Molecular Pathology Unit

Saint-Jean-Brévelay, France

Molecular Pathology Unit

Saint-Jean-Brévelay, France
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Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.Leukemia advance online publication, 3 February 2017; doi:10.1038/leu.2016.394. © 2017 Macmillan Publishers Limited, part of Springer Nature.


PubMed | National Cancer Institute INCa, Hospital Charles Nicolle, Institute Bergonie, Nice University Hospital Center and 15 more.
Type: Journal Article | Journal: The Journal of molecular diagnostics : JMD | Year: 2016

Personalized medicine has gained increasing importance in clinical oncology, and several clinically important biomarkers are implemented in routine practice. In an effort to guarantee high quality of molecular testing in France, three subsequent external quality assessment rounds were organized at the initiative of the National Cancer Institute between 2012 and 2014. The schemes included clinically relevant biomarkers for metastatic colorectal (KRAS, NRAS, BRAF, PIK3CA, microsatellite instability) and non-small cell lung cancer (EGFR, KRAS, BRAF, PIK3CA, ERBB2), and they represent the first multigene/multicancer studies throughout Europe. In total, 56 laboratories coordinated by 28 regional molecular centers participated in the schemes. Laboratories received formalin-fixed, paraffin-embedded samples and were asked to use routine methods for molecular testing to predict patient response to targeted therapies. They were encouraged to return results within 14 calendar days after sample receipt. Both genotyping and reporting were evaluated separately. During the three external quality assessment rounds, mean genotype scores were all above the preset standard of 90% for all biomarkers. Participants were mainly challenged in case of rare insertions or deletions. Assessment of the written reports showed substantial progress between the external quality assessment schemes on multiple criteria. Several essential elements such as the clinical interpretation of test results and the reason for testing still require improvement by continued external quality assessment education.


A team of Massachusetts General Hospital (MGH) investigators has shown that a method they developed to improve the usefulness and precision of the most common form of the gene-editing tools CRISPR-Cas9 RNA-guided nucleases can be applied to Cas9 enzymes from other bacterial sources. In a paper receiving advance online publication in Nature Biotechnology, the team reports evolving a variant of SaCas9 - the Cas9 enzyme from the Streptococcus aureus bacteria - that recognizes a broader range of nucleotide sequences, allowing targeting of genomic sites previously inaccessible to CRISPR-Cas9 technology. "The development of Cas9 variants with a broader targeting range is particularly important for applications requiring precise targeting of genomic sequences," says Benjamin Kleinstiver, PhD, a research fellow in the MGH Molecular Pathology Unit and lead and co-corresponding author of the Nature Biotechnology paper. "In addition, the coding sequence of SaCas9 is 23 percent smaller than that of SpCas9 - the version derived from Streptococcus pyogenes - a size difference that makes SaCas9 advantageous for potential therapeutic applications requiring delivery by viruses." CRISPR-Cas9 nucleases are comprised of a short RNA molecule, 20 nucleotides of which match the target DNA sequence, and a Cas9 bacterial enzyme that cuts the DNA in the desired location. Along with the match between the RNA and DNA sequences, Cas9 needs to recognize an adjacent nucleotide sequence called a protospacer adjacent motif (PAM). In a previous study reported earlier this year in Nature, the MGH team described a genetic system that enabled them to rapidly evolve SpCas9 to recognize different PAM sequences. While the naturally occurring SpCas9 recognizes a PAM sequence of the form NGG - in which N signifies any nucleotide and G is a guanine molecule - the MGH team was able to evolve versions of SpCas9 that recognize a broader range of PAM sequences, essentially doubling the range of targetable sites. In their current study, the MGH team turned to SaCas9, which naturally requires the PAM sequence NNGRRT - in which R can be either adenine or guanine and T must be thymine - adjacent to its target DNA. Using an advanced form of the molecular evolution system described in the previous report, the MGH team succeeded in developing a variant they call KKH SaCas9 that recognizes PAM sequences with any nucleotide in the third position, increasing their targeting range two- to four-fold. The system was able to engineer these changes in PAM specificity without requiring advance knowledge of the precise structure of the SaCas9 enzyme, something that was unknown at the time this study was taking place. "We now have shown that our directed evolution approach can be used to modify the PAM specificity of SaCas9, greatly expanding the number of genomic sites that can be accessed by this important Cas9 nuclease," says J. Keith Joung, MD, PhD, associate chief of Research in the MGH Department of Pathology and co-corresponding author of the Nature Biotechnology paper. "The ability to precisely target sites is important for researchers interested in disrupting small genetic elements or performing studies involving DNA repair by homologous recombination, an exchange of nucleotides between DNA strands, which best can be accomplished when the DNA break is close to the site of interest. We believe that our directed evolution approach provides an important blueprint for altering the recognition properties of the wealth of Cas9 nucleases that exist in many bacteria." Joung is a professor of Pathology at Harvard Medical School Explore further: New genome-editing platform significantly increases accuracy of CRISPR-based systems More information: Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition, Nature Biotechnology, DOI: 10.1038/nbt.3404


"Our creation of a Cas9 variant that brings off-target effects to levels where we can no longer detect them, even with the most sensitive methods, provides a substantial advance for therapeutic applications in which you want to accurately hit your target without causing damage anywhere else in the genome," says J. Keith Joung, MD, PhD, associate chief for Research and the Jim and Ann Orr MGH Research Scholar in the MGH Department of Pathology, senior author of the Nature paper. "But its impact will also be incredibly important for research applications because off-target effects can potentially confound the results of any experiment. As a result, we envision that our high-fidelity variant will supplant the use of standard Cas9 for many research and therapeutic applications." Used to create targeted DNA breaks at which genetic changes can be introduced, CRISPR-Cas9 nucleases combine a bacterial DNA-cutting enzyme called Cas9 with a short guide RNA sequence that can bind to the target DNA sequence. While easier to use than previous gene-editing tools, CRISPR-Cas9 nucleases have a well-characterized and significant limitation. As described in 2013 studies led by Joung and others, CRISPR-Cas9 nucleases can induce off-target DNA breaks at sites that resemble the on-target sequence. Subsequent investigations by Joung's team and others have reduced but never completely and consistently eliminated these off-target effects. Joung and his colleagues hypothesized that reducing interactions between Cas9 and the target DNA might more completely eliminate off-target effects while still retaining the desired on-target interaction. The MGH team focused on the fact that certain portions of the Cas9 enzyme itself can interact with the backbone of the target DNA molecule. Pursuing an observation originally made by co-lead author Vikram Pattanayak, MD, PhD, of MGH Pathology, the team altered four of these Cas9-mediated contacts by replacing the long amino acid side-chains that bind to the DNA backbone with shorter ones unable to make those connections. "Our previous work suggested that Cas9 might bind to its intended target DNA site with more energy than it needs, enabling unwanted cleavage of imperfectly matched off-target sites," says Pattanayak. "We reasoned that, by making substitutions at these four positions, we could remove some of that energy to eliminate off-target effects while still retaining full on-target activities." Co-lead author Benjamin Kleinstiver, PhD, of the MGH Molecular Pathology Unit and Michelle Prew, a research technician in Joung's lab, then tested all 15 possible variants in which any combination of one, two, three or four of those amino acid side-chains were altered and found that one three-substitution and one four-substitution variant appeared to show the greatest promise in discriminating against mismatched target sites while retaining full on-target activities in human cells. The researchers then more fully characterized the four-substitution variant, which they called SpCas9-HF1 (Sp for the Streptococcus pyogenes bacteria, which is the source of this widely used Cas9, and HF for high-fidelity). They found that this variant induced on-target effects comparable to those observed with the original unaltered SpCas9 when used with more than 85 percent of 37 different guide RNAs they tested. Using GUIDE-Seq, a highly sensitive system Joung's lab developed in 2014 to detect off-target CRISPR-Cas9 effects across the genome, the team found that, while nucleases combining unaltered SpCas9 with seven different guide RNAs induced as many as 25 off-target mutations, use of SpCas9-HF1 produced no detectable off-target effects with six of those guide RNAs and only one off-target site with the seventh. These results were further confirmed using targeted deep-sequencing experiments. Joung's team also found that SpCas9-HF1 could reduce off-target effects when targeting atypical DNA sites characterized by repeat sequences of one or two nucleotides - sites that are typically subject to many off-target mutations. They developed additional derivatives of SpCas9-HF1 - called HF2, HF3 and HF4 - which could eliminate the few residual off-target effects that persisted with the HF1 variant and a small number of guide RNAs. "If SpCas9-HF1 using a certain guide RNA still produces a handful of off-target effects that are particularly difficult to eliminate, it may be possible to engineer new variants that get rid of even those effects," says Joung, who is a professor of Pathology at Harvard Medical School. The researchers also showed that SpCas9-HF1, like its naturally occurring counterpart, could be combined with other useful alterations that extend its utility. Previous work from the Joung lab published last summer in Nature had shown that introducing a series of amino acid substitutions could expand the targeting range of unaltered SpCas9. In the current study, the authors show that introducing these same alterations into SpCas9-HF1 also extended the targeting range of the high-fidelity variant. "These results show that these variants should be broadly useful to anyone currently using CRISPR-Cas9 technology," says Kleinstiver. "They can easily be used in place of wild-type SpCas9 and provide a highly effective method for reducing off-target mutations to undetectable levels." Explore further: New genome-editing platform significantly increases accuracy of CRISPR-based systems More information: Benjamin P. Kleinstiver et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects, Nature (2016). DOI: 10.1038/nature16526


Giannoni P.,University of Genoa | Cutrona G.,Molecular Pathology Unit | de Totero D.,Molecular Pathology Unit
Current Molecular Medicine | Year: 2017

Chronic lymphocytic leukemia (CLL), the most common leukemia among adults in the western world, is characterized by a progressive accumulation of relatively mature CD5+ B cells in peripheral blood, lymph nodes and bone marrow. Despite much recent advancement in therapy, CLL is still incurable. Lymph nodes and bone marrow represent sanctuary sites preserving leukemic cells from spontaneous or drug-induced apoptosis, and infiltration of leukemic cells in these districts correlates with clinical stages and prognosis. The central role played by the microenvironment in the disease has become increasingly clear. Different chemokines (CXCL12, CXCL13, CCL19, CCL21) may in fact participate in attracting CLL cells into bone marrow and lymph nodes, where various factors, such as IL-15 and CXCL12, enhance leukemic cells survival. Recently, we have suggested that hepatocyte growth factor (HGF), produced by microenvironmental stromal cells, can contribute to CLL pathogenesis. We have demonstrated that HGF exerts a double effect on CLL B cells through the interaction with its receptor c- MET; HGF, infact, protects CLL B cells, which are c-MET+, from apoptosis, and also polarizes mono/macrophages towards the M2 phenotype, thus facilitating the evasion of the CLL clone from immune control. This double effect appears mediated by the activation of two major signaling pathways: STAT3TYR705 and AKT. The aim of this review is to summarize data on HGF and c-MET expression in normal B cells and in B cell malignancies, with a particular emphasis on our results obtained in CLL. Altogether, the observations described here suggest that the HGF/c-MET axis may have a prominent role in malignancy progression further indicating novel potential therapeutic options aimed to block HGF-induced signaling pathways in B lymphoproliferative disorders. © 2017 Bentham Science Publishers.


De Cesare M.,Fondazione IRCCS Instituto Nazionale per Lo Studio e la Cura Dei Tumori | Lauricella C.,Molecular Pathology Unit | Marco Veronese S.,Molecular Pathology Unit | Cominetti D.,Fondazione IRCCS Instituto Nazionale per Lo Studio e la Cura Dei Tumori | And 5 more authors.
Clinical Cancer Research | Year: 2014

Purpose: Despite the frequent overexpression of epidermal growth factor receptor (EGFR) in squamous cell carcinoma (SCC), the efficacy of cetuximab alone is limited. Given the marked activity of namitecan, a hydrophilic camptothecin, against SCC models, the present study was performed to explore the efficacy of the cetuximab-namitecan combination in a panel of SCC models. Experimental Design: We examined the antiproliferative and antitumor activities of the cetuximab- namitecan combination in four SCC models characterized by a different EGFR gene copy number/EGFR protein level.Wealso assessed the effects of the combination on EGFR expression at bothmRNAand protein levels and investigated the molecular basis of the interaction between the two agents. Results: Cetuximab and namitecan exhibited synergistic effects, resulting in potentiation of cell growth inhibition and, most importantly, enhanced therapeutic efficacy, with high cure rates in three SCC models characterized by high EGFR gene copy number, without increasing toxicity. The synergistic antitumor effect was also observed with the cetuximab-irinotecan combination. At the molecular level, the two agents produced a cooperative effect resulting in complete downregulation of EGFR. Interestingly, when singly administered, the camptothecin was able to strongly decrease EGFR expression mainly by transcriptional inhibition. Conclusions: Our results (i) demonstrate a marked efficacy of the cetuximab-namitecan combination, which reflects a complete abrogation of EGFR expression as a critical determinant of the therapeutic improvement, in SCC preclinical models, and (ii) suggest EGFR gene copy number as a possible marker to be used for patient selection in the clinical setting. © 2014 American Association for Cancer Research.


PubMed | Hematology Unit, University of Genoa, University of Ferrara, IRCCS A.O.U. San Martino IST and 6 more.
Type: | Journal: Leukemia | Year: 2017

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, [del(13)(q14)] involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,cnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1 specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA based therapeutic strategy.Leukemia accepted article preview online, 05 January 2017. doi:10.1038/leu.2016.394.


PubMed | Molecular Pathology Unit, Fondazione IRCCS Instituto Nazionale dei Tumori and University of Milan
Type: | Journal: Journal of translational medicine | Year: 2016

Diffuse malignant peritoneal mesothelioma (DMPM) is a rare and locally aggressive disease. DMPM prognosis is dismal, mainly due to the lack of effective treatment options and the development of new therapeutic strategies is urgently needed. In this context, novel immunotherapy approaches can be explored in an attempt to improve DMPM patients survival.We tested the efficacy of CpG-oligodeoxynucleotides (CpG-ODN), synthetic DNA sequences recognized by Toll-like receptor 9 and able to induce innate/adaptive immune response, in two DMPM orthotopic xenografts (MesoII and STO), which properly recapitulate the dissemination pattern of the disease in the peritoneal cavity. Severe combined immunodeficiency mice carrying DMPM xenografts were treated at different stages of tumor development with i.p. delivered CpG-ODN1826 for 4 weeks. CpG-ODN1826-induced modulation in the composition of peritoneal immune infiltrate was assessed by flow cytometry.When administered to early-stage tumors (i.e., 4 days after i.p. DMPM cell injection in mice), the agent exhibited impressive efficacy against MesoII by completely inhibiting tumor take and ascites development (no evidence of tumor masses and ascites in 6/6 mice at necropsy), and also impaired STO tumor take and growth (4/6 tumor-free mice; i.p. tumor masses reduced by 94 % in the 2 remaining mice, P = 0.00005). Interestingly, when tested against late-stage STO tumors (i.e., 11 days after i.p. DMPM cell injection in mice), CpG-ODN1826 was still able to reduce the growth of i.p. tumor masses by 66 % (P = 0.0009). Peritoneal washings of tumor-bearing mice revealed a strong increase of macrophage infiltration together with a decrease in the presence of B-1 cells and a reduced IgM concentration after CpG-ODN1826 treatment.Our results indicate that locally administered CpG-ODN1826 is able to markedly affect the growth of both early- and late-stage DMPM orthotopic xenografts in the absence of severe side effects, and suggest a possible clinical role for the agent in the therapy of DMPM.


Cade L.,Massachusetts General Hospital | Reyon D.,Molecular Pathology Unit | Reyon D.,Massachusetts General Hospital | Reyon D.,Harvard University | And 16 more authors.
Nucleic Acids Research | Year: 2012

Transcription activator-like effector nucleases (TALENs) are powerful new research tools that enable targeted gene disruption in a wide variety of model organisms. Recent work has shown that TALENs can induce mutations in endogenous zebrafish genes, but to date only four genes have been altered, and larger-scale tests of the success rate, mutation efficiencies and germline transmission rates have not been described. Here, we constructed homodimeric TALENs to 10 different targets in various endogenous zebrafish genes and found that 7 nuclease pairs induced targeted indel mutations with high efficiencies ranging from 2 to 76%. We also tested obligate heterodimeric TALENs and found that these nucleases induce mutations with comparable or higher frequencies and have better toxicity profiles than their homodimeric counterparts. Importantly, mutations induced by both homodimeric and heterodimeric TALENs are passed efficiently through the germline, in some cases reaching 100% transmission. For one target gene sequence, we observed substantially reduced mutagenesis efficiency for a variant site bearing two mismatched nucleotides, raising the possibility that TALENs might be used to perform allele-specific gene disruption. Our results suggest that construction of one to two heterodimeric TALEN pairs for any given gene will, in most cases, enable researchers to rapidly generate knockout zebrafish. © 2012 The Author(s).

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