Elen K. Møller

Next generation sequencing of disseminated tumor cells

Disseminated tumor cells (DTCs) detected in the bone marrow have been shown as an independent prognostic factor for women with breast cancer. However, the mechanisms behind the tumor cell dissemination are still unclear and more detailed knowledge is needed to fully understand why some cells remain dormant and others metastasize. Sequencing of single cells has opened for the possibility to dissect the genetic content of subclones of a primary tumor, as well as DTCs. Previous studies of genetic changes in DTCs have employed single-cell array comparative genomic hybridization which provides information about larger aberrations. To date, next-generation sequencing provides the possibility to discover new, smaller, and copy neutral genetic changes. In this study, we performed whole-genome amplification and subsequently next-generation sequencing to analyze DTCs from two breast cancer patients. We compared copy-number profiles of the DTCs and the corresponding primary tumor generated from sequencing and SNP-comparative genomic hybridization (CGH) data, respectively. While one tumor revealed mostly whole-arm gains and losses, the other had more complex alterations, as well as subclonal amplification and deletions. Whole-arm gains or losses in the primary tumor were in general also observed in the corresponding DTC. Both primary tumors showed amplification of chromosome 1q and deletion of parts of chromosome 16q, which was recaptured in the corresponding DTCs. Interestingly, clear differences were also observed, indicating that the DTC underwent further evolution at the copy-number level. This study provides a proof-of-principle for sequencing of DTCs and correlation with primary copy-number profiles. The analyses allow insight into tumor cell dissemination and show ongoing copy-number evolution in DTCs compared to the primary tumors.

Interaction between p53 Mutation and a Somatic HDMX Biomarker Better Defines Metastatic Potential in Breast Cancer

TP53 gene mutation is associated with poor prognosis in breast cancer, but additional biomarkers that can further refine the impact of the p53 pathway are needed to achieve clinical utility. In this study, we evaluated a role for the HDMX-S/FL ratio as one such biomarker, based on its association with other suppressor mutations that confer worse prognosis in sarcomas, another type of cancer that is surveilled by p53. We found that HDMX-S/FL ratio interacted with p53 mutational status to significantly improve prognostic capability in patients with breast cancer. This biomarker pair offered prognostic utility that was comparable with a microarray-based prognostic assay. Unexpectedly, the utility tracked independently of DNA-damaging treatments and instead with different tumor metastasis potential. Finally, we obtained evidence that this biomarker pair might identify patients who could benefit from anti-HDM2 strategies to impede metastatic progression. Taken together, our work offers a p53 pathway marker, which both refines our understanding of the impact of p53 activity on prognosis and harbors potential utility as a clinical tool.

Integrative clustering reveals a novel split in the luminal A subtype of breast cancer with impact on outcome

BackgroundBreast cancer is a heterogeneous disease at the clinical and molecular level. In this study we integrate classifications extracted from five different molecular levels in order to identify integrated subtypes.MethodsTumor tissue from 425 patients with primary breast cancer from the Oslo2 study was cut and blended, and divided into fractions for DNA, RNA and protein isolation and metabolomics, allowing the acquisition of representative and comparable molecular data. Patients were stratified into groups based on their tumor characteristics from five different molecular levels, using various clustering methods. Finally, all previously identified and newly determined subgroups were combined in a multilevel classification using a “cluster-of-clusters” approach with consensus clustering.ResultsBased on DNA copy number data, tumors were categorized into three groups according to the complex arm aberration index. mRNA expression profiles divided tumors into five molecular subgroups according to PAM50 subtyping, and clustering based on microRNA expression revealed four subgroups. Reverse-phase protein array data divided tumors into five subgroups. Hierarchical clustering of tumor metabolic profiles revealed three clusters. Combining DNA copy number and mRNA expression classified tumors into seven clusters based on pathway activity levels, and tumors were classified into ten subtypes using integrative clustering. The final consensus clustering that incorporated all aforementioned subtypes revealed six major groups. Five corresponded well with the mRNA subtypes, while a sixth group resulted from a split of the luminal A subtype; these tumors belonged to distinct microRNA clusters. Gain-of-function studies using MCF-7 cells showed that microRNAs differentially expressed between the luminal A clusters were important for cancer cell survival. These microRNAs were used to validate the split in luminal A tumors in four independent breast cancer cohorts. In two cohorts the microRNAs divided tumors into subgroups with significantly different outcomes, and in another a trend was observed.ConclusionsThe six integrated subtypes identified confirm the heterogeneity of breast cancer and show that finer subdivisions of subtypes are evident. Increasing knowledge of the heterogeneity of the luminal A subtype may add pivotal information to guide therapeutic choices, evidently bringing us closer to improved treatment for this largest subgroup of breast cancer.

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.

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.

Abstract LB-051: Tumor heterogeneity and dissemination in breast cancer: Deep sequencing of single disseminated cells from bone marrow compared to primary tumor and lymph node metastases

Metastasis is the main cause of death amongst breast cancer patients. Our knowledge of the metastatic cascade and how to inhibit it is limited. Here we dissect the genetic profile of multiple single disseminated tumor cells (DTCs) taken at various time points after diagnosis, and compare them to their matched primary tumors and lymph node metastasis. We have previously published a method for studying CNAs in single DTCs by whole genome sequencing, where we compared two primary breast carcinomas to two corresponding DTCs. Copy number profiles from whole genome sequencing (WGS) from 40 DTCs were analyzed. The single cell whole genome amplified (WGA) DNA was used to generate WGS libraries, and the DTCs were subsequently sequenced on the Illumina HiSeq 2000. The WGS reads were trimmed for WGA adapters and aligned to GRCh37 human reference using Burrows-Wheeler Aligner (BWA). LogR values were calculated for genomic bins and corrected for% GC-bias and segmented using the piecewise constant fitting (PCF) algorithm (the penalty parameter, γ, was set to 25). Copy number was estimated per segment as 2logR × Ψ, where Ψ is the average ploidy. B allele frequency (BAF) was calculated for each known SNP position from dbSNP (dbSNP build 135) and somatic mutations read-outs generated. In this study we compared the mutation spectre and CNAs in six primary tumors, one with corresponding lymph node metastasis and single DTCs. In total, CN profile from 40 DTCs showed evidence of dissemination at both early and late stage of disease progression. At large, the copy number profile of the examined DTCs exhibited either a limited number of alterations, or a pattern similar to the primary tumor and lymph node metastasis suggesting continuous dissemination of single tumor cells throughout the tumor evolution. By demonstrating sub-clonality in the lymph node metastasis we provide novel insight into the metastatic process. Further, the correlation in aberration pattern between the lymph node metastasis and multiple DTCs, implies that cells found in the bone marrow may have originated from the lymph node metastasis. The DTCs exhibited common aberrations typically found in breast carcinomas, and several DTCs had deletion of 16q and17p, and gain of 1q, 8q. Certain DTCs exhibited CNAs not visible in the primary tumor or lymph node including gain of 9q, 14q, 19q and Xq, and loss of 2p, 6p, 8p, 18p and 19p. Two DTCs from time of diagnosis exhibited gain of the whole chromosome 5 that was not observed in the primary tumor or the lymph node. These results reveal the importance of assessing the sub-clonal genetic alterations in the primary tumor, as well as in the lymph node metastasis and DTCs, in order to evaluate patient treatment and prognosis. Citation Format: Elen Moller, Parveen Kumar, Silje Nord, David Wedge, Peter van Loo, April Peterson, Randi R. Mathiesen, Renathe Fjelldal, Masoud Z. Esteki, Jason A. Grundstad, Elin Borgen, Lars O. Baumbusch, Anne-Lise Borresen-Dale, Kevin P. White, Thierry Voet, Bjorn Naume, Vessela N. Kristensen. Tumor heterogeneity and dissemination in breast cancer: Deep sequencing of single disseminated cells from bone marrow compared to primary tumor and lymph node metastases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-051. doi:10.1158/1538-7445.AM2015-LB-051

Abstract A72: Next-generation sequencing of disseminated tumor cells

Disseminated tumor cells (DTCs) detected in the bone marrow have been shown as an independent prognostic factor for women with breast cancer. However, the mechanisms behind the tumor cell dissemination are still unclear and more detailed knowledge is needed to fully understand why some cells remain dormant and others metastasize. Sequencing of single cells has opened for the possibility to dissect the genetic content of subclones of a primary tumor, as well as DTCs. Previous studies of genetic changes in DTCs have employed single-cell array comparative genomic hybridization which provides information about larger aberrations. To date, next generation sequencing provides the possibility to discover new, smaller and copy neutral genetic changes. In this study, we performed whole genome amplification and subsequently next generation sequencing to analyze DTCs from two breast cancer patients. We compared copy number profiles of the DTCs and the corresponding primary tumor generated from sequencing and SNP-CGH data, respectively. While one tumor revealed mostly whole arm gains and losses, the other had more complex alterations, as well as subclonal amplification and deletions. Whole arm gains or losses in the primary tumor were in general also observed in the corresponding DTC. Both primary tumors showed amplification of chromosome 1q and deletion of parts of chromosome 16q, which was recaptured in the corresponding DTCs. Interestingly, clear differences were also observed, indicating that the DTC underwent further evolution at the copy number level. This study provides a proof-of-principle for sequencing of DTCs and correlation with primary copy number profiles. The analyses allow insight into tumor cell dissemination and show ongoing copy number evolution in DTCs compared to the primary tumors. Note: This abstract was not presented at the conference. Citation Format: Elen K. Moller, Parveen Kumar, Thierry Voet, April Peterson, Peter Van Loo, Randi Mathiesen, Renate Fjelldal, Jason Grundstad, Elin Borgen, Lars O. Baumbusch, Bjorn Naume, Anne-Lise Borresen-Dale, Silje Nord, Vessela N. Kristensen. Next-generation sequencing of disseminated tumor cells. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A72. doi:10.1158/1538-7445.CHTME14-A72

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

Abstract P4-11-14: Molecular response in breast cancer treated with neoadjuvant chemotherapy with and without bevacizumab: Results from NeoAva - a randomized phase II study

The NeoAva study is a phase II clinical trial of patients with HER2 negative primary tumors of ≥25 mm 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. Tumor response were evaluable in 131 patients; of which 66 received bevacizumab in addition to chemotherapy. Tumor material was obtained at screening, 12 weeks into treatment and at surgical removal of tumors at 25 weeks. mRNA expression profiling was performed on Agilent 8x60K platform and the tumors were classified into LuminalA, LuminalB, Her2-enriched, Basal and Normal-like subtypes using the PAM50 classifier. Ratio of the tumor size at final pathology assessment, and at inclusion (by radiology assessment) was calculated to obtain a continuous scale of response reflecting the percentage of tumor shrinkage in response to therapy. Genomic Grade Index (GGI scores) based on expression profiles of 97 genes (including cell-cycle and proliferation genes) were calculated. There were no significant differences in the tumor size, lymph node, hormone receptor status or PAM50 subtypes between the treatment arms. pCR in breast and axilla were obtained in 14 (21.1%) patients in the chemo+bev arm, and in 7 (10.6%) patients in the chemo-only arm. Tumors that obtained pCR were in higher number ER negative and TP53 mutated and exhibited Basal-like phenotype. The overall pCR rates were higher in the ER negative tumors compared to ER positive tumors {39.1% (9 of 23) vs 11.1% (12 of 108)}. However, addition of bevacizumab seemed to improve pCR in the ER positive patient group (9 vs 3) and not in ER negative patient group (5 vs 4). On evaluating the continuous response variable, ER status, TP53 mutation status and PAM50 subtypes were significantly associated to response (p Next, we evaluated a shift in PAM50 subtypes across the timepoints. A shift towards a better prognosis group, i.e Luminal A or Normal-like profile was observed in response to therapy. Distribution of Luminal A and Normal-like tumors at week 25, (and not at screening or week 12) was significantly different in the chemo+bev vs chemo-only group (p = 0.026, Fisher’s exact test). GGI scores regressed across timepoints reflecting the loss of aggressive and proliferating component of the tumors in response to therapy. GGI scores in the chemo+bev group became significantly lower (p These results, with potentially important clinical relevance will be further investigated with respect to subtypes and the molecular changes induced by antiangiogenic therapy. Citation Format: Olav Engebraaten, Laxmi Silwal-Pandit, Marit Krohn, Elen K Moller, Silje Nord, Thomas Fleischer, Hedda von der Lippe Gythfelt, Elin Borgen, Oystein Garred, Anne Fangberget, Marit Muri Holmen, Ellen Schlichting, Helle Skjerven, Steinar Lundgren, Vessela N Kristensen, Ole Christian Lingjaerde, Erik Wist, Bjorn Naume, Anne-Lise Borresen-Dale. Molecular response in breast cancer treated with neoadjuvant chemotherapy with and without bevacizumab: Results from NeoAva - a randomized phase II study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-14.