Molecular screening program to select molecular-based recommended therapies for metastatic cancer patients: analysis from the ProfiLER trial.

BACKGROUND: Antitumor activity of molecular-targeted agents is guided by the presence of documented genomic alteration in specific histological subtypes. We aim to explore the feasibility, efficacy and therapeutic impact of molecular profiling in routine setting. PATIENTS AND METHODS: This multicentric prospective study enrolled adult or pediatric patients with solid or hematological advanced cancer previously treated in advanced/metastatic setting and noneligible to curative treatment. Each molecular profile was established on tumor, relapse or biopsies, and reviewed by a molecular tumor board (MTB) to identify molecular-based recommended therapies (MBRT). The main outcome was to assess the incidence rate of genomic mutations in routine setting, across specific histological types. Secondary objectives included a description of patients with actionable alterations and for whom MBRT was initiated, and overall response rate. RESULTS: Four centers included 2579 patients from February 2013 to February 2017, and the MTB reviewed the molecular profiles achieved for 1980 (76.8%) patients. The most frequently altered genes were CDKN2A (N = 181, 7%), KRAS (N = 177, 7%), PIK3CA (N = 185, 7%), and CCND1 (N = 104, 4%). An MBRT was recommended for 699/2579 patients (27%), and only 163/2579 patients (6%) received at least one MBRT. Out of the 182 lines of MBRT initiated, 23 (13%) partial responses were observed. However, only 0.9% of the whole cohort experienced an objective response. CONCLUSION: An MBRT was provided for 27% of patients in our study, but only 6% of patients actually received matched therapy with an overall response rate of 0.9%. Molecular screening should not be used at present to guide decision-making in routine clinical practice outside of clinical trials.This trial is registered with ClinicalTrials.gov, number NCT01774409.

The immune microenvironment of HPV-negative oral squamous cell carcinoma from never-smokers and never-drinkers patients suggests higher clinical benefit of IDO1 and PD1/PD-L1 blockade.

BACKGROUND: Never-smokers and never-drinkers patients (NSND) suffering from oral squamous cell carcinoma (OSCC) are epidemiologically different from smokers drinkers (SD). We therefore hypothesized that they harbored distinct targetable molecular alterations. PATIENTS AND METHODS: Data from The Cancer Genome Atlas (TCGA) (discovery set), Gene Expression Omnibus and Centre Léon Bérard (CLB) (three validation sets) with available gene expression profiles of HPV-negative OSCC from NSND and SD were mined. Protein expression profiles and genomic alterations were also analyzed from TCGA, and a functional pathway enrichment analysis was carried out. Formalin-fixed paraffin-embedded samples from 44 OSCC including 20 NSND and 24 SD treated at CLB were retrospectively collected to perform targeted-sequencing of 2559 transcripts (HTG EdgeSeq system), and CD3, CD4, CD8, IDO1, and PD-L1 expression analyses by immunohistochemistry (IHC). Enrichment of a six-gene interferon-γ signature of clinical response to pembrozulimab (PD-1 inhibitor) was evaluated in each sample from all cohorts, using the single sample gene set enrichment analysis method. RESULTS: A total of 854 genes and 29 proteins were found to be differentially expressed between NSND and SD in TCGA. Functional pathway analysis highlighted an overall enrichment for immune-related pathways in OSCC from NSND, especially involving T-cell activation. Interferon-γ response and PD1 signaling were strongly enriched in NSND. IDO1 and PD-L1 were overexpressed and the score of response to pembrolizumab was higher in NSND than in SD, although the mutational load was lower in NSND. IHC analyses in the CLB cohort evidenced IDO1 and PD-L1 overexpression in tumor cells that was associated with a higher rate of tumor-infiltrating T-cells in NSND compared with SD. CONCLUSION: The main biological and actionable difference between OSCC from NSND and SD lies in the immune microenvironment, suggesting a higher clinical benefit of PD-L1 and IDO1 inhibition in OSCC from NSND.

EZH2 alterations in follicular lymphoma: biological and clinical correlations.

The histone methyltransferase EZH2 has an essential role in the development of follicular lymphoma (FL). Recurrent gain-of-function mutations in EZH2 have been described in 25% of FL patients and induce aberrant methylation of histone H3 lysine 27 (H3K27). We evaluated the role of EZH2 genomic gains in FL biology. Using RNA sequencing, Sanger sequencing and SNP-arrays, the mutation status, copy-number and gene-expression profiles of EZH2 were assessed in a cohort of 159 FL patients from the PRIMA trial. Immunohistochemical (IHC) EZH2 expression (n=55) and H3K27 methylation (n=63) profiles were also evaluated. In total, 37% of patients (59/159) harbored an alteration in the EZH2 gene (mutation n=46, gain n=23). Both types of alterations were associated with highly similar transcriptional changes, with increased proliferation programs. An H3K27me3/me2 IHC score fully distinguished mutated from wild-type samples, showing its applicability as surrogate for EZH2 mutation analysis. However, this score did not predict the presence of gains at the EZH2 locus. The presence of an EZH2 genetic alteration was an independent factor associated with a longer progression-free survival (hazard ratio 0.58, 95% confidence interval 0.36-0.93, P=0.025). We propose that the copy-number status of EZH2 should also be considered when evaluating patient stratification and selecting patients for EZH2 inhibitor-targeted therapies.

Translation in Bacillus subtilis: roles and trends of initiation and termination, insights from a genome analysis.

We analysed the Bacillus subtilis protein coding sequences termini, and compared it to other genomes. The analysis focused on signals, com-positional biases of nucleotides, oligonucleotides, codons and amino acids and mRNA secondary structure. AUG is the preferred start codon in all genomes, independent of their G+C content, and seems to induce less stable mRNA structures. However, it is not conserved between homologous genes neither is it preferred in highly expressed genes. In B.subtilis the ribosome binding site is very strong. We found that downstream boxes do not seem to exist either in Escherichia coli or in B.subtilis. UAA stop codon usage is correlated with the G+C content and is strongly selected in highly expressed genes. We found less stable mRNA structures at both termini, which we related to mRNA-ribosome and mRNA-release-factor interactions. This pattern seems to impose a peculiar A-rich nucleotide and codon usage bias in these regions. Finally the analysis of all proteins from B.subtilis revealed a similar amino acid bias near both termini of proteins consisting of over-representation of hydrophilic residues. This bias near the stop codon is partially release-factor specific.

The complete genome sequence of the murine respiratory pathogen Mycoplasma pulmonis.

Mycoplasma pulmonis is a wall-less eubacterium belonging to the Mollicutes (trivial name, mycoplasmas) and responsible for murine respiratory diseases. The genome of strain UAB CTIP is composed of a single circular 963 879 bp chromosome with a G + C content of 26.6 mol%, i.e. the lowest reported among bacteria, Ureaplasma urealyticum apart. This genome contains 782 putative coding sequences (CDSs) covering 91.4% of its length and a function could be assigned to 486 CDSs whilst 92 matched the gene sequences of hypothetical proteins, leaving 204 CDSs without significant database match. The genome contains a single set of rRNA genes and only 29 tRNAs genes. The replication origin oriC was localized by sequence analysis and by using the G + C skew method. Sequence polymorphisms within stretches of repeated nucleotides generate phase-variable protein antigens whilst a recombinase gene is likely to catalyse the site-specific DNA inversions in major M.pulmonis surface antigens. Furthermore, a hemolysin, secreted nucleases and a glyco-protease are predicted virulence factors. Surprisingly, several of the genes previously reported to be essential for a self-replicating minimal cell are missing in the M.pulmonis genome although this one is larger than the other mycoplasma genomes fully sequenced until now.

Searching genomes for sequences with the potential to form intrastrand triple helices.

The canonical double-helix form of DNA is thought to predominate both in dilute solution and in living cells. Sequence-dependent fluctuations in local DNA shape occur within the double helix. Besides these relatively modest variations in shape, more extreme and remarkable structures have been detected in which some bases become unpaired. Examples include unusual three-stranded structures such as H-DNA. Certain RNA and DNA strands can also fold onto themselves to form intrastrand triplexes. Although they have been extensively studied in vitro, it remains unknown whether nucleic acid triplexes play natural roles in cells. If natural nucleic acid triplexes were identified in cells, much could be learned by examining the formation, stabilization, and function of such structures. With these goals in mind, we adapted a pattern-recognition program to search genetic databases for a type of potential triplex structure whose presence in genomes has not been previously investigated. We term these sequences Potential Intrastrand Triplex (PIT) elements. The formation of an intrastrand triplex requires three consecutive sequence domains with appropriate symmetry along a single nucleic acid strand. It is remarkable that we discovered multiple copies of sequence elements with the potential to form one particular class of intrastrand triplexes in the fully sequenced genomes of several bacteria. We then focused on the characterization of the 25 copies of a particular approximately 37 nt PIT sequence detected in Escherichia coli. Through biochemical studies, we demonstrate that an isolated DNA strand from this family of E. coli PIT elements forms a stable intrastrand triplex at physiological temperature and pH in the presence of physiological concentrations of Mg(2+).

Analysis of a Bacillus subtilis genome fragment using a co-operative computer system prototype.

Analysis of the huge volume of data generated by large scale sequencing projects requires the construction of new, sophisticated computer systems. These systems should be able to manage the biological data as well as the results of their analysis. They should also help the user to choose the most appropriate methods, and to string them together in order to solve a global analysis task. In this paper we present the prototype of a software system providing an environment for the analysis of large-scale sequence data. As a first step toward this end, this environment has been put to the test within the Bacillus subtilis genome sequencing project. This system integrates both the descriptive knowledge of the entities involved (genes, regulatory signals and the like) and the methodological knowledge comprising an extensible set of analytical methods. A knowledge representation based on two existing object-oriented models is used to implement this integrated system. In addition, the present prototype provides a suitable user interface both for displaying simultaneously the results generated by several methods and for interacting with the objects. We present in this paper the analysis of a B. subtilis genome fragment, present in data libraries but not annotated. Annotation of the genes present in the fragment allowed us to combine the results of several methods used for predicting coding sequences, and to characterize it as comprising a cryptic phage, the skin element. Comparison between the annotation of the skin element and a standard region of the chromosome indicated that local features of the nucleotide sequence could discriminate between phage and non-phage DNA sequence.

Pairwise and multiple identification of three-dimensional common substructures in proteins.

In this paper, we present an algorithm to find three-dimensional substructures common to two or more molecules. The basic algorithm is devoted to pairwise structural comparison. Given two sets of atomic coordinates, it finds the largest subsets of atoms which are "similar" in the sense that all internal distances are approximately conserved. The basic idea of the algorithm is to recursively build subsets of increasing sizes, combining two sets of size k to build a set of size k + 1. The algorithm can be used "as is" for small molecules or local parts of proteins (about 30 atoms). When a high number of atoms is involved, we use a two step procedure. First we look for common "local" fragments by using the previous algorithm, and then we gather these fragments by using a Branch and Bound technique. We also extend the basic algorithm to perform multiple comparisons, by using one of the structures as a reference point (pivot) to which all other structures are compared. The solution is the largest subsets of atoms common to the pivot and at least q other structures. Although both algorithms are theoretically exponential in the number of atoms, experiments performed on biological data and using realistic parameters show that the solution is obtained within a few minutes. Finally, an application to the determination of the structural core of seven globins is presented.

Functional and evolutionary roles of long repeats in prokaryotes.

Most recently published complete bacterial genomes have revealed unexpectedly high numbers of long strict repeats. In this article we discuss the various functional and evolutionary roles of these repeats, focusing in particular on their role in terms of genome stability, gene transfer, and antigenic variation.

Implication of gene distribution in the bacterial chromosome for the bacterial cell factory.

As bacterial genome sequences accumulate, more and more pieces of data suggest that there is a significant correlation between the distribution of genes along the chromosome and the physical architecture of the cell, suggesting that the map of the cell is in the chromosome. Considering sequences and experimental data indicative of cell compartmentalisation, mRNA folding and turnover, as well as known structural features of protein and membrane complexes, we show that preliminary in silico analysis of whole genome sequences strongly substantiates this hypothesis. If there is a correlation between the genome sequence and the cell architecture, it must derive from some selection pressure in the organisms growing in the wild. As a consequence, the underlying constraints should be optimised in genetically modified organisms if one is to expect high product yields. Consequences in terms of gene expression for biotechnology are straightforward: knocking genes out and in genomes should not be randomly performed, but should follow the rules of chromosome organisation.

Saponins from Steganotaenia araliacea.

Six saponins have been isolated and identified from the leaves of Steganotaenia araliacea. They were identified as 3-O-[beta-D-galactopyranosyl(1----2)-(beta-D-galactopyranosyl (1----3))-beta-D-glucuronopyranosyl]-21-O-tigloyl and -21-O-angeloyl-R1-barrigenol, 3-O-[beta-D-glucopyranosyl(1----2)-(beta-D-xylopyranosyl (1----3))-beta-D-glucuronopyranosyl]-21-O-tigloyl and -21-O-angeloyl-R1-barrigenol, 3-O-[beta-D-glucopyranosyl(1----2)-(beta-D-glucopyranosyl-(1----3))-(alp ha-L- rhamnopyranosyl(1----4))-beta-D-glucopyranosyl] steganogenin and 3-O-[(beta-D-galactopyranosyl(1----2)-beta-D-glucuronopyranosyl]-2 8-O- beta-D-glucopyranosyl olean-12-ene-28-oic acid. Steganogenin is a new 17,22-seco-oleanolic acid derivative. The structures of the saponins were established by analysis of their 1H and 13C NMR spectra with the help of 2D-experiments and by Californium Plasma Desorption Mass Spectrometry.

Saponins from stem bark of Petersianthus macrocarpus.

Two bioactive saponins were isolated from the stem bark of Petersianthus macrocarpus. Their structures were elucidated by chemical degradations and by a combination of 2D NMR techniques and by Californium plasma desorption mass spectrometry. They are 3-O-([beta-D-galactopyranosyl (1-->2)][beta-D-galactopyranosyl (1-->3)]- beta-D-glucuronopyranosyl)-21-O-[3-(3-tigloyloxynilic acid)-4-tigloyloxy- alpha-L-arabinopyranosyl] barringtogenol C and 3-O-([beta-D-galactopyranosyl (1-->2)][beta-D-galactopyranosyl (1-->3)]-beta-D-glucuronopyranosyl)-28-O-alpha-L-rhamnopyranosyl barringtogenol C-21-O-benzoate. The absolute configuration of nilic acid was determined by partial synthesis. 3,3'-Dimethoxy ellagic acid and 3,3'-dimethoxy-4-O-beta-D- glucopyranosyl ellagic acid were also isolated.

Identification of the two cis-syn [2 + 2] cycloadducts resulting from the photoreaction of 3-carbethoxypsoralen with 2'-deoxycytidine and 2'-deoxyuridine.

Near-ultraviolet photolysis of 2'-deoxycytidine (dCyd) and 3-carbethoxypsoralen (3-CPs) in the dry state was found to generate two main stable photoadducts which were separated by thin-layer and high-performance liquid chromatography. Fast atom bombardment and plasma desorption mass spectrometry analyses suggested that the bound molecule to 3-CPs is dCyd. These two compounds were found to produce the corresponding 2'-deoxyuridine (dUrd) derivatives through a deamination process when left in aqueous solutions with a lifetime close to 24 h at 20 degrees C. The chemical structure of the deaminated photoadducts was confirmed by photochemical synthesis using dUrd as the substrate. UV and fluorescent measurements indicated that the furan moiety of 3-CPs is involved in the photobinding reaction. The cyclobutane type structure of the modified dUrd derivatives was established on the basis of its photoreversibility and detailed 1H NMR analysis. The cis-syn stereoconfiguration of the two photocycloadducts was inferred from coupling constant considerations and on the basis of the complete assignment of the cyclobutyl protons, requiring the synthesis of deuterated nucleosides at pyrimidine carbon C(6). Further confirmation of the diastereoisomeric relationship between the two cis-syn dUrd <54' 65'> 3-CPs was provided by circular dichroism measurements.

A new class of psoralen photoadducts to DNA components: isolation and characterization of 8-MOP adducts to the osidic moiety of 2'-deoxyadenosine.

The near-UV-induced photoreaction of the bifunctional 8-methoxypsoralen (8-MOP) with 2'-deoxyadenosine (dAdo) was investigated in the dry state. Four main monoadducts of 8-MOP to 2'-deoxyadenosine were separated by high performance liquid chromatography and subsequently characterized by soft ionization mass spectrometry (fast atom bombardment and plasma desorption mass spectrometries) and extensive 1H NMR analysis including nuclear Overhauser effect (NOE) measurements. These new types of furocoumarin-nucleic acid component which appear to be specific to 2'-deoxyadenosine were shown to result from recombination of the 3,4-dihydropyron-4-yl radical of 8-MOP with 2'-deoxyadenosyl radical either at the 1' or the 5' position.

Palingol: a declarative programming language to describe nucleic acids' secondary structures and to scan sequence database.

At the DNA/RNA level, biological signals are defined by a combination of spatial structures and sequence motifs. Until now, few attempts had been made in writing general purpose search programs that take into account both sequence and structure criteria. Indeed, the most successful structure scanning programs are usually dedicated to particular structures and are written using general purpose programming languages through a complex and time consuming process where the biological problem of defining the structure and the computer engineering problem of looking for it are intimately intertwined. In this paper, we describe a general representation of structures, suitable for database scanning, together with a programming language, Palingol, designed to manipulate it. Palingol has specific data types, corresponding to structural elements-basically helices-that can be arranged in any way to form a complex structure. As a consequence of the declarative approach used in Palingol, the user should only focus on 'what to search for' while the language engine takes care of 'how to look for it'. Therefore, it becomes simpler to write a scanning program and the structural constraints that define the required structure are more clearly identified.

'Multifrequency' location and clustering of sequence patterns from proteins.

In previous work, we have shown that a set of characteristics, defined as (code frequency) pairs, can be derived from a protein family by the use of a signal-processing method. This method enables the location and extraction of sequence patterns by taking into account each (code frequency) pair individually. In the present paper, we propose to extend this method in order to detect and visualize patterns by taking into account several pairs simultaneously. Two 'multifrequency' methods are described. The first one is based on a rewriting of the sequences with new symbols which summarize the frequency information. The second method is based on a clustering of the patterns associated with each pair. Both methods lead to the definition of significant consensus sequences. Some results obtained with calcium-binding proteins and serine proteases are also discussed.

A scale-independent signal processing method for sequence analysis.

In this paper, we present methods to detect and localize patterns in biologically related protein sequences (family). The patterns common to the sequences of the family are detected by using Fourier analysis. No previous scales (codes) are needed, they are actually produced as a result of the analysis procedure, together with the frequencies of the Fourier decompositions. Characteristic features of the family are thus expressed as (code-frequency) pairs. Various tools are proposed in order to localize the patterns, to compare the codes, and to evaluate the proximity of an arbitrary sequence to the investigated family. The general strategy is illustrated on a family composed of calcium-binding proteins.

Analysis of intrachromosomal duplications in yeast Saccharomyces cerevisiae: a possible model for their origin.

The complete genome of the yeast Saccharomyces cerevisiae was investigated for intrachromosomal duplications at the level of nucleotide sequences. The analysis was performed by looking for long approximate repeats (from 30 to 3,885 bp) present on each of the chromosomes. We show that direct and inverted repeats exhibit very different characteristics: the two copies of direct repeats are more similar and longer than those of inverted repeats. Furthermore, contrary to the inverted repeats, a large majority of direct repeats appear to be closely spaced. The distance (delta) between the two copies is generally smaller than 1 kb. Further analysis of these "close direct repeats" shows a negative correlation between delta and the percentage of identity between the two copies, and a positive correlation between delta and repeat length. Moreover, contrary to the other categories of repeats, close direct repeats are mostly located within coding sequences (CDSs). We propose two hypotheses in order to interpret these observations: first, the deletion/conversion rate is negatively correlated with delta; second, there exists an active duplication mechanism which continuously creates close direct repeats, the other intrachromosomal repeats being the result, by chromosomal rearrangements of these "primary repeats."

Finding flexible patterns in a text: an application to three-dimensional molecular matching.

Finding certain regularities in a text is an important problem in many areas, e.g. in the analysis of biological molecules such as nucleic acids or proteins. In the latter case, the text may be sequences of amino acids or a linear coding of three-dimensional structures, and the regularities then correspond to lexical or structural motifs common to two, or more, proteins. We first recall an earlier algorithm that found these regularities in a flexible way. Then we introduce a generalized version of this algorithm designed for the particular case of protein three-dimensional structures, since these structures present a few peculiarities that make them computationally harder to process. Finally, we give some applications of our new algorithm on concrete examples.