Isoform-level gene signature improves prognostic stratification and accurately classifies glioblastoma subtypes.

Molecular stratification of tumors is essential for developing personalized therapies. Although patient stratification strategies have been successful; computational methods to accurately translate the gene-signature from high-throughput platform to a clinically adaptable low-dimensional platform are currently lacking. Here, we describe PIGExClass (platform-independent isoform-level gene-expression based classification-system), a novel computational approach to derive and then transfer gene-signatures from one analytical platform to another. We applied PIGExClass to design a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) based molecular-subtyping assay for glioblastoma multiforme (GBM), the most aggressive primary brain tumors. Unsupervised clustering of TCGA (the Cancer Genome Altas Consortium) GBM samples, based on isoform-level gene-expression profiles, recaptured the four known molecular subgroups but switched the subtype for 19% of the samples, resulting in significant (P = 0.0103) survival differences among the refined subgroups. PIGExClass derived four-class classifier, which requires only 121 transcript-variants, assigns GBM patients' molecular subtype with 92% accuracy. This classifier was translated to an RT-qPCR assay and validated in an independent cohort of 206 GBM samples. Our results demonstrate the efficacy of PIGExClass in the design of clinically adaptable molecular subtyping assay and have implications for developing robust diagnostic assays for cancer patient stratification

Intratumor genetic heterogeneity and clonal evolution to decode endometrial cancer progression

Analyzing different tumor regions by next generation sequencing allows the assessment of intratumor genetic heterogeneity (ITGH), a phenomenon that has been studied widely in some tumor types but has been less well explored in endometrial carcinoma (EC). In this study, we sought to characterize the spatial and temporal heterogeneity of 9 different ECs using whole-exome sequencing, and by performing targeted sequencing validation of the 42 primary tumor regions and 30 metastatic samples analyzed. In addition, copy number alterations of serous carcinomas were assessed by comparative genomic hybridization arrays. From the somatic mutations, identified by whole-exome sequencing, 532 were validated by targeted sequencing. Based on these data, the phylogenetic tree reconstructed for each case allowed us to establish the tumors’ evolution and correlate this to tumor progression, prognosis, and the presence of recurrent disease. Moreover, we studied the genetic landscape of an ambiguous EC and the molecular profile obtained was used to guide the selection of a potential personalized therapy for this patient, which was subsequently validated by preclinical testing in patient-derived xenograft models. Overall, our study reveals the impact of analyzing different tumor regions to decipher the ITGH in ECs, which could help make the best treatment decisionWe thank all those at the Translational Research Laboratory of the MD Anderson Cancer Center Madrid for their invaluable help with this study. Tissue samples were obtained with the support of the MD Anderson Foundation Biobank (recordnumber B.0000745, ISCIII National Biobank Record), the “Xarxa Catalana de Bancs de Tumors” and “Plataforma de Biobancos” ISCIII (PT13/0010/0014, B.000609). This study has been supported by the Spanish Ministry of Economy and Innovation (PID2019-104644RB-I00 (GMB), the Instituto de Salud Carlos III (ISCIII, CIBERONC, CB16/12/00295 - GMB-, CB16/12/00328 -EC, AGM- and CB16/12/00231 -XMG- [all partly supported by FEDER funds]) and by the AECC Scientific Foundation (FC_AECC PROYE19036MOR -GMB- and Coordinated groups 2018 -XMG, AGM, GMB-). SO is funded by an AECC-postdoctoral grant (2020). JSR-F and BW are funded in part by the Breast Cancer Research Foundation and in part by the NIH/ NCI P50 CA247749 01 grant. Research reported in this publication was supported in part by a Cancer Center Support Grant of the NIH/NCI (Grant No. P30CA008748; MSK). We thank the Eurofins Megalab laboratory for helping us to perform the analysis of DNA HPV detection

Intratumor genetic heterogeneity and clonal evolution to decode endometrial cancer progression

Endometrial cancer; Clonal evolution; MutationCáncer endometrial; Evolución clonal; MutaciónCàncer d'endometri; Evolució clonal; MutacióAnalyzing different tumor regions by next generation sequencing allows the assessment of intratumor genetic heterogeneity (ITGH), a phenomenon that has been studied widely in some tumor types but has been less well explored in endometrial carcinoma (EC). In this study, we sought to characterize the spatial and temporal heterogeneity of 9 different ECs using whole-exome sequencing, and by performing targeted sequencing validation of the 42 primary tumor regions and 30 metastatic samples analyzed. In addition, copy number alterations of serous carcinomas were assessed by comparative genomic hybridization arrays. From the somatic mutations, identified by whole-exome sequencing, 532 were validated by targeted sequencing. Based on these data, the phylogenetic tree reconstructed for each case allowed us to establish the tumors’ evolution and correlate this to tumor progression, prognosis, and the presence of recurrent disease. Moreover, we studied the genetic landscape of an ambiguous EC and the molecular profile obtained was used to guide the selection of a potential personalized therapy for this patient, which was subsequently validated by preclinical testing in patient-derived xenograft models. Overall, our study reveals the impact of analyzing different tumor regions to decipher the ITGH in ECs, which could help make the best treatment decision.We thank all those at the Translational Research Laboratory of the MD Anderson Cancer Center Madrid for their invaluable help with this study. Tissue samples were obtained with the support of the MD Anderson Foundation Biobank (record number B.0000745, ISCIII National Biobank Record), the “Xarxa Catalana de Bancs de Tumors” and “Plataforma de Biobancos” ISCIII (PT13/0010/0014, B.000609). This study has been supported by the Spanish Ministry of Economy and Innovation (PID2019-104644RB-I00 (GMB), the Instituto de Salud Carlos III (ISCIII, CIBERONC, CB16/12/00295 - GMB-, CB16/12/00328 -EC, AGM- and CB16/12/00231 -XMG- [all partly supported by FEDER funds]) and by the AECC Scientific Foundation (FC_AECC PROYE19036MOR -GMB- and Coordinated groups 2018 -XMG, AGM, GMB-). SO is funded by an AECC-postdoctoral grant (2020). JSR-F and BW are funded in part by the Breast Cancer Research Foundation and in part by the NIH/NCI P50 CA247749 01 grant. Research reported in this publication was supported in part by a Cancer Center Support Grant of the NIH/NCI (Grant No. P30CA008748; MSK). We thank the Eurofins Megalab laboratory for helping us to perform the analysis of DNA HPV detection

Genetic Analysis of Axillary Meristem Development in Arabidopsis: Roles of MIR164, CUC1, CUC2, CUC3 and LAS, and identification of novel regulators.

Aerial architecture and reproductive success in higher plants is determined by the formation of secondary axes of growth which are formed by axillary meristems initiated post-embryonically in the axils of leaves. Among the genetic modulators of axillary meristem fate in Arabidopsis is LATERAL SUPPRESSOR, a putative transcription factor belonging to the GRAS family, which specifically regulates the initiation of axillary meristems during the vegetative phase of development. The aim of this work was to study the mechanism of LAS function in the meristem and to identify new regulators of axillary meristem initiation in Arabidopsis. To study the spatio-temporal specification of its function, LAS was misexpressed from promoters of meristematic genes possessing adjoining or overlapping expression domains in the SAM. Analysis of STM::LAS, KNAT1::LAS and UFO::LAS transgenic plants in las-4 background revealed partial to complete complementation of the las-4 branching phenotype, but did not lead to the formation of ectopic meristems. These results imply a function for LAS in maintaining the meristematic potential in axillary cells which can later initiate axillary meristems upon activation by other developmental cues. A potential mechanism of LAS function in axillary meristems was investigated by GA spraying experiments and complementation analysis of LAS::GAI and LAS::GAI DELLA transgenic plants in las-4 mutant background. Preliminary results indicate a role for LAS as a regulator of GA signaling in axillary meristems. To identify new regulators of axillary meristem development, two approaches were employed. Firstly, an EMS mutagenesis screen was carried out to identify supperssors of the las-4 max1-1 phenotype. Characterisation of three suppressor of las-4 (sol) candidates, sol2, sol6 and sol7, revealed three novel loci that regulate axillary meristem development. sol2, sol6 and sol7 complemented the branching defect in las-4 max1-1 to different degrees and were found to be non-allelic to each other. Their phenotypes were dependent on the las-4 mutation. Molecular mapping of two of these loci is underway. Secondly, the NAC domain transcription factors CUP-SHAPED COTYLEDON1, CUC2 and CUC3, exhibiting a characteristic expression pattern in the axils of leaf primordia, were investigated for potential roles in the development of axillary meristems. Investigation of loss-of-function mutants of these genes revealed that cuc3-2 is impaired in axillary bud formation, and that the severity of this phenotype is day length dependent. Transcripts of the other two CUC genes, CUC1 and CUC2, are targeted for degradation by miR164. Overexpression of MIR164A or MIR164B in the cuc3-2 mutant caused an almost complete block in axillary bud development. Conversely, plants harbouring miR164-resistant alleles of CUC1 and CUC2 developed accessory buds in rosette and cauline leaf axils, revealing redundant functions of CUC1 and CUC2 in axillary meristem development. Development of accessory buds was also observed in mir164 mutants. Thus, the role of CUC genes and miR164 in regulation of axillary meristem development was unveiled in this study

SNiPlay: a web-based tool for detection, management and analysis of SNPs. Application to grapevine diversity projects

<p>Abstract</p> <p>Background</p> <p>High-throughput re-sequencing, new genotyping technologies and the availability of reference genomes allow the extensive characterization of Single Nucleotide Polymorphisms (SNPs) and insertion/deletion events (indels) in many plant species. The rapidly increasing amount of re-sequencing and genotyping data generated by large-scale genetic diversity projects requires the development of integrated bioinformatics tools able to efficiently manage, analyze, and combine these genetic data with genome structure and external data.</p> <p>Results</p> <p>In this context, we developed SNiPlay, a flexible, user-friendly and integrative web-based tool dedicated to polymorphism discovery and analysis. It integrates:</p> <p>1) a pipeline, freely accessible through the internet, combining existing softwares with new tools to detect SNPs and to compute different types of statistical indices and graphical layouts for SNP data. From standard sequence alignments, genotyping data or Sanger sequencing traces given as input, SNiPlay detects SNPs and indels events and outputs submission files for the design of Illumina's SNP chips. Subsequently, it sends sequences and genotyping data into a series of modules in charge of various processes: physical mapping to a reference genome, annotation (genomic position, intron/exon location, synonymous/non-synonymous substitutions), SNP frequency determination in user-defined groups, haplotype reconstruction and network, linkage disequilibrium evaluation, and diversity analysis (Pi, Watterson's Theta, Tajima's D).</p> <p>Furthermore, the pipeline allows the use of external data (such as phenotype, geographic origin, taxa, stratification) to define groups and compare statistical indices.</p> <p>2) a database storing polymorphisms, genotyping data and grapevine sequences released by public and private projects. It allows the user to retrieve SNPs using various filters (such as genomic position, missing data, polymorphism type, allele frequency), to compare SNP patterns between populations, and to export genotyping data or sequences in various formats.</p> <p>Conclusions</p> <p>Our experiments on grapevine genetic projects showed that SNiPlay allows geneticists to rapidly obtain advanced results in several key research areas of plant genetic diversity. Both the management and treatment of large amounts of SNP data are rendered considerably easier for end-users through automation and integration. Current developments are taking into account new advances in high-throughput technologies.</p> <p>SNiPlay is available at: <url>http://sniplay.cirad.fr/</url>.</p

W-Curve Alignments for HIV-1 Genomic Comparisons

The W-curve was originally developed as a graphical visualization technique for viewing DNA and RNA sequences. Its ability to render features of DNA also makes it suitable for computational studies. Its main advantage in this area is utilizing a single-pass algorithm for comparing the sequences. Avoiding recursion during sequence alignments offers advantages for speed and in-process resources. The graphical technique also allows for multiple models of comparison to be used depending on the nucleotide patterns embedded in similar whole genomic sequences. The W-curve approach allows us to compare large numbers of samples quickly.We are currently tuning the algorithm to accommodate quirks specific to HIV-1 genomic sequences so that it can be used to aid in diagnostic and vaccine efforts. Tracking the molecular evolution of the virus has been greatly hampered by gap associated problems predominantly embedded within the envelope gene of the virus. Gaps and hypermutation of the virus slow conventional string based alignments of the whole genome. This paper describes the W-curve algorithm itself, and how we have adapted it for comparison of similar HIV-1 genomes. A treebuilding method is developed with the W-curve that utilizes a novel Cylindrical Coordinate distance method and gap analysis method. HIV-1 C2-V5 env sequence regions from a Mother/Infant cohort study are used in the comparison.The output distance matrix and neighbor results produced by the W-curve are functionally equivalent to those from Clustal for C2-V5 sequences in the mother/infant pairs infected with CRF01_AE.Significant potential exists for utilizing this method in place of conventional string based alignment of HIV-1 genomes, such as Clustal X. With W-curve heuristic alignment, it may be possible to obtain clinically useful results in a short time-short enough to affect clinical choices for acute treatment. A description of the W-curve generation process, including a comparison technique of aligning extremes of the curves to effectively phase-shift them past the HIV-1 gap problem, is presented. Besides yielding similar neighbor-joining phenogram topologies, most Mother and Infant C2-V5 sequences in the cohort pairs geometrically map closest to each other, indicating that W-curve heuristics overcame any gap problem

Rapid identification of BCR/ABL1-like acute lymphoblastic leukaemia patients using a predictive statistical model based on quantitative real time-polymerase chain reaction: clinical, prognostic and therapeutic implications.

BCR/ABL1-like acute lymphoblastic leukaemia (ALL) is a subgroup of B-lineage acute lymphoblastic leukaemia that occurs within cases without recurrent molecular rearrangements. Gene expression profiling (GEP) can identify these cases but it is expensive and not widely available. Using GEP, we identified 10 genes specifically overexpressed by BCR/ABL1-like ALL cases and used their expression values - assessed by quantitative real time-polymerase chain reaction (Q-RT-PCR) in 26 BCR/ABL1-like and 26 non-BCR/ABL1-like cases to build a statistical "BCR/ABL1-like predictor", for the identification of BCR/ABL1-like cases. By screening 142 B-lineage ALL patients with the "BCR/ABL1-like predictor", we identified 28/142 BCR/ABL1-like patients (19·7%). Overall, BCR/ABL1-like cases were enriched in JAK/STAT mutations (P < 0·001), IKZF1 deletions (P < 0·001) and rearrangements involving cytokine receptors and tyrosine kinases (P = 0·001), thus corroborating the validity of the prediction. Clinically, the BCR/ABL1-like cases identified by the BCR/ABL1-like predictor achieved a lower rate of complete remission (P = 0·014) and a worse event-free survival (P = 0·0009) compared to non-BCR/ABL1-like ALL. Consistently, primary cells from BCR/ABL1-like cases responded in vitro to ponatinib. We propose a simple tool based on Q-RT-PCR and a statistical model that is capable of easily, quickly and reliably identifying BCR/ABL1-like ALL cases at diagnosis

In Silico Identification of MYB and bHLH Families Reveals Candidate Transcription Factors for Secondary Metabolic Pathways in Cannabis sativa L

Plant secondary metabolic pathways are finely regulated by the activity of transcription factors, among which members of the bHLH and MYB subfamilies play a main role. Cannabis sativa L. is a unique officinal plant species with over 600 synthesized phytochemicals having diverse scale-up industrial and pharmaceutical usage. Despite comprehensive knowledge of cannabinoids\u2019 metabolic pathways, very little is known about their regulation, while the literature on flavonoids\u2019 metabolic pathways is still scarce. In this study, we provide the first genome-wide analysis of bHLH and MYB families in C. sativa reference cultivar CBDRx and identification of candidate coding sequences for these transcription factors. Cannabis sativa bHLHs and MYBs were then classified into functional subfamilies through comparative phylogenetic analysis with A. thaliana transcription factors. Analyses of gene structure and motif distribution confirmed that CsbHLHs and CsMYBs belonging to the same evolutionary clade share common features at both gene and amino acidic level. Candidate regulatory genes for key metabolic pathways leading to flavonoid and cannabinoid synthesis in Cannabis were also retrieved. Furthermore, a candidate gene approach was used to identify structural enzyme-coding genes for flavonoid and cannabinoid synthesis. Taken as a whole, this work represents a valuable resource of candidate genes for further investigation of the C. sativa cannabinoid and flavonoid metabolic pathways for genomic studies and breeding programs