immunogenicity. The nature of interactions across tissues (i.e. which biological processes are associated and their patterns of expression) varies highly with tumor subtype. For example, aspects of the immune SR are underexpressed proportionally to the level of expression of defined molecular processes specific to basal tumors. The catalog of subtype-specific interactions across tissues from BC patients provides promising new ways to tackle or monitor the disease by exploiting the patient SR.

Abstract 1813: Bevacizumab potentiates the proteomic response to neoadjuvant chemotherapy in breast cancer patients: Rppa exploration of consecutive tumor samples in the NeoAva randomized phase II trial

Antiangiogenic therapy using bevacizumab has proven effective for a number of cancers; however, in breast cancer there is an unmet need to identify patients that benefit from such treatment. Sampling of tumor biopsies before and during treatment, as well as at the time of surgery enables the assessment of response at multiple molecular levels. At the proteomic level reverse phase protein analysis (RPPA) support expression of numerous cancer associated proteins simultaneously, which can further be used to unravel molecular mechanisms associated with clinical response to bevacizumab treatment. In this phase II clinical trial, patients with HER2 negative primary tumors of ≥25 mm were treated with neoadjuvant chemotherapy (4 x FEC100 + 12 weeks of taxane-based therapy) and randomized (1:1) to receive bevacizumab or not. Mammography, ultrasound and MR imaging were used for response evaluation, in addition to final pathology assessment. Tumor responses were evaluable in 132 patients; of which 66 received bevacizumab. Ratio of the tumor size at final pathology assessment, and at inclusion was calculated to obtain a continuous scale of response reflecting the percentage of tumor shrinkage in response to therapy. Tumor biopsies were removed before start of treatment, at week 12 at the start of taxane-based tharapy and at the time of surgery. Lysates from each sample was analyzed on reverse phase protein arrays (RPPA) for expression levels of 210 proteins of which 54 were phospho-specific. The addition of bevacizumab to the chemotherapy do not alter proteomic response from week 0 to 25 to such extent that this patient group cluster naturally together. While the proteomic response from week 0 to 12 in both treatment arms had an overall similar profile regarding up- and down-regulated proteins, the combination treatment (FEC100 + bevacizumab) induced substantially more effect on the regulation of each protein. This suggests that bevacizumab treatment have the capability to potentiate the effects of the anthracyclin based chemotherapy from week 0 to 12. Conversely, from week 12-25 (taxane-based therapy + bevacizumab) this effect was lost or even reversed, possibly due to a de-vascularized and less accessible tumor. An exception to this observation was a few phospho-proteins that do seem to have sustained stronger regulation over the whole treatment period. We are in the process of analyzing in more detail the impact of phosphorylation and thus protein activation states on treatment response. Deciphering molecular response and activity regulation at the proteomic level is a promising approach and may reveal novel knowledge with potential important clinical relevance. Citation Format: Mads H. Haugen, Ole Christian Lingjaerde, Marit Krohn, Wei Zhao, Evita M. Lindholm, Laxmi Silwal-Pandit, Elin Borgen, Oystein Garred, Anne Fangberget, Marit M. Holmen, Ellen Schlichting, Helle K. Skjerven, Steinar Lundgren, Erik Wist, Bjorn Naume, Gunhild M. Maelandsmo, Yiling Lu, Anne-Lise Boerresen-Dale, Gordon B. Mills, Olav Engebraaten. Bevacizumab potentiates the proteomic response to neoadjuvant chemotherapy in breast cancer patients: Rppa exploration of consecutive tumor samples in the NeoAva randomized phase II trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1813. doi:10.1158/1538-7445.AM2017-1813

Abstract 3268: Proteomic response in breast cancer treated with neoadjuvant chemotherapy with and without bevacizumab: Reverse phase protein array (RPPA) results from NeoAva - a randomized phase II study

BACKGROUND: In this phase II clinical trial, patients with HER2 negative primary tumors of ≥25 mm were treated with neoadjuvant chemotherapy (4 x FEC100 + 12 weeks of taxane-based therapy) and randomized (1:1) to receive bevacizumab or no bevacizumab. Mammography, ultrasound and MR imaging were used for response evaluation, in addition to final pathology assessment. HYPOTHESIS: RPPA proteomic analyses support identification of molecular mechanisms associated with clinical response to bevacizumab treatment. METHODS: Tumor responses were evaluable in 132 patients; of which 66 received bevacizumab. Ratio of the tumor size at final pathology assessment, and at inclusion was calculated to obtain a continuous scale of response reflecting the percentage of tumor shrinkage in response to therapy. Tumor material was obtained at screening, 12 weeks into treatment and at surgical removal of tumors at 25 weeks. Lysates from each sample was analyzed on reverse phase protein arrays (RPPA) for expression levels of 210 proteins of which 54 were phospho-specific. Data from protein analyses was compared to previously generated mRNA expression data. RESULTS: Several proteins were found for which expression prior to treatment (week 0) reflected a better response on tumor shrinkage in the combination treatment arm (chemotherapy+bevacizumab): E.g. good responders had lower PDGFR-beta expression, and this was also observed at the mRNA level, while this result was not identified in the mono treatment arm (chemotherapy alone) on either level. The proteomic response from week 0 to 12 in both treatment arms had an overall similar profile regarding up- and down-regulated proteins; however, the combination treatment (FEC100 + bevacizumab) induced substantially more effect on regulation of each protein. This might reflect the capability of bevacizumab treatment to potentiate the effects of the anthracyclin based chemotherapy from week 0 to 12. Conversely, from week 12-25 (taxane-based therapy + bevacizumab) this effect was lost or even reversed, and reveals a possible need for further studies investigating changes in protein expression and correlation to response of a given treatment. Of particular interest were proteins that switched direction of regulation between the FEC and taxane-based regimes, however, these effects were not confined to the combination treatment and thus probably not due to the added bevacizumab. We are in the process of analyzing the impact of phosphorylation and thus protein activation states on treatment response. The above mentioned results have potentially important clinical relevance and will be further investigated with respect to subtypes and the biological pathways affected by antiangiogenic therapy. Citation Format: Mads H. Haugen, Ole Christian Lingjaerde, Marit Krohn, Evita M. Lindholm, Laxmi Silwal-Pandit, Elin Borgen, Oystein Garred, Anne Fangberget, Marit M. Holmen, Ellen Schlichting, Helle Skjerven, Steinar Lundgren, Erik Wist, Bjoern Naume, Gunhild M. Maelandsmo, Yiling Lu, Anne-Lise Boerresen-Dale, Gordon B. Mills, Olav Engebraaten. Proteomic response in breast cancer treated with neoadjuvant chemotherapy with and without bevacizumab: Reverse phase protein array (RPPA) results from NeoAva - a randomized phase II study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3268.

Abstract LB-337: Systemic shift in genomic aberrations in breast carcinomas during neoadjuvant chemotherapy in combination with bevacizumab

A time course study was designed to investigate the dynamics of copy number aberrations in tumor DNA during treatment of breast cancer patients. A phase II randomized clinical trial of Her2 negative breast cancer patients was conducted, with patients being treated with neoadjuvant chemotherapy (FEC and taxane), with or without bevacizumab. Serial biopsies were obtained at time of diagnosis, at 12 weeks after treatment with FEC +/- bevacizumab, and at 25 weeks after treatment with taxane +/- bevacizumab. Tumor DNA alterations and tumor percentage were studied over time, and substantial differences were observed with some tumors changing mainly between diagnosis and 12 weeks (after the FEC cycle), others between 12 and 25 weeks (taxanes), and still others changing in both time periods. In both treatment arms, good responders (GR) and non-responders (NR) displayed significant difference in genomic instability index (GII) at time of diagnosis. In the Combination arm at time of diagnosis, 25 loci harbored copy number alterations, which were significantly different between the GR and NR. An inverse aberration pattern was observed between the two extreme response groups at 6p22-p12 for patients in the Combination arm. In most cases, tumors that retained aberrations at all time points did not decrease in size. Signs of subclonal reduction were observed, with some aberrations disappearing and others being retained during treatment. In both treatment arms an increase in subclonal amplification was observed at 6p21.1, the locus which contains the VEGFA gene targeted by bevacizumab and was associated with good response. Significant decrease of frequency of subclones carrying gains at 17q21.32-q22 was observed at 12 week, with the peak occurring at TMEM100 , an ALK1 receptor signaling-dependent gene essential for vasculogenesis. This implies that cells bearing amplifications of TMEM100 are particularly sensitive to the treatment regime. Taken together, these results suggest that heterogeneity and subclonal architecture influence the response to targeted treatment in combination with chemotherapy, with possible implications for clinical decision making and monitoring of treatment efficacy. Citation Format: Elen K. Moller, Silje Nord, David Wedge, Ole Christian Lngjaerde, Laxmi Silwal-Pandit, Hedda vdL Gythfeld, Hans Kristian M. Vollan, Thomas Fleischer, Marit Krohn, Ellen Schlichting, Elin Borgen, Oystein Garred, Marit M. Holmen, Erik Wist, Bjorn Naume, Peter V. Loo, Anne-Lise Borresen-Dale, Olav Engebraten, Vessela Kristensen. Systemic shift in genomic aberrations in breast carcinomas during neoadjuvant chemotherapy in combination with bevacizumab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-337.

Peripheral blood cells inform on the presence of breast cancer

Tumor–host interactions extend beyond the local microenvironment and cancer development largely depends on the ability of malignant cells to hijack and exploit the normal physiological processes of the host. Here, we established that many genes within peripheral blood cells show differential expression when an untreated breast cancer (BC) is present, and harnessed this fact to construct a 50‐gene signature that distinguish BC patients from population‐based controls. Our results were derived from a series of large datasets within our unique population‐based Norwegian Women and Cancer cohort that allowed us to investigate the influence of medications and tumor characteristics on our blood‐based test, and were further tested in two external datasets. Our 50‐gene signature contained cytostatic signals including the specific suppression of the immune response and medications influencing transcription involved in those processes were identified as confounders. Through analysis of the biological processes differentially expressed in blood, we were able to provide a rationale as to why the systemic response of the host may be a reliable marker of BC, characterized by the underexpression of both immune‐specific pathways and “universal” cell programs driven by MYC (i.e., metabolism, growth and cell cycle). In conclusion, gene expression of peripheral blood cells is markedly perturbed by the specific presence of carcinoma in the breast and these changes simultaneously engage a number of systemic cytostatic signals emerging connections with immune escape of BC.

Peripheral blood cells inform on the presence of breast cancer: A population-based case-control study: Peripheral blood cells inform on the presence of breast cancer

Tumor–host interactions extend beyond the local microenvironment and cancer development largely depends on the ability of malignant cells to hijack and exploit the normal physiological processes of the host. Here, we established that many genes within peripheral blood cells show differential expression when an untreated breast cancer (BC) is present, and harnessed this fact to construct a 50‐gene signature that distinguish BC patients from population‐based controls. Our results were derived from a series of large datasets within our unique population‐based Norwegian Women and Cancer cohort that allowed us to investigate the influence of medications and tumor characteristics on our blood‐based test, and were further tested in two external datasets. Our 50‐gene signature contained cytostatic signals including the specific suppression of the immune response and medications influencing transcription involved in those processes were identified as confounders. Through analysis of the biological processes differentially expressed in blood, we were able to provide a rationale as to why the systemic response of the host may be a reliable marker of BC, characterized by the underexpression of both immune‐specific pathways and “universal” cell programs driven by MYC (i.e., metabolism, growth and cell cycle). In conclusion, gene expression of peripheral blood cells is markedly perturbed by the specific presence of carcinoma in the breast and these changes simultaneously engage a number of systemic cytostatic signals emerging connections with immune escape of BC.

Abstract P4-14-01: A time course study of genomic instability in breast cancer patients receiving neoadjuvant therapy with or without bevacizumab

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tumor heterogeneity is an area of intense research, revealing tumors with high complexity consisting of different subclones and infiltrating cells. Identification of subclones that are resistant to therapy may be critical to improve treatment outcome. The NeoAva study is a randomized phase II, clinical trial of Her2 negative breast cancer patients treated in a neoadjuvant setting with chemotherapy (FEC and taxane) +/- bevacizumab. Core needle biopsies were obtained at screening and after 12 weeks, and the tumor was surgically removed after 25 weeks. DNA copy number changes in the tumors were analyzed using Affymetrix SNP Array 6.0. Allele specific copy number changes were assessed using the Allele-Specific Copy number Analysis of Tumors (ASCAT) algorithm (Van Loo, Norgard et al., PNAS 2010) and allele-specific Piecewise Constant Fitting (asPCF) algorithms (Nilsen, Liestol et al., BMC Genomics 2012). Measures of genomic instability were obtained through the complex arm-wise aberration index (CAAI) that captures local rearrangements (‘firestorms’) (Russnes, Vollan et al., Sci Transl Med 2010). Changes in copy number aberrations between the three different time points were observed in almost all tumors. Some tumors showed a decrease in tumor percentage and aberrations after just 12 weeks of treatment, where others showed loss of aberrations only at the time of surgery (25 weeks). Most of the tumors that did retain aberrations at all time points during treatment, did not demonstrate any decrease in tumor size. Other profiles indicated subclonal reduction, where some aberrations are kept throughout treatment and others disappear. Many of the tumors shrinking in size showed fewer whole arm aberrations than before treatment, but retained their focal amplicons. Some of the tumor aberrations seem to disappear after 12 weeks, but to reappear after 25 weeks, but with the addition of novel aberration. Complex rearrangements were identified in 67% of tumors before treatment. The most frequent ‘firestorms’ were found on 20p, 11q and 8p. Some events were persistent through therapy, but the majority changed. An association between complex tumor genomes and patients having progressive disease/non-responders were observed. These results show the complex structure of a tumor and suggest that heterogeneity will influence the response to treatment. The subclonal patterns of tumors may be of great importance for clinical decision-making, as well as for monitoring treatment efficacy. Citation Format: Elen K. Moller, Silje Nord, Hans Kristian Moen Vollan, Hedda von der Lippe Gythfeldt, Hege Edvardsen, Laxmi Silwal- Pandit, Marit Krohn, Thomas Fleischer, Ellen Schlitchting, Elin Borgen, Oystein Garred, Anne Fangberget, Marit M. Holmen, Helle Skjerven, Steinar Lundgren, Erik Wist, Bjorn Naume, Anne-Lise Borresen-Dale, Ole Christian Lingjaerde, Olav Engebraten, Vessela N. Kristensen. A time course study of genomic instability in breast cancer patients receiving neoadjuvant therapy with or without bevacizumab. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1533. doi:10.1158/1538-7445.AM2014-1533