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The use of conventional methods (immunohistochemistry, pentaplex PCR) for detecting microsatellite instability (MSI), a predictive biomarker of immunotherapy efficacy, is debated for cancers with low MSI prevalence, such as breast cancer (BC). We developed two multiplex drop-off droplet digital PCR (ddPCR) assays targeting four microsatellites, initially identified from public BC whole-genome sequencing dataset. Performances of the assays were investigated and 352 tumor DNA and 28 circulating cell-free DNA from BC patients, with unknown MSI status were blindly screened. Cross-validation of ddPCR MSI status with other MSI detection methods was performed. We then monitored circulating tumor DNA (ctDNA) dynamics before and during pembrolizumab immunotherapy in one patient with MSI-high (MSI-H) metastatic BC. The assays showed high analytical specificity and sensitivity (limit of detection = 0.16%). Among N = 380 samples, seven (1.8%) were found as MSI-H by ddPCR with six of them confirmed by next-generation sequencing (NGS). Specificity was 100% in N = 133 microsatellite stable BC submitted to NGS. In the patient with MSI-H metastatic BC, ctDNA monitoring revealed an early decrease of microsatellite mutant allelic frequencies during immunotherapy. These results demonstrated MSI detection by ddPCR, a non-invasive, fast and cost-effective approach, allowing for large pre-screening of BC patients who may benefit from immunotherapy.

Transposable elements (TEs) are mobile parasitic sequences that have been repeatedly coopted during evolution to generate new functions and rewire gene regulatory networks. Yet, the contribution of active TEs to the creation of heritable mutations remains unknown. Using TE accumulation lines in Arabidopsis thaliana we show that once initiated, transposition produces an exponential spread of TE copies, which rapidly leads to high mutation rates. Most insertions occur near or within genes and targets differ between TE families. Furthermore, we uncover an essential role of the histone variant H2A.Z in the preferential integration of Ty1/copia retrotransposons within environmentally responsive genes and away from essential genes. We also show that epigenetic silencing of new Ty1/copia copies can affect their impact on major fitness-related traits, including flowering time. Our findings demonstrate that TEs are potent episodic (epi)mutagens that, thanks to marked chromatin tropisms, limit the mutation load and increase the potential for rapid adaptation., The contribution of transposable elements (TEs) to the creation of heritable mutations is unknown. Here the authors show in Arabidopsis that TEs accumulate exponentially once mobilized and that COPIA retrotransposons preferentially integrate in environmental response genes in a H2A.Z-dependent manner.

Transposable elements (TEs) are repetitive DNA sequences that have the ability to mobilize in the genome and create major effect mutations. Despite the importance of transposition as a source of genetic novelty, we still know little about the rate, landscape, and consequences of TE mobilization. This situation stems in large part from the repetitive nature of TEs, which complicates their analysis. Moreover, TE mobilization is typically rare and therefore new TE (i.e., non-reference) insertions tend to be missed in small-scale population studies. This chapter describes a TE-sequence capture approach designed to identify transposition events for most of the TE families that are potentially active in Arabidopsis thaliana. We show that our TE-sequence capture design provides an efficient means to detect with high sensitivity and specificity insertions that are present at a frequency as low as 1/1000 within a DNA sample.

International audience; Transposable elements (TEs) are repetitive DNA sequences that have the ability to mobilize in the genome and create major effect mutations. Despite the importance of transposition as a source of genetic novelty, we still know little about the rate, landscape, and consequences of TE mobilization. This situation stems in large part from the repetitive nature of TEs, which complicates their analysis. Moreover, TE mobilization is typically rare and therefore new TE (i.e., non-reference) insertions tend to be missed in small-scale population studies. This chapter describes a TE-sequence capture approach designed to identify transposition events for most of the TE families that are potentially active in Arabidopsis thaliana. We show that our TE-sequence capture design provides an efficient means to detect with high sensitivity and specificity insertions that are present at a frequency as low as 1/1000 within a DNA sample.

Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are two cancer-derived blood biomarkers that inform on patient prognosis and treatment efficacy in breast cancer. We prospectively evaluated the clinical validity of quantifying both CTCs (CellSearch) and ctDNA (targeted next-generation sequencing). Their combined value as prognostic and early monitoring markers was assessed in 198 HER2-negative metastatic breast cancer patients. All patients were included in the prospective multicenter UCBG study COMET (NCT01745757) and treated by first-line chemotherapy with weekly paclitaxel and bevacizumab. Blood samples were obtained at baseline and before the second cycle of chemotherapy. At baseline, CTCs and ctDNA were respectively detected in 72 and 74% of patients and were moderately correlated (Kendall’s τ = 0.3). Only 26 (13%) patients had neither detectable ctDNA nor CTCs. Variants were most frequently observed in TP53 and PIK3CA genes. KMT2C/MLL3 variants detected in ctDNA were significantly associated with a lower CTC count, while the opposite trend was seen with GATA3 alterations. Both CTC and ctDNA levels at baseline and after four weeks of treatment were correlated with survival. For progression-free and overall survival, the best multivariate prognostic model included tumor subtype (triple negative vs other), grade (grade 3 vs other), ctDNA variant allele frequency (VAF) at baseline (per 10% increase), and CTC count at four weeks (≥5CTC/7.5 mL). Overall, this study demonstrates that CTCs and ctDNA have nonoverlapping detection profiles and complementary prognostic values in metastatic breast cancer patients. A comprehensive liquid-biopsy approach may involve simultaneous detection of ctDNA and CTCs.

BackgroundMicrosatellite instability (MSI) has recently emerged as a predictive pan-tumor biomarker of immunotherapy efficacy, stimulating the development of diagnostic tools compatible with large-scale screening of patients. In this context, noninvasive detection of MSI from circulating tumor DNA stands as a promising diagnostic and posttreatment monitoring tool.MethodsWe developed drop-off droplet-digital PCR (ddPCR) assays targeting BAT-26, activin A receptor type 2A (ACVR2A), and defensin beta 105A/B (DEFB105A/B) microsatellite markers. Performances of the assays were measured on reconstitution experiments of various mutant allelic fractions, on 185 tumor samples with known MSI status, and on 72 blood samples collected from 42 patients with advanced colorectal or endometrial cancers before and/or during therapy.ResultsThe 3 ddPCR assays reached analytical sensitivity ConclusionsThis innovative approach for MSI detection provides a noninvasive, cost-effective, and fast diagnostic tool, well suited for large-scale screening of patients that may benefit from immunotherapy agents, as well as for monitoring treatment responses.

A comprehensive phenotypic analysis in five Arabidopsis accessions combined with methylome and transcriptome analyses in the Col-0 reference accession indicate plasticity to mild drought but no transgenerational effects or inherited stress memory., There is renewed interest in whether environmentally induced changes in phenotypes can be heritable. In plants, heritable trait variation can occur without DNA sequence mutations through epigenetic mechanisms involving DNA methylation. However, it remains unknown whether this alternative system of inheritance responds to environmental changes and if it can provide a rapid way for plants to generate adaptive heritable phenotypic variation. To assess potential transgenerational effects induced by the environment, we subjected four natural accessions of Arabidopsis thaliana together with the reference accession Col-0 to mild drought in a multi-generational experiment. As expected, plastic responses to drought were observed in each accession, as well as a number of intergenerational effects of the parental environments. However, after an intervening generation without stress, except for a very few trait-based parental effects, descendants of stressed and non-stressed plants were phenotypically indistinguishable irrespective of whether they were grown in control conditions or under water deficit. In addition, genome-wide analysis of DNA methylation and gene expression in Col-0 demonstrated that, while mild drought induced changes in the DNA methylome of exposed plants, these variants were not inherited. We conclude that mild drought stress does not induce transgenerational epigenetic effects.

Background: In metastatic breast cancer (MBC), monitoring circulating tumor DNA (ctDNA) can detect mutation associated with resistance to treatment and its variations reflect changes in tumor burden. We prospectively monitored CTC and ctDNA during first line chemotherapy for MBC. Methods: The French cohort COMET is a prospective study including first line HER2- negative pts receiving weekly paclitaxel and bevacizumab. Blood samples were obtained at baseline (BL) and before the second cycle of chemotherapy (C2). ctDNA was analyzed by targeted resequencing (custom panel 220kb) SNV (28 genes + 8 promoters and CNA (18 genes). Results: For ctDNA, out of 196 pts analyzed, 147 had at least one somatic mutation (SNV) detected in plasma (75%). Despite no complete overlap, 24 pts (12%) had no CTC nor ctDNA detected at baseline. Including Copy Number Variation analysis (CNV), the number of patients without detectable ctDNA at baseline could be reduced to 18 (9%). The average number of mutations per pt was 2.4 (range 1 to 9). Median Allelic Frequency (MAF) was 9.1%. Most commonly mutated genes were TP53 and GATA3 with 14% and 7% of all mutations, respectively. TP53 mutations were detected in 31 % of patients and were associated with a shorter progression-free and overall survival. PI3KCA mutations were detected in 23.2% of the pts, were correlated with presence of bone metastasis and had no prognostic value. ESR1 was mutated in 10.6% of the pts, restricted to the ER+ subgroup and had no impact on overall survival. At baseline, CTC and ctDNA levels were correlated (r = 0.40, p < 0.0001). CTC detection was correlated with TP53 mutations detection but not with mutations PI3KCA or ESR1. Only 68 pts (36%) had detectable ctDNA at C2 with a MAF of 2% (0.3%-40%). 52.8% of patients positive for ctDNA at D1C1 became negative at D1C2 and only one negative at baseline became positive at C2. Correlation rate with CTC was the same after one cycle of chemotherapy (r = 0.40). Median follow-up was 53 months and median OS was 32 months. At multivariate analysis, triple negative status, detectable ctDNA at C2, CTC ≥5 at C2 and grade 3 on primary tumor were independent prognostic for OS. Conclusion: Early decrease of CTC and/or ctDNA after one cycle of chemotherapy are independent predictive markers of favorable outcome. Compared to CTC, ctDNA allows monitoring of tumor burden during chemotherapy and specific detection of targetable mutations as PI3KCA, HER2 or BRCA. Citation Format: Jean-Yves Pierga, Amanda Silveira, Elodie Girard, Veronique Lorgis, Marie-Laure Tanguy, Benoit Albaud, Olivier Tredan, Coraline Dubot, Caroline Hego, William Jacot, Anthony Goncalves, Marc Debled, Christelle Levy, Jean-Marc Ferrero, Christelle Jouannaud, Marie-Ange Mouret-Reynier, Florence Dalenc, Sylvain Baulande, Jerome Lemonnier, Frederique Berger, Francois-Clement Bidard, Charlotte Proudhon. Predictive and prognostic value of circulating tumor DNA (ctDNA) compared to circulating tumor cells (CTC) in a prospective cohort of metastatic breast cancer patients: The UCBG COMET trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3390.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

SummaryWhether environmentally induced changes in phenotypes can be heritable is a topic with revived interest, in part because of observations in plants that heritable trait variation can occur without DNA sequence mutations. This other system of inheritance, called transgenerational epigenetics, typically involves differences in DNA methylation that are stable across multiple generations. However, it remains unknown if such a system responds to environmental changes and if it could therefore provide a rapid way for plants to generate adaptive heritable phenotypic variation. Here, we used a well-controlled phenotyping platform and whole-genome bisulfite sequencing to investigate potential heritable effects of mild drought applied over two successive generations in Arabidopsis thaliana. Plastic phenotypic responses were observed in plants exposed to drought. After an intervening generation without stress, descendants of stressed and non-stressed plants were phenotypically indistinguishable, except for very few trait-based parental effects, and irrespective of whether they were grown in control conditions or under water deficit. Moreover, while mild drought induced changes to the DNA methylome of exposed plants, DNA methylation variants were not inherited. These findings add to the growing body of evidence indicating that transgenerational epigenetics is not a common response of plants to environmental changes.

Background: Increased levels of CTC and a persistent elevated level after just one cycle of chemotherapy are very strong and independent markers of worse progression-free survival (PFS) and overall survival (OS) in patients (pts) with metastatic breast cancer (MBC) (Bidard et al, Lancet Oncol 2014). ctDNA can be used to detect mutation associated with resistance to treatment. It has also been shown that dynamic changes in ctDNA levels closely reflect changes in tumor burden. We prospectively monitored CTC and ctDNA early variations during first line chemotherapy for MBC. Patients & methods: The French cohort COMET is a prospective study including first line HER2 negative patients (pts) receiving weekly paclitaxel and bevacizumab according to EMA approved combination. The aim of this cohort is to evaluate clinical, biological and radiological parameters associated with pts outcome (CTC, serum markers, ctDNA, pharmacogenomic polymorphisms, metabolomic parameters, visceral fat, serum estradiol level and quality of life). We present here the first planned analysis on pts evaluated for CTC (CellSearch) and ctDNA using targeted sequencing (Roche SeqCap technology) of a panel of 46 genes and 8 promoters, using unique molecular identifiers to increase ctDNA detection sensitivity. Blood samples were obtained at baseline (BL) and before the second cycle of chemotherapy (C2). Results: From 09/2012 to 5/2014, 218 pts were included in this substudy. Median age was 55 years and 22% of pts had triple negative BC. At BL, 70% of pts had ≥1 detectable CTC per 7.5 ml of blood (median 4 CTC, range 1- 30,000) and 37% at C2. With a threshold of ≥5 CTC, 47% of pts were positive at BL and 22% at C2. For ctDNA, out of the first 141 pts analyzed, 105 had at least one somatic mutation detected in plasma (74%). The average number of mutations per pt was 2.7 and most commonly mutated genes were TP53 and PIK3CA. ESR1 was found mutated in 9% of all cases and restricted to the ER+ subgroup. Median Allelic Frequency was 10% (range 0.6-83%). Only 33% of pts had detectable ctDNA at C2. At BL, CTC and ctDNA levels were correlated (r=0.46, p Conclusion: This is the largest prospective cohort assessing the respective prognostic values of early CTC and ctDNA changes in homogenously treated first line MBC patients. Analysis of mutations profile variations and comparison with primary tumor and metastasis biopsies are ongoing and may reveal early mechanisms of resistance. Citation Format: Pierga J-Y, Silveira A, Lorgis V, Tanguy M-L, Tredan O, Dubot C, Jacot W, Goncalves A, Debled M, Levy C, Ferrero J-M, Jouannaud C, Luporsi E, Mouret-Reynier M-A, Dalenc F, Lemonnier J, Berger F, Proudon C, Bidard F-C. Circulating tumor DNA (ctDNA) and circulating tumor cells (CTC) predictive value in HER2 negative metastatic breast cancer patients treated with first line weekly paclitaxel and bevacizumab: Results of a prospective cohort from the French Breast Cancer InterGroup Unicancer (UCBG): COMET study [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-03.

3019 Background: Circulating Tumor Cells (CTC) are independent markers of progression-free survival (PFS) and overall survival (OS) in patients (pts) with metastatic breast cancer (MBC). Monitoring circulating tumor DNA (ctDNA) can detect mutation associated with resistance to treatment and its variations reflect changes in tumor burden. We prospectively monitored CTC, Circulating Endothelial Cells (CEC), serum markers and ctDNA during first line chemotherapy for MBC. Methods: The French cohort COMET is a prospective study including first line HER2 negative pts receiving weekly paclitaxel and bevacizumab . Blood samples were obtained at baseline (BL) and before the second cycle of chemotherapy (C2).We present here the final planned analysis. Results: From 09/2012 to 11/2014, 286 patients were included: 198 for ctDNA, 251 for CEC and 283 for CTC. Median age was 56 years and 23% of pts had triple negative BC. At baseline, 71% of pts had ≥1 detectable CTC per 7.5 ml of blood (median 4 CTC, range 1- 30,000). With a threshold of ≥5 CTC, 49% of pts were positive at baseline and 22% at C2. For ctDNA, out of the first 196 pts analyzed, 147 had at least one somatic mutation (SNV) detected in plasma (75%). The average number of mutations per pt was 2.4 (range 1 to 9). Most commonly mutated genes were TP53 and GATA3. ESR1 was mutated in 10.6% of the pts and restricted to the ER+ subgroup. PIK3CA was mutated in 23.2% of the pts. Median Allelic Frequency was 9.1% . Only 68 pts (36%) had detectable ctDNA at C2. At baseline, CTC and ctDNA levels were correlated (r = 0.40, p < 0.0001). Despite no complete overlap, 24 pts (12%) had no CTC nor ctDNA detected at baseline. Median follow-up was 53 months and median OS was 32 months. Detectable CTC and ctDNA at baseline and at C2 were significantly associated with decreased PFS and OS. CEC and serum markers level had no prognostic value. At multivariate analysis, triple negative status, detectable ctDNA at C2, CTC ≥5 at C2 and grade 3 on primary tumor were independent prognostic factors. Conclusions: This is the largest prospective cohort assessing the respective prognostic values of early CTC and ctDNA changes in homogenously treated first line MBC pts. Early decrease of CTC and ctDNA after one cycle of chemotherapy are independent predictive markers of favorable outcome, with a stronger value for ctDNA compared to CTC. Clinical utility of early ctDNA variations monitoring and changes in mutation profile remain to be demonstrated. Clinical trial information: NCT01745757.

BACKGROUND Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. RESULTS The KRAS and EGFR assays were highly specific and both reached a limit of detection of CONCLUSIONS This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy.

Transposable elements (TEs) are powerful motors of genome evolution yet a comprehensive assessment of recent transposition activity at the species level is lacking for most organisms. Here, using genome sequencing data for 211 Arabidopsis thaliana accessions taken from across the globe, we identify thousands of recent transposition events involving half of the 326 TE families annotated in this plant species. We further show that the composition and activity of the 'mobilome' vary extensively between accessions in relation to climate and genetic factors. Moreover, TEs insert equally throughout the genome and are rapidly purged by natural selection from gene-rich regions because they frequently affect genes, in multiple ways. Remarkably, loci controlling adaptive responses to the environment are the most frequent transposition targets observed. These findings demonstrate the pervasive, species-wide impact that a rich mobilome can have and the importance of transposition as a recurrent generator of large-effect alleles. DOI: http://dx.doi.org/10.7554/eLife.15716.001

Background: Following the PALOMA-3 study results, the combination of palbociclib, a cdk4/6 inhibitor, with fulvestrant has become a standard therapy in women with estrogen receptor-positive (ER+) HER2-negative (HER2-) metastatic breast cancer (MBC) whose disease progressed under hormone therapy. Palbociclib increased the progression-free survival (PFS) in all patient subgroups, and no predictive biomarker of palbociclib efficacy has been validated so far. In that context, we evaluated whether early changes of circulating tumor DNA (ctDNA) levels are associated with the efficacy of palbociclib plus fulvestrant. Methods: After providing written informed consent, ER+ HER2- MBC patients were included in a prospective observational cohort (“ALCINA”, NCT02866149) prior to the initiation of palbociclib plus fulvestrant. Tumor response was assessed by a radiological evaluation (RECIST v1.1) every 3 months. Fresh plasma samples were collected at baseline, before the initiation of treatment, at day 15, at day 30 and at disease progression. For each patient, DNA from archived tumor tissue was subjected to targeted NGS to identify a driver mutation. In patients with identified driver mutation, circulating cell-free tumor DNA extracted from plasma was subjected to digital droplet PCR (ddPCR). Ratios of ctDNA levels (J15/baseline and J30/baseline) were correlated with prospectively registered patient characteristics and outcomes. For the current analysis, “responders” were defined as patients who experienced a PFS longer than 3 months. Results: 61 MBC patients have been included and 26 were eligible for ctDNA mutation detection, after the characterization of single nucleotide variations in PIK3CA (N=21 patients), TP53 (N=3) and AKT (N=2). At baseline, 21 patients (81%) had detectable ctDNA levels, with a median level of ctDNA of 381 copies/ml of plasma; There was no significant correlation between ctDNA levels at baseline and PFS (p=0,06). ctDNA levels decreased under therapy, with 82% and 68% of patients displaying detectable levels of ctDNA at day 15 and day 30. Patients with undetectable ctDNA levels at day 15 and, principally, at 30 were more likely to experience a PFS > 3 month (HR=5,8, 95% IC=1,5-22,5, p=0.004). We explored ctDNA level ratios (day 15/baseline and day 30/baseline) and found that all patients whom ctDNA increases at day 30 (31%) will not respond to treatment. Moreover, among all patients, ctDNA detection at day 30 is correlated with shorter PFS [HR=2,634 IC95 (1,1 to 6,5)]. At time of tumor progression, all patients presented increased ctDNA levels. In addition to tumor tissue sequencing, activating ESR1 mutations were found at baseline in 5 patients (19%). Conclusion: Our study suggests that the efficacy of palbociclib and fulvestrant may be monitored by serial analyses of ctDNA levels, before radiological evaluation and that early ctDNA levels and dynamics are prognostics factors for PFS. A large randomized trial, PADA-1 (NCT03079011), is currently testing the utility of real time resistant subclones detection in ctDNA from ER+ HER2- MBC treated with palbociclib and aromatase inhibitor. Citation Format: Darrigues L, Pierga J-Y, Bernard A, Bièche I, Silveira A, Michel M, Sablin M-P, Cottu P, Dubot C, Geiss R, Ricci F, Proudhon C, Bidard F-C. Circulating tumor DNA as a predictive biomarker of response to palbociclib-fulvestrant in patients with estrogen receptor-positive, HER2-negative metastatic breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-20.

Background: Microsatellite instability (MSI) is a hypermutator phenotype occurring in gastrointestinal, endometrial and colorectal tumors, and more rarely, in urinary tract, ovarian, breast, prostate, lung, head and neck, liver and glioblastoma tumors. The diagnosis of MSI phenotype has recently emerged as the first pan-tumor biomarker likely to predict clinical benefit from immune-checkpoint blockade therapy, making its precise identification primordial for treatment decisions and disease monitoring. Current diagnosis of MSI is performed by multiplex PCR of five microsatellite markers, followed by capillary electrophoresis to detect shifts in allele size. An important limitation of this method is its low sensitivity preventing its use when tumor content is below 10%. Methods: We designed ddPCR assays examining 3 microsatellites. Analytical performance was assessed in vitro. Clinical performance was evaluated in a series of FFPE and body fluid DNA samples obtained from patients with Stage IV colorectal or endometrial carcinomas, previously classified as MSI-H or MSS using the standard pentaplex PCR method. Mutant allele frequencies (MAF) quantified with the ddPCR MSI assay were also compared with the MAFs reported by ddPCR assays targeting specific BRAF or PIK3CA mutations. Results: The ddPCR assays for the 3 microsatellites were found to have high specificity and reached a limit of detection of Conclusion: This technique allows a cost-effective, sensitive and large scale screening of the MSI phenotype, as well as the follow-up of MSI patients using liquid biopsies. Citation Format: Marc-Henri Stern, Amanda B. Silveira, Ivan Bieche, Samia Melaabi, Luc Cabel, Bruno Buecher, Jean-Yves Pierga, Francois-Clement Bidard, Charlotte Proudhon. Detecting MSI phenotype in circulating blood DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4599.

The Arabidopsis group C bZIP transcriptional regulatory factors includes four members that are homologous to the maize Opaque-2 regulatory locus. These four Arabidopsis bZIP were organized into three groups of orthologues each of which possibly representing an ancestral angiosperm function. To better define the evolution of group C functions we initiated the characterization of AtbZIP9, a single Arabidopsis gene corresponding to one of the three group C ancestral functions and for which little functional information is available. Promoter fusion with GUS revealed that AtbZIP9 expression is restricted to the phloem of all organs analyzed and in situ hybridization confirmed this conclusion. AtbZIP9 mRNA accumulation was also shown to be repressed by glucose and induced by abscissic acid and cytokinin. Knockout T-DNA mutant or transgenic lines overexpressing AtbZIP9 mRNA were undistinguishable from the wild type indicating that post-transcriptional regulation and/or genetic redundancy act on AtbZIP9. To overcome the redundancy aspect, we produced transgenic plants expressing a fusion between AtbZIP9 cDNA and the VP16 transcriptional activator domain. These plants displayed leaf developmental defects and accumulation of phenolic compounds in the mesophyl. These alterations may be the consequence of changes in the phloem developmental process.

Biological diversity within species can be an important driver of population and ecosystem functioning. Until now, such within-species diversity effects have been attributed to underlying variation in DNA sequence. However, within-species differences, and thus potentially functional biodiversity, can also be created by epigenetic variation. Here, we show that epigenetic diversity increases the productivity and stability of plant populations. Epigenetically diverse populations of Arabidopsis thaliana produce up to 40% more biomass than epigenetically uniform populations. The positive epigenetic diversity effects are strongest when populations are grown together with competitors and infected with pathogens, and they seem to be partly driven by complementarity among epigenotypes. Our study has two implications: first, we may need to re-evaluate previous within-species diversity studies where some effects could reflect epigenetic diversity; second, we need to incorporate epigenetics into basic ecological research, by quantifying natural epigenetic diversity and testing for its ecological consequences across many different species.

Epigenetic variation, such as heritable changes of DNA methylation, can affect gene expression and thus phenotypes, but examples of natural epimutations are few and little is known about their stability and frequency in nature. Here, we report that the gene Qua-Quine Starch (QQS) of Arabidopsis thaliana, which is involved in starch metabolism and that originated de novo recently, is subject to frequent epigenetic variation in nature. Specifically, we show that expression of this gene varies considerably among natural accessions as well as within populations directly sampled from the wild, and we demonstrate that this variation correlates negatively with the DNA methylation level of repeated sequences located within the 5′end of the gene. Furthermore, we provide extensive evidence that DNA methylation and expression variants can be inherited for several generations and are not linked to DNA sequence changes. Taken together, these observations provide a first indication that de novo originated genes might be particularly prone to epigenetic variation in their initial stages of formation., Author Summary Epigenetics is defined as the study of heritable changes in gene expression that are not linked to changes in the DNA sequence. In plants, these heritable variations are often associated with differences in DNA methylation. So far, very little is known about the extent and stability of epigenetic variation in nature. In this study, we report a case of extensive epigenetic variation in natural populations of the flowering plant Arabidopsis thaliana, which concerns a gene involved in starch metabolism, named Qua-Quine Starch (QQS). We show that in the wild QQS expression varies extensively and concomitantly with DNA methylation of the gene promoter. We also demonstrate that these variations are independent of DNA sequence changes and are stably inherited for several generations. In view of the recent evolutionary origin of QQS, we speculate that genes that emerge from scratch could be particularly prone to epigenetic variation. This would in turn endow epigenetic variation with a unique adaptive role in enabling de novo originated genes to adjust their expression pattern.

Glucose modulates plant metabolism, growth, and development. In Arabidopsis (Arabidopsis thaliana), Hexokinase1 (HXK1) is a glucose sensor that may trigger abscisic acid (ABA) synthesis and sensitivity to mediate glucose-induced inhibition of seedling development. Here, we show that the intensity of short-term responses to glucose can vary with ABA activity. We report that the transient (2 h/4 h) repression by 2% glucose of AtbZIP63, a gene encoding a basic-leucine zipper (bZIP) transcription factor partially involved in the Snf1-related kinase KIN10-induced responses to energy limitation, is independent of HXK1 and is not mediated by changes in ABA levels. However, high-concentration (6%) glucose-mediated repression appears to be modulated by ABA, since full repression of AtbZIP63 requires a functional ABA biosynthetic pathway. Furthermore, the combination of glucose and ABA was able to trigger a synergistic repression of AtbZIP63 and its homologue AtbZIP3, revealing a shared regulatory feature consisting of the modulation of glucose sensitivity by ABA. The synergistic regulation of AtbZIP63 was not reproduced by an AtbZIP63 promoter-5′-untranslated region::β-glucuronidase fusion, thus suggesting possible posttranscriptional control. A transcriptional inhibition assay with cordycepin provided further evidence for the regulation of mRNA decay in response to glucose plus ABA. Overall, these results indicate that AtbZIP63 is an important node of the glucose-ABA interaction network. The mechanisms by which AtbZIP63 may participate in the fine-tuning of ABA-mediated abiotic stress responses according to sugar availability (i.e., energy status) are discussed.

Orientador: Michel Georges Albert Vincentz Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: O crescimento e o desenvolvimento dos organismos são baseados na capacidade celular de expressão gênica diferencial que resulta, principalmente, do controle da taxa de iniciação da transcrição por fatores reguladores de transcrição (FTs). FTs do tipo Basic Leucine QQjJer(bZIP) fQram descritos em todos os eucariotos. Seu domínio conservado é constituído de uma região de ligação ao DNA rica em aminoácidos básicos, flanqueada a um zíper de leucinas responsável pela dimerização. Em angiospermas, os bZIPs são reguladores importantes de processos específicos como fotomorfogênese, desenvolvimento de órgãos, elongação celular, controle do balanço de carbono/nitrogênio, mecanismos de defesa, via de sinalização de hormônios e sacarose, controle osmótico e florescimento. Mostramos que os genomas de Arabidopsis thaliana e Orysa sativa codificam para um conjunto completo e não redundante de 76 e 113 fatores bZIP respectivamente, que foram organizados em 11 grupos de proteínas evolutivamente relacionadas e 33 Possíveis Grupos de Genes Ortólogos (PoGO) de mono e eudicotiledôneas, o que deve permitir racionalizar o processo de caracterização funcional destes fatores em angiospermas. O Grupo C, que inclui genes homólogos ao lócus de regulação Opaco-2 (02) de milho, está organizado em três PoGOS, que possivelmente desempenham três funções ancestrais de angiospermas. Em Arabidopsis estas três possíveis funções ancestrais estão representadas por quatro genes (bZIP' 02 !1omologous, Bzo2h), Bzo2h3/AtbZIP63 (PoGO C1), Bzo2h1/AtbZIP10 e Bzo2h4/ AtbZIP25 (PoGO C2) e Bzo2h2/AtbZIP9 (PoGO C3). Visando um melhor conhecimento sobre a evolução das funções dos fatores bZIP de angiospermas do Grupo C, iniciamos a caracterização funcional de~tes quatro reguladores, focando principalmente em AtbZIP9, um gene único representativo de uma função ancestral de angiospermas e cujo papel ainda é desconhecido. Notamos que a expressão de AtbZIP9 é restrita as células do floema e regulada por glicose, ácido abscísico e citocinina, sugerindo que este gene integra as vias de sinalização destes sinais metabólicos e hormonais no floema. Abordagens de genética reversa como RNAi, knockout e superexpressão não permitiram elucidar de maneira clara a atuação de AtbZIP9 no ciclo de vida de Arabidopsis, indicando que mecanismos de regulação pós-transcricional e/ou redundância genética atuam sobre este gene. Visando dar continuidade e ampliar o estudo funcional de AtbZIP9, foram obtidas linhagens transgênicas de Arabidopsis expressando versões modificadas deste gene que codificam para proteínas ativadoras constitutivas fortes da transcrição. Estas novas versões de AtbZIP9 são teoricamente capazes de ativar de maneira constitutiva a expressão dos genes alvos de AtbZIP9, contornando assim, as dificuldades decorrentes da análise de famílias gênicas que apresentam redundância funcional. Quando comparados a plantas selvagens, transformantes primários para ativadores constitutivos fortes apresentaram diversas alterações de morfologia foliar, além de mudanças metabólicas e fisiológicas como acúmulo de compostos fenólicos em folhas, sintomas de morte celular e senescência. A análise destes transformantes ainda sugere uma possível participação de AtbZIP9 no controle do desenvolvimento do sistema vascular de raízes e folhas. Suspeitamos que as alterações de morfologia foliar e fisiologia observadas possivelmente representem conseqüências de mudanças nas propriedades funcionais de transporte do floema, decorrentes de defeitos no processo de diferenciação e organização das células do cilindro vascular Abstract: Transcriptional regulatory factors (TFs) play an important role in controlling growth and development of ali organisms. bZIPs TFs have been described in ali eukaryotes and are characterized by a basic aminoacid rich DNA binding domain and a leucine zipper, responsible for dimerization. bZIPs have been reported to act in several different plantspecific processes such as organ development, cell elongation, defense mechanism, hormones and sucrose signalization, light response, control of nitrogen/carbon balance, osmotic control and flowering. We showed that Arabidopsis thaliana and Orysa sativa genomes encode a complete and non-redundant set of 76 and 113 bZIP transcription factors, respectively, which were divided into 11 unique groups of homologous genes. More detailed phylogenetic analysis led to the identification of 33 Possible Groups of Monocot and Eudicot Orthologous Genes (PoGO), which allows rationalizing functional studies in angiosperms. Group C, which includes genes homologous to the maize Opaque-2 locus, is formed by three PoGOs, suggesting that this group represents three ancestral functions among angiosperms. In Arabidopsis these three possible ancestral functions may be represented by the bZIP Qpaque-2. homologous genes (Bzo2h), Bzo2h3/AtbZIP63 (PoGO C1), Bzo2h1/AtbZIP10 and Bzo2h4/AtbZIP25 (PoGO C2) and Bzo2h2/AtbZIP9 (PoGO C3). To get insight into the evolution pattern and function of Group C members, we have iniciated the functional characterization of the Bzo2h genes concentrating initially on AtbZIP9, a unique gene that represents an ancestral function and for which no functional informational is available. We showed that AtbZIP9 expression is restricted to phloem cells and regulated by glucose, abscisic acid and cytokinin, suggesting that this gene is an element of the signalization pathways of these metabolic and hormonal signals in the phloem. Reverse genetic approaches such as RNAi, knockout and superexpression failed to reveal the biological function of AtbZIP9 in Arabidopsis life cycle and suggested that post-transcriptional regulation and/or functional redundancy may act on AtbZIP9. In order to improve our Rnowledge on AtbZIP9 function, Arabidopsis transgenic lines expressing constitutive transcriptional activator versions of AtbZIP9 were obtained. Since such modified versions of AtbZIP9 are theoretically able to promote the superexpression of AtbZIP9 target genes, this strategy should be independent of functional redundancy. When compared to wild type plants, primary transformants for constitutive transcriptional activator versions of AtbZIP9 showed alterations of leaf morphology, as well as metabolic and physiologic modifications, such as phenolic compound accumulation in leaves, cell death and senescence symptoms. Analyses of this transformants also suggest that AtbZIP9 is possibly involved in the control of leaf and root vascular system development. We suspect that the alteration of leaf morphology and physiology observed in primary transformants possibly reflects consequences of changes in phloem transport functional properties, due to defects in vascular cylinder cell differentiation and organization Mestrado Genética Vegetal e Melhoramento Mestre em Genética e Biologia Molecular