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PDF file - 36K, Supplementary Table 1: Protein regulation after Bevacizumab monotherapy.

PDF file - 37K, Supplementary Table 3: Protein regulation in basal‐like tumors after bevacizumab and BEZ235 treatment.

Table S1: Genes significantly differentially expressed in samples with pathological complete response compared to those without in the Combination arm. Table S2: DAVID analysis of 720 differentially expressed genes in tumors that achieved pathological complete reponse compared to those that did not, in the combination arm. Table S3 : Genes significantly differentially expressed in samples with pathological complete response compared to those without in the Chemotherapy arm. Table S4: DAVID analysis of 1243 differentially expressed genes in tumors that achieved pathological complete reponse compared to those that did not, in the chemotherapy arm. Table S5 : List of genes with significant lower expression in week 12 samples treated with combination therapy compared to those treated with chemotherapy Table S6: DAVID analysis of 42 differentially expressed genes in week-12 tumors in the combination arm compared to chemotherapy arm. Table S7 : Differentially expressed genes between week-0 and week-12 in Basal-like tumors treated with combination therapy Table S8 : Differentially expressed genes between week-0 and week-12 Luminal B tumors treated with combination therapy Table S9 : Differentially expressed genes between week-0 and week-12 Luminal A tumors treated with combination therapy Table S10: Differentially expressed genes between week-0 and week-12 Basal-like tumors treated with chemotherapy Table S11 : Differentially expressed genes between week-0 and week-12 Luminal B tumors treated with chemotherapy Table S12 : Differentially expressed genes between week-0 and week-12 Luminal A tumors treated with chemotherapy Table S13: Pathways significantly altered between Chemotherapy and Combination therapy arms from week-0 to week-12 LuminalB samples Table S14: Pathways significantly altered between anthracycline and taxane treatment in the Luminal A samples in chemotherapy arm

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.

Supplementary figures as referred to in the manuscript

PDF file - 36K, Supplementary Table 2: Protein regulation after bevacizumab + doxorubicin combination treatment, compared to doxorubicin monotherapy.

Purpose: Neoangiogenesis is an important feature in tumor growth and progression, and combining chemotherapy and antiangiogenic drugs have shown clinical efficacy. However, as treatment-induced resistance often develops, our goal was to identify pathways indicating response and/or evolving resistance to treatment and inhibit these pathways to optimize the treatment strategies.Experimental Design: To identify markers of response and/or resistance, reverse-phase protein array (RPPA) was used to characterize treatment-induced changes in a bevacizumab-responsive and a nonresponsive human breast cancer xenograft. Results were combined with bioinformatic modeling to predict druggable targets for optimization of the treatment.Results: RPPA analysis showed that both tumor models responded to bevacizumab with an early (day 3) upregulation of growth factor receptors and downstream signaling pathways, with persistent mTOR signaling until the end of the in vivo experiment. Adding doxorubicin to bevacizumab showed significant and superior growth inhibition of basal-like tumors, whereas no additive effect was seen in the luminal-like model. The combination treatment corresponded to a continuous late attenuation of mTOR signaling in the basal-like model, whereas the inhibition was temporary in the luminal-like model. Integrating the bevacizumab-induced dynamic changes in protein levels with bioinformatic modeling predicted inhibition of phosphoinositide 3-kinase (PI3K) pathway to increase the efficacy of bevacizumab monotherapy. In vivo experiments combining bevacizumab and the PI3K/mTOR inhibitor BEZ235 confirmed their significant and additive growth-inhibitory effect in the basal-like model.Conclusions: Treatment with bevacizumab caused compensatory upregulation of several signaling pathways. Targeting such pathways increased the efficacy of antiangiogenic therapy. Clin Cancer Res; 20(2); 404–12. ©2013 AACR.

Background Chemotherapeutic agents such as anthracyclines and taxanes are commonly used in the neoadjuvant setting. Bevacizumab is an antibody which binds to vascular endothelial growth factor A (VEGFA) and inhibits its receptor interaction, thus obstructing the formation of new blood vessels. Methods A phase II randomized clinical trial of 123 patients with Her2-negative breast cancer was conducted, with patients treated with neoadjuvant chemotherapy (fluorouracil (5FU)/epirubicin/cyclophosphamide (FEC) and taxane), with or without bevacizumab. Serial biopsies were obtained at time of diagnosis, after 12 weeks of treatment with FEC ± bevacizumab, and after 25 weeks of treatment with taxane ± bevacizumab. A time course study was designed to investigate the genomic landscape at the three time points when tumor DNA alterations, tumor percentage, genomic instability, and tumor clonality were assessed. Substantial differences were observed with some tumors changing mainly between diagnosis and at 12 weeks, others between 12 and 25 weeks, and still others changing in both time periods. Results 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, copy number alterations at 25 loci at the time of diagnosis were significantly different between the GR and NR. An inverse aberration pattern was also observed between the two extreme response groups at 6p22-p12 for patients in the combination arm. Signs of subclonal reduction were observed, with some aberrations disappearing and others being retained during treatment. Increase in subclonal amplification was observed at 6p21.1, a locus which contains the VEGFA gene for the protein which are targeted by the study drug bevacizumab. Of the 13 pre-treatment samples that had a gain at VEGFA, 12 were responders. Significant decrease of frequency of subclones carrying gains at 17q21.32-q22 was observed at 12 weeks, with the peak occurring at TMEM100, an ALK1 receptor signaling-dependent gene essential for vasculogenesis. This implies that cells bearing amplifications of VEGFA and TMEM100 are particularly sensitive to this treatment regime. Conclusions 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. Trial registration NCT00773695 . Registered 15 October 2008

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 < 0.001). GGI scores were highly correlated to response (p< 0.001), i. e tumors with higher GGI scores showed better response. Importantly, when the chemo+bev and the chemo-only arms were evaluated separately, although similar trend of associations was observed in both arms, the associations were found to be enhanced in the chemo+bev arm. 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 < 0.01) already at week 12. This suggests that the removal of the proliferating component of the tumors by chemotherapy is accelerated and improved by addition of bevacizumab. 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 Møller, Silje Nord, Thomas Fleischer, Hedda von der Lippe Gythfelt, Elin Borgen, Øystein Garred, Anne Fangberget, Marit Muri Holmen, Ellen Schlichting, Helle Skjerven, Steinar Lundgren, Vessela N Kristensen, Ole Christian Lingjaerde, Erik Wist, Bjørn Naume, Anne-Lise Børresen-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.

BACKGROUND:Breast 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.METHODS:Tumor 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.RESULTS:Based 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.CONCLUSIONS:The 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. BACKGROUND: Breast 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.METHODS: Tumor 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.RESULTS: Based 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.CONCLUSIONS: The 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.

SummarySmall noncoding miRNAs represent underexplored targets of genomic aberrations and emerging therapeutic targets. The 3q26.2 amplicon is among the most frequent genomic aberrations in multiple cancer lineages including ovarian and breast cancers. We demonstrate that hsa-miR-569 (hereafter designated as miR569), which is overexpressed in a subset of ovarian and breast cancers, at least in part due to the 3q26.2 amplicon, alters cell survival and proliferation. Downregulation of TP53INP1 expression by miR569 is required for the effects of miR569 on survival and proliferation. Targeting miR569 sensitizes ovarian and breast cancer cells overexpressing miR569 to cisplatin by increasing cell death both in vitro and in vivo. Thus targeting miR569 could potentially benefit patients with the 3q26.2 amplicon and subsequent miR569 elevation.

Background:B7-H3, an immunoregulatory protein, is overexpressed in several cancers and is often associated with metastasis and poor prognosis. Here, our aim was to identify microRNAs (miRNAs) regulating B7-H3 and assess their potential prognostic implications in breast cancer. Methods:MicroRNAs targeting B7-H3 were identified by transfecting two breast cancer cell lines with a library of 810 miRNA mimics and quantifying changes of B7-H3 protein levels using protein lysate microarrays. For validations we used western immunoblotting and 3'-UTR luciferase assays. Clinical significance of the miRNAs was assayed by analysing whether their expression levels correlated with outcome in two cohorts of breast cancer patients (142 and 81 patients). Results:We identified nearly 50 miRNAs that downregulated B7-H3 protein levels. Western immunoblotting validated the impact of the 20 most effective miRNAs. Thirteen miRNAs (miR-214, miR-363*, miR-326, miR-940, miR-29c, miR-665, miR-34b*, miR-708, miR-601, miR-124a, miR-380-5p, miR-885-3p, and miR-593) targeted B7-H3 directly by binding to its 3'-UTR region. Finally, high expression of miR-29c was associated with a significant reduced risk of dying from breast cancer in both cohorts. Conclusions:We identified miRNAs efficiently downregulating B7-H3 expression. The expression of miR-29c correlated with survival in breast cancer patients, suggesting a tumour suppressive role for this miRNA.British Journal of Cancer advance online publication, 27 February 2014; doi:10.1038/bjc.2014.113 www.bjcancer.com

Withdrawn by Author Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-17-02.

Inhibitory mechanisms called checkpoints regulate progression of the cell cycle in the presence of DNA damage or when a previous cell-cycle event is not finished. In fission yeast exposed to ultraviolet light the G1-S transition is regulated by a novel checkpoint that depends on the Gcn2 kinase. The molecular mechanisms involved in checkpoint induction and maintenance are not known. Here we characterise the checkpoint further by exposing the cells to a variety of DNA-damaging agents. Exposure to methyl methane sulphonate and hydrogen peroxide induce phosphorylation of eIF2α, a known Gcn2 target, and an arrest in G1 phase. By contrast, exposure to psoralen plus long-wavelength ultraviolet light, inducing DNA adducts and crosslinks, or to ionizing radiation induce neither eIF2α phosphorylation nor a cell-cycle delay. We conclude that the G1-S checkpoint is not a general DNA-damage checkpoint, in contrast to the one operating at the G2-M transition. The tight correlation between eIF2α phosphorylation and the presence of a G1-phase delay suggests that eIF2α phosphorylation is required for checkpoint induction. The implications for checkpoint signalling are discussed.

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, Øystein Garred, Anne Fangberget, Marit M. Holmen, Ellen Schlichting, Helle K. Skjerven, Steinar Lundgren, Erik Wist, Bjørn 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

Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the 25 new ovarian tumour cell lines described here retain the genomic landscape, histopathology and molecular features of the original tumours. Furthermore, the molecular profile and drug response of these cell lines correlate with distinct groups of primary tumours with different outcomes. Thus, tumour cell lines derived using this methodology represent a significantly improved platform to study human tumour pathophysiology and response to therapy., Despite methodological improvements, it remains extremely difficult to routinely establish cell lines from human primary tumours. Here, the authors develop a culture medium that allows efficient cell line establishment and use this to generate 25 ovarian cancer cell lines.

Purpose: Neoangiogenesis is an important feature in tumor growth and progression, and combining chemotherapy and antiangiogenic drugs have shown clinical efficacy. However, as treatment-induced resistance often develops, our goal was to identify pathways indicating response and/or evolving resistance to treatment and inhibit these pathways to optimize the treatment strategies. Experimental Design: To identify markers of response and/or resistance, reverse-phase protein array (RPPA) was used to characterize treatment-induced changes in a bevacizumab-responsive and a nonresponsive human breast cancer xenograft. Results were combined with bioinformatic modeling to predict druggable targets for optimization of the treatment. Results: RPPA analysis showed that both tumor models responded to bevacizumab with an early (day 3) upregulation of growth factor receptors and downstream signaling pathways, with persistent mTOR signaling until the end of the in vivo experiment. Adding doxorubicin to bevacizumab showed significant and superior growth inhibition of basal-like tumors, whereas no additive effect was seen in the luminal-like model. The combination treatment corresponded to a continuous late attenuation of mTOR signaling in the basal-like model, whereas the inhibition was temporary in the luminal-like model. Integrating the bevacizumab-induced dynamic changes in protein levels with bioinformatic modeling predicted inhibition of phosphoinositide 3-kinase (PI3K) pathway to increase the efficacy of bevacizumab monotherapy. In vivo experiments combining bevacizumab and the PI3K/mTOR inhibitor BEZ235 confirmed their significant and additive growth-inhibitory effect in the basal-like model. Conclusions: Treatment with bevacizumab caused compensatory upregulation of several signaling pathways. Targeting such pathways increased the efficacy of antiangiogenic therapy. Clin Cancer Res; 20(2); 404–12. ©2013 AACR.

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. Møller, Silje Nord, David Wedge, Ole Christian Lngjærde, Laxmi Silwal-Pandit, Hedda vdL Gythfeld, Hans Kristian M. Vollan, Thomas Fleischer, Marit Krohn, Ellen Schlichting, Elin Borgen, Øystein Garred, Marit M. Holmen, Erik Wist, Bjørn Naume, Peter V. Loo, Anne-Lise Børresen-Dale, Olav Engebråten, 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.

Entry into S phase is carefully regulated and, in most organisms, under the control of a G 1 -S checkpoint. We have previously described a G 1 -S checkpoint in fission yeast that delays formation of the prereplicative complex at chromosomal replication origins after exposure to UV light (UVC). This checkpoint absolutely depends on the Gcn2 kinase. Here, we explore the signal for activation of the Gcn2-dependent G 1 -S checkpoint in fission yeast. If some form of DNA damage can activate the checkpoint, deficient DNA repair should affect the length of the checkpoint-induced delay. We find that the cell-cycle delay differs in repair-deficient mutants from that in wild-type cells. However, the duration of the delay depends not only on the repair capacity of the cells, but also on the nature of the repair deficiency. First, the delay is abolished in cells that are deficient in the early steps of repair. Second, the delay is prolonged in repair mutants that fail to complete repair after the incision stage. We conclude that the G 1 -S delay depends on damage to the DNA and that the activating signal derives not from the initial DNA damage, but from a repair intermediate(s). Surprisingly, we find that activation of Gcn2 does not depend on the processing of DNA damage and that activated Gcn2 alone is not sufficient to delay entry into S phase in UVC-irradiated cells. Thus, the G 1 -S delay depends on at least two different inputs.

Ultraviolet irradiation of fission yeast cells in G1 phase induced a delay in chromatin binding of replication initiation factors and, consistently, a transient delay in S-phase entry. The cell cycle delay was totally dependent on the Gcn2 kinase, a sensor of the nutritional status, and was accompanied by phosphorylation of the translation initiation factor eIF2α and by a general depression of translation. However, the G1-specific synthesis of factors required for DNA replication was not reduced by ultraviolet radiation. The cell cycle delay represents a novel checkpoint with a novel mechanism of action that is not activated by ionizing radiation.

2523 Background: The molecular characteristics of responding and non-responding breast cancers when treated with antiangiogenic therapy are largely unknown. Methods: To investigate molecular altera...

Antibody microarrays, two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (2-DE/MALDI-TOF-MS) were used to examine protein changes in 56 oral cancers (OCs)/normal controls (NCs) from Sudanese (41 OCs vs. 31 NCs) and Sri Lankan (15 OCs vs. 15 NCs) patients. Pools of extracted proteins were prepared and used for microarrays/2-DE/MALDI-TOF-MS. From 2-DE, protein spots (differentially-expressed) were cut and identified with peptide mass fingerprinting based on MALDI-TOF-MS, and the proteins were identified by submitting peptide mass profiles to the NCBInr database. By microarrays, 6 and 8 proteins demonstrated significant differences in their abundance values as differentially-expressed in OCs examined from Sudan and Sri Lanka, respectively. For some of the proteins found, like p56dok2 and NEK2, this is the first report in OCs. By MALDI-TOF-MS/2-DE, patterns of OCs/NCs were acquired and tumour-associated proteins, like psoriasin, calgranulin-B and glutathione transferase, were found to be altered in OCs compared to NCs. The proteins found in this work (by two different methods) represent a global protein change specific to OCs from two populations. This might indicates involvement of multiple pathways in the process of tumorgenesis; thus, multiple proteins should be simultaneously targeted in OCs. The finding of few common proteins might suggest involvement of different pathways, which may parallel differences in ethnicity and/or lifestyle.

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. Møller, Silje Nord, Hans Kristian Moen Vollan, Hedda von der Lippe Gythfeldt, Hege Edvardsen, Laxmi Silwal- Pandit, Marit Krohn, Thomas Fleischer, Ellen Schlitchting, Elin Borgen, Øystein Garred, Anne Fangberget, Marit M. Holmen, Helle Skjerven, Steinar Lundgren, Erik Wist, Bjørn Naume, Anne-Lise Børresen-Dale, Ole Christian Lingjærde, Olav Engebråten, 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

One of the challenging features of cancer is intratumoral heterogeneity (ITH). The existence of ITH can have profound consequences on tumor progression and drug sensitivity. However it is been essentially impossible to monitor the extent to which individual clonal populations contribute to and affect tumor behavior because of our inability to culture tumor cells from patients in vitro without loosing initial heterogeneity resulting from clonal selection under culture conditions. Through our collaboration with Dr. Tan Ince, we have access to cell culture medium (WIT-OC media) that allows primary tumor cells to undergo continuous population doublings without selecting for or against specific clones under culture conditions. By utilizing this media we have generated 80 single cell clones from a Clear cell Carcinoma (CCC) patient sample and monitored the extent of phenotypic and genomic heterogeneity among 12 of the clonal populations. These clones showed striking heterogeneity in copy number alterations as well as phenotypic heterogeneity in vitro as assessed by anoikis, growth in soft agar, doubling time, and growth in 3D reconstituted basement membrane cultures. We also examined correlations between in vitro properties and tumor formation in vivo using clones tagged with Gaussia-Luciferase (Gluc), a secreted luciferase that is secreted from cells and can be assayed in whole blood. We found that only three clones were transplantable by themselves; however, the clone which showed the most rapid expansion in the mouse was limited in it tumorigenic capacity, generating only ascitic tumor populations. To address whether clonal heterogeneity could contribute to tumor progression, we transplanted mixtures of clones and utilized barcoded clones to analyze the clonal composition of tumor masses. Interestingly, mixtures of the 12 clones resulted in the generation of more aggressive tumors, including the formation of solid tumors and metastases to lungs, liver and brain and allow in vivo growth; these metastatic growths contain distinct mixtures of individual clonal populations. Moreover, clones that could not initiate tumor formation individually could generate metastatic tumors, suggesting that there is cooperativity between the clones to enhance growth and induce metastasis. The study has provided evidence that cell lines generated from individual clones vary significantly in their functional activities in vitro and in vivo. Moreover, by studying the single cell clones, we have found evidence for the existence of interclonal crosstalk during tumor progression and are currently studying the nature and impact of such crosstalk on tumor progression and metastasis. Citation Format: Suha Naffar-Abu Amara, Laura Selfors, Marit Krohn, Tan Ince, Gordon Mills, Joan Brugge. Intratumoral cellular heterogeneity of epithelial ovarian carcinoma and its impact on tumor behavior. [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 2976. doi:10.1158/1538-7445.AM2014-2976

One of the most challenging aspects of cancer treatment is intratumoral heterogeneity (ITH). However it is been extremely difficult to monitor the extent to which individual clonal populations contribute to and affect tumor behavior because of the inability to culture tumor cells from patients in vitro without clonal selection under standard culture conditions, which lead to a loss in initial heterogeneity. Using a medium developed by Dr. Tan Ince that allows primary tumor cells to undergo continuous population doublings with minimal, if any, selection for or against specific clones, we generated 80 clonal populations from a Clear Cell Carcinoma (CCC) patient sample and monitored the extent of phenotypic and genomic heterogeneity among 12 of the clonal populations. These clones showed striking heterogeneity in copy number alterations as well as morphologic and phenotypic heterogeneity in vitro in assays such as anoikis, growth in soft agar, population doubling time, and growth in 3D reconstituted basement membrane cultures. To examine correlations between in vitro properties and tumor formation in vivo, we tagged the clones with Gaussia-Luciferase (Gluc), a secreted luciferase, which allowed us to monitor tumor burden over time using a blood-based assay. Based on tumor growth at 10 weeks, only two clonal populations were able to grow in the peritoneum when injected alone, one of which (#31) showed a much more rapid growth rate than the other; however #31 was still limited in its tumorigenic capacity, generating only ascitic tumor populations. To address whether functional crosstalk between clones affects tumor progression, we injected mixtures of the clones. Interestingly, mixtures of the 12 clones resulted in the generation of more aggressive in vivo phenotypes, e.g. the formation of solid peritoneal tumors and metastasis to lungs and brain, behaviors not detected with any individual clones. These results suggest that there is cooperativity between the clones to enhance growth and induce metastasis. The study has provided evidence that cell lines generated from individual clones vary significantly in their functional activities in vitro and in vivo. Moreover, by studying the single cell clones, we have found evidence for the existence of interclonal crosstalk during tumor progression and are currently studying the nature and impact of such cross talks on tumor progression and metastasis. This abstract is also presented as Poster A57. Citation Format: Suha Naffar-Abu Amara, Laura M. Selfors, Marit Krohn, Tan A. Ince, Bo R. Rueda, Rosemary Foster, Gordon B. Mills, Joan S. Brugge. Cellular heterogeneity of ovarian carcinoma cells and its impact on tumor behavior. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR09.