David Gentien

Chromatin regulators as a guide for cancer treatment choice

The limited capacity to predict a patients response to distinct chemotherapeutic agents is a major hurdle in cancer management. The efficiency of a large fraction of current cancer therapeutics (radio- and chemotherapies) is influenced by chromatin structure. Reciprocally, alterations in chromatin organization may affect resistance mechanisms. Here, we explore how the misexpression of chromatin regulators—factors involved in the establishment and maintenance of functional chromatin domains—can inform about the extent of docetaxel response. We exploit Affymetrix and NanoString gene expression data for a set of chromatin regulators generated from breast cancer patient-derived xenograft models and patient samples treated with docetaxel. Random Forest classification reveals specific panels of chromatin regulators, including key components of the SWI/SNF chromatin remodeler, which readily distinguish docetaxel high-responders and poor-responders. Further exploration of SWI/SNF components in the comprehensive NCI-60 dataset reveals that the expression inversely correlates with docetaxel sensitivity. Finally, we show that loss of the SWI/SNF subunit BRG1 (SMARCA4) in a model cell line leads to enhanced docetaxel sensitivity. Altogether, our findings point toward chromatin regulators as biomarkers for drug response as well as therapeutic targets to sensitize patients toward docetaxel and combat drug resistance. Mol Cancer Ther; 15(7); 1768–77. ©2016 AACR.

A gene-expression profiling score for prediction of outcome in patients with follicular lymphoma: a retrospective training and validation analysis in three international cohorts

Background Patients with follicular lymphoma (FL) have heterogeneous outcomes. Predictor models able to distinguish, at diagnosis, patients at high versus low risk of progression are still needed. Methods The primary objective of this study was to use gene-expression profiling data to build a signature predictive of outcome in patients treated in the rituximab era. In a retrospectively assembled training cohort of 134 pretreatment FL patients from the prospective randomized PRIMA trial, we developed an expression-based predictor of progression-free survival (PFS) that was further evaluated in FFPE samples obtained from three independent international cohorts, using NanoString technology. The validation cohorts comprised a distinct set of patients from the PRIMA trial (n=178), a cohort from the University of Iowa/Mayo Clinic Lymphoma SPORE (n=201) and the Hospital Clinic University of Barcelona (n=109). All tissue samples consisted of pretreatment diagnostic biopsies and were confirmed as FL grade 1-3a. The patients were all treated with regimens containing rituximab and chemotherapy, possibly followed by either rituximab maintenance or ibritumomab-tiuxetan consolidation. Findings The expression levels of 395 genes were associated with a risk of progression. Twenty-three genes reflecting both B-cell biology and tumor microenvironment were retained to build a predictive model, which identified a population at an increased risk of progression (p<0.0001). In a multivariate Cox model for PFS adjusted on rituximab maintenance treatment and FLIPI-1, this predictor was found to independently predict progression (adjusted hazard ratio (HR) of the high-risk compared to the low-risk group: 3.68; 95%CI: 2.19-6.17). The digital gene expression data met quality criteria for 460/488 (94%) FFPE samples of the validation cohorts. The predictor performances were confirmed in each of the individual validation cohorts (adjusted HR [95%CI] comparing high risk to low risk groups were respectively 2.57 [1.65-4.01], 2.12 [1.32-3.39] and 2.11 [1.01-4.41]). In the combined validation cohort, the median PFS values were 3.1 (95%CI: 2.4-2.8) and 10.8 (95%CI: 10.1-NR) years in the high- and low-risk groups, respectively. The risk of lymphoma progression at 2 years was twice as high in the high-risk group (38% (95%CI: 29-46) versus 19% (95%CI: 15-24)). In a multivariate analysis, the score predicted PFS independently of anti-CD20 maintenance treatment and of the FLIPI score (hazard ratio for the combined cohort, 2.30; 95%CI, 1.72-3.07). Interpretation We developed a robust 23-gene expression-based predictor of PFS, applicable to routinely available FFPE biopsies from FL patients at diagnosis. This score may allow individualizing therapy for patients with FL according to the patient risk category. Funding Roche Company, SIRIC Lyric, LYSARC, NIH and the Henry J. Predolin Foundation, Spanish Plan Nacional de Investigacion SAF2015-64885-R.

Novel combinatorial screening identifies neurotrophic factors for selective classes of motor neurons.

Significance Neurotrophic factors are endogenous survival factors for developing neurons during their programmed cell death, and represent therapeutic candidates in several neurodegenerative diseases. Studies in the developing spinal cord suggest that neurotrophic factors promote the survival of motor neurons in a combinatorial manner. To better understand this, we systematically assayed pairwise combinations of neurotrophic factors (NTFs) on highly standardized motor neuron cultures prepared by a unique FACS technique. Our data unravel potent additivity of three neurotrophic factors due to their specific survival effects on distinct classes of motor neurons innervating different targets. Further analyses are required to better understand combinatorial NTF effects in adulthood and to define optimized NTF combinations for degenerative motor neuron diseases. Numerous neurotrophic factors promote the survival of developing motor neurons but their combinatorial actions remain poorly understood; to address this, we here screened 66 combinations of 12 neurotrophic factors on pure, highly viable, and standardized embryonic mouse motor neurons isolated by a unique FACS technique. We demonstrate potent, strictly additive, survival effects of hepatocyte growth factor (HGF), ciliary neurotrophic factor (CNTF), and Artemin through specific activation of their receptor complexes in distinct subsets of lumbar motor neurons: HGF supports hindlimb motor neurons through c-Met; CNTF supports subsets of axial motor neurons through CNTFRα; and Artemin acts as the first survival factor for parasympathetic preganglionic motor neurons through GFRα3/Syndecan-3 activation. These data show that neurotrophic factors can selectively promote the survival of distinct classes of embryonic motor neurons. Similar studies on postnatal motor neurons may provide a conceptual framework for the combined therapeutic use of neurotrophic factors in degenerative motor neuron diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy, and spinobulbar muscular atrophy.

Nanobodies against surface biomarkers enable the analysis of tumor genetic heterogeneity in uveal melanoma patient‐derived xenografts

Monoclonal antibodies specific for biomarkers expressed on the surface of uveal melanoma (UM) cells would simplify the immune capture and genomic characterization of heterogeneous tumor cells originated from patient‐derived xenografts (PDXs). Antibodies against four independent tumor antigens were isolated by panning a nanobody synthetic library. Such antibodies enabled flow cytometry‐based sorting of distinct cell subpopulations from UM PDXs and to analyze their genomic features. The complexity and specificity of the biochemical and genomic biomarker combinations mirrored the UM tumor polyclonality. The data showed that MUC18 is highly and universally displayed on the surface of UM cells with different genetic background and consequently represents a reliable pan‐biomarker for their identification and purification. In contrast, the other three biomarkers were detected in very variable combinations in UM PDX cells. The availability of the identified nanobodies will be instrumental in developing clone‐specific drug evaluation and rational clinical strategies based on accurate genomic profiling.

Reliable subtype classification of diffuse large B-cell lymphoma samples from GELA LNH2003 trials using the Lymph2Cx gene expression assay

Diffuse large B-cell lymphoma (DLBCL) comprises two molecular subgroups originally defined according to gene expression profiling (GEP) of frozen tissue samples: the germinal center B-cell-like (GCB) and activated B-cell-like (ABC) lymphomas, which show major differences in essential biological processes. This classification has a strong prognostic impact and was predictive of response to specific therapies in early phase trials. There is a need for a test that can be used to stratify patients’ treatment in future clinical trials and that can be translated into a routine lab procedure as a companion diagnostic test when molecular hallmarks of DLBCL will be used to decide upfront therapy. The Lymphoma/Leukemia Molecular Profiling Project (LLMPP) described a digital gene expression-based assay using NanoString technology (Lymph2Cx) which can be performed using formalin fixed paraffin embedded (FFPE) tissue samples, and which shows a strong correlation with the original cellof-origin (COO) classification. In a retrospective series of DLBCL patients included in the Groupe dEtude des Lymphomes de lAdulte (GELA) clinical trials, we used FFPE tissue samples to compare the COO classification obtained with the Lymph2Cx assay to the gold standard classification, based on Wright’s predictor using Affymetrix data on matched frozen samples. Our findings indicate that this assay, performed in an independent series and a non-LLMPP laboratory, proved to be reliable to classify FFPE samples of a large series of DLBCL. This study was based on lymphoma tissue samples of patients who had been included in the GELA/LYSA sponsored LNH-2003 program, which enrolled more than 1500 patients into different clinical trials combining rituximab and chemotherapy, or who had been included in the GELA LNH01-5B trial (Online Supplementary Table S1). All the patients included in these trials had signed an informed consent to the use of their samples for research, in agreement with French regulations. The histological diagnosis of de novo DLBCL had been confirmed by a panel of expert hemato-pathologists. Gene expression profiling (GEP) data from frozen tissues were available for 221 patients. RNA expression had been analyzed with HGU133+2.0 Affymetrix GeneChip arrays. Raw feature normalization and quality check were handled using Bioconductor software (affy, affyQCReport, GCRMA), excluding multi-gene probesets (x). The Affymetrix COO classification into GCB, ABC or Unclassified was made according to Wright’s predictor. Primary mediastinal B-cell lymphomas (PMBL) were identified with hierarchical clustering (complete distance, Ward agglomeration) according to a previously published gene signature, excluding E2F2 probe sets (which did not fit the PMBL Lymphochip profile) and TCL1A probe sets which induced a strong classification bias. The genetic features of this series of cases, according to the COO, have been recently described. After careful review of the FFPE material, matched FFPE tumor samples were available for RNA extraction for 168 of these 221 patients. The 168 cases included: 64 ABC, 63 GCB, and 26 Unclassified DLBCL, as well as 15 cases identified as PMBL, diagnosed between March 2002 and April 2011. Total RNA were extracted from FFPE tumor samples with a fully automated method (Siemens Healthcare Diagnostics), using 1-3 10 μm tissue scrolls of 0.4±0.3 cm, 2-12 years after diagnosis. Fifty ng RNA were hybridized to the Lymph2Cx CodeSet, using high sensitivity setting and analyzed with the Nanostring nCounter Analyzer (Generation 2; maximum resolution: 555 FOV). We only used 30 ng RNA for 4 samples for which there was no more RNA available. Digital counts were used to calculate the normalization and linear predictor score (LPS), ABC likelihood and “raw” subgroup prediction. It has been recognized that lot-to-lot variability introduces bias between lots and that, in one study, this bias was reduced from 52 points to 26 points (on a scale of approximately 4000 units) when synthetic reference oligonucleotides were used to correct for hybridization differences. In the current study, the LPS was adjusted using the same method with counts from reference oligonucleotides on the new code set; this adjustment reduced the number of unclassified samples and was used in the analyses that follow. Despite heterogeneity in fixatives, the experiments were successful in 157 of 168 (93%) FFPE samples (Figure 1). For 11 cases, the normalization score was below the threshold (20) required for reproducible results, and the experiments were considered as failed. These 11 cases included 2 cases with low RNA amounts, 2 cases extracted from AFA (acid acetic/formol/alcohol) blocks, and 5 cases with unknown fixative. Still, it is noteworthy that successful analysis was achieved in tumor samples fixed in AFA (n=13), Bouin’s fixative (n= 4), or unspecified fixative (n=44) (Figure 1) and in 2 cases with low RNA amounts (30 ng), showing that the assay was robust even when different fixatives were used. In 5 cases, RNA was extracted twice or 3 times from the same block and the classification was concordant for each of these extractions. In 2 cases, the second RNA extraction shifted the classification from GCB to Unclassified. The histological control of these 2 samples showed that the paraffin block had been exhausted by the first extraction and no longer contained a significant amount of tumor cells. In one case, the first RNA extraction (with low RNA content)

The iron chelator deferasirox synergises with chemotherapy to treat triple-negative breast cancers: Iron deprivation for breast cancer treatment

To ensure their high proliferation rate, tumor cells have an iron metabolic disorder causing them to have increased iron needs, making them more susceptible to iron deprivation. This vulnerability could be a therapeutic target. In breast cancers, the development of new therapeutic approaches is urgently needed for patients with triple‐negative tumors, which frequently relapse after chemotherapy and suffer from a lack of targeted therapies. In this study, we demonstrated that deferasirox (DFX) synergises with standard chemotherapeutic agents such as doxorubicin, cisplatin and carboplatin to inhibit cell proliferation and induce apoptosis and autophagy in triple‐negative breast cancer (TNBC) cells. Moreover, the combination of DFX with doxorubicin and cyclophosphamide delayed recurrences in breast cancer patient‐derived xenografts without increasing the side‐effects of chemotherapies alone or altering the global iron storage of mice. Antitumor synergy of DFX and doxorubicin seems to involve downregulation of the phosphoinositide 3‐kinase and nuclear factor‐κB pathways. Iron deprivation in combination with chemotherapy could thus help to improve the effectiveness of chemotherapy in TNBC patients without increasing toxicity. Copyright

BRCA2 Loss-of-Function and High Sensitivity to Cisplatin-Based Chemotherapy in a Patient With a Pleomorphic Soft Tissue Sarcoma: Effect of Genomic Medicine

&NA; We report the case of a patient with a BRCA2 germline mutation who developed a localized pleomorphic soft tissue sarcoma of the leg with poor prognostic features. BRCA2 germline mutations were not previously reported to be associated with pleomorphic sarcoma. BRCA2 loss‐of‐heterozygosity was found in the tumor, resulting in a complete BRCA2 loss‐of‐function. BRCA2 deficiency is associated with sensitivity to cisplatin‐based chemotherapy in breast and ovarian cancer patients. We used a cisplatin‐based chemotherapy. A rapid major partial response was obtained, which allowed a curative and conservative surgical resection of the sarcoma followed by adjuvant irradiation. This case illustrates that sarcoma patients may present unexpected but targetable genetic abnormalities and that BRCA2 loss‐of‐function may be targetable in sarcoma as it is associated with enhanced sensitivity to cisplatin. Our observation emphasizes the input of genomic medicine in clinical practice, its importance for treatment decisions, and the overlap between constitutional and somatic genetics.

Genome-wide identification of genic and intergenic neuronal DNA regions bound by Tau protein under physiological and stress conditions

Abstract Tauopathies such as Alzheimers Disease (AD) are neurodegenerative disorders for which there is presently no cure. They are named after the abnormal oligomerization/aggregation of the neuronal microtubule-associated Tau protein. Besides its role as a microtubule-associated protein, a DNA-binding capacity and a nuclear localization for Tau protein has been described in neurons. While questioning the potential role of Tau-DNA binding in the development of tauopathies, we have carried out a large-scale analysis of the interaction of Tau protein with the neuronal genome under physiological and heat stress conditions using the ChIP-on-chip technique that combines Chromatin ImmunoPrecipitation (ChIP) with DNA microarray (chip). Our findings show that Tau protein specifically interacts with genic and intergenic DNA sequences of primary culture of neurons with a preference for DNA regions positioned beyond the ±5000 bp range from transcription start site. An AG-rich DNA motif was found recurrently present within Tau-interacting regions and 30% of Tau-interacting regions overlapped DNA sequences coding for lncRNAs. Neurological processes affected in AD were enriched among Tau-interacting regions with in vivo gene expression assays being indicative of a transcriptional repressor role for Tau protein, which was exacerbated in neurons displaying nuclear pathological oligomerized forms of Tau protein.

Abstract P5-09-07: Identification of resistance-specific gene expression signatures in a breast cancer patient-derived xenograft with acquired resistance to different endocrine therapies

Background :nnAcquired resistance to endocrine treatments (ET) occurs in more than 70% of cases of luminal breast cancer (LBC). We used patient derived xenografts (PDX) models of LBC to study molecular changes associated with acquired resistance to different ET modalities.nnMethods :nnA PDX model of LBC, established from an early stage BRCA2-mutated breast cancer, was treated with different ET (tamoxifen, fulvestrant, oophorectomy and letrozole) during several months. Tumors escaping to therapies were re-engrafted and maintained under therapy. ET-resistant and parental hormono-responders tumors were analyzed with immunohistochemistry (IHC), RT-PCR and Affymetrix Gene Expression Arrays. Hormono-resistant tumors were additionally studied for their in vivo response to ET, mTOR and PARP inhibitors.nnResults :nnFrom the initially ET sensitive HBCx22 xenograft model (Cottu, BCRT 2012) two resistant models were obtained respectively to tamoxifen (HBCx22-TamR) and to estrogen deprivation (HBCx22-OvaR). Unsupervised clustering of gene expression showed a clear cut separation between parental, TamR and OvaR tumors. Genes differentially expressed in TamR and OvaR tumors compared to parental HBCx22 were only partially overlapping. Up-Regulated genes in both TamR and OvaR tumors (n = 302) were involved in response to wounding, nucleotide metabolism, immune system, adhesion and cell growth. Biological Processes (BP) specifically deregulated in OvaR tumors (n = 380) included embryonic development, antigen presentation, amino acid and lipid metabolism. The top BP specifically regulated in TamR tumors (n = 1059) were response to estrogen and steroid hormones, TGF-b signaling, hypoxia, regulation of cell proliferation, with several strongly up-regulated genes of the histone clusters 1 and 3. Ingenuity Transcription Factor Analysis predicted activation of NFKB, SP1, AP-1 and JUN, and inhibition of ESR1. RT-PCR and IHC analyses confirmed the down regulation of ER controlled genes in the TamR tumors. Expression of ER co-regulators determined by RT-PCR showed that GREB1 was strongly reduced in TamR, while PBX1, GATA3 and FOXA1 were inhibited in OvaR. IHC analysis showed a loss of PTEN expression in HBCx22, with high levels of p-AKT and p-RPS6 in both parental and TamR and OvaR tumors. In vivo ET showed that the TamR xenograft was resistant to all modalities of ET, while OvaR was resistant to estrogen deprivation while retaining some sensitivity to tamoxifen and fulvestrant. Treatment with the mTOR inhibitor RAD001 arrested tumor growth but did not show any additive effect when combined to ET in TamR or OvaR tumors. Conversely, the combination of RAD001 with Olaparib was highly synergistic and induced complete tumor response in 70% of mice.nnConclusions :nnAccording to the therapeutic selection, tumors derived from a PDX model of ER+ breast cancer show specific resistance patterns and gene expression profiles including disruption in the ER transcriptional program. The analysis of additional resistant tumors established from a second ER+ PDX will be presented at the meeting.nnCitation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-07.