Absence Of Somatic Mutations Research Articles

Somatic mutations in FAT cadherin family members constitute an underrecognized subtype of colorectal adenocarcinoma with unique clinicopathologic features.

The FAT cadherin family members (FAT1, FAT2, FAT3 and FAT4) are conserved tumor suppressors that are recurrently mutated in several types of human cancers, including colorectal carcinoma (CRC). To characterize the clinicopathologic features of CRC patients with somatic mutations in FAT cadherin family members. We analyzed 526 CRC cases from The Cancer Genome Atlas PanCancer Atlas dataset. CRC samples were subclassified into 2 groups based on the presence or absence of somatic mutations in FAT1, FAT2, FAT3 and FAT4. Individual clinicopathological data were collected after digital slide review. Statistical analysis was performed using t tests and chi-square tests. This CRC study cohort had frequent mutations in the FAT1 (10.5%), FAT2 (11.2%), FAT3 (15.4%) and FAT4 (23.4%) genes. Two hundred CRC patients (38.0%) harbored somatic mutations in one or more of the FAT family genes and were grouped into the FAT mutated CRC subtype. The FAT-mutated CRC subtype was more commonly located on the right side of the colon (51.0%) than in the rest of the cohort (30.1%, P < 0.001). It showed favorable clinicopathologic features, including a lower rate of positive lymph nodes (pN1-2: 33.5% vs 46.4%, P = 0.005), a lower rate of metastasis to another site or organ (pM1: 7.5% vs 16.3%, P = 0.006), and a trend toward an early tumor stage (pT1-2: 25.0% vs 18.7%, P = 0.093). FAT somatic mutations were significantly enriched in microsatellite instability CRC (28.0% vs 2.1%, P < 0.001). However, FAT somatic mutations in microsatellite stable CRC demonstrated similar clinicopathologic behaviors, as well as a trend of a better disease-free survival rate (hazard ratio = 0.539; 95% confidence interval: 0.301-0.967; log-rank P = 0.073). FAT cadherin family genes are frequently mutated in CRC, and their mutation profile defines a subtype of CRC with favorable clinicopathologic characteristics.

Abstract 1692: Single-cell DNA sequencing from frozen endometrial tumors to address clonal evolution of somatic mutations

Abstract Background: Single-cell DNA sequencing with droplet microfluidics has enabled the characterization of genetic heterogeneity in hematologic malignancies and fresh solid tumors through the direct DNA sequencing of thousands of single cells from individual samples without the need of whole-genome amplification. The application of this technology in banked solid tumor samples has been rather limited to date, however. Here, we describe and validate a method to characterize clonal evolution of somatic mutations from frozen tumor specimens, utilizing endometrial carcinomas (ECs) as an example. Methods: Tissue cores were obtained from frozen ECs and the extracellular matrix was enzymatically digested. A subset of extracted nuclei were used for bulk DNA extraction and targeted sequencing. The remaining nuclei were encapsulated with cell lysis buffer, and the DNA was primed and barcoded with hydrogel beads containing 317 cancer gene amplicons using a Tapestri instrument (Mission Bio). Following droplet PCR, Illumina libraries were generated and sequenced on a NovaSeq6000. Sequencing reads were trimmed and mapped to the reference genome. Barcode sequences were error-corrected and cells were detected from barcodes based on number of reads assigned to each barcode and amplicon read completeness. Genotyping was performed using GATK and the allele drop-out (ADO) rate was calculated. Non-neoplastic cells were defined by the absence of somatic mutations. Allele-specific copy number (ASCN) profiles for each tumor cell were estimated by normalizing mean reads per cell per amplicon to reads of non-neoplastic cells. Results: We performed single-cell genotyping and ASCN profiling on two ECs with matched bulk sequencing data. For EC1, 5,004 tumor and 132 non-neoplastic cells were sequenced, for a 97% tumor purity. For EC2, 2,981 tumor and 165 non-neoplastic cells were obtained, for a 95% tumor purity. The average number of reads per amplicon per cell for EC1 and EC2 was 116 and 146 with an ADO rate of 6% and 8%, respectively. The data obtained from single-cell analysis were concordant with the matched bulk sequencing data in terms of clonal and subclonal mutations, and their respective cancer cell fraction. Single-cell sequencing further revealed the presence of loss of heterozygosity and emergence of minor subclones as late events in tumor evolution, which could not be inferred by bulk sequencing. In EC2, branch mutations displayed a spectrum consistent with microsatellite instability (MSI), reflecting its acquired MSI phenotype. Conclusions: Here we describe a robust method to perform single-cell targeted DNA sequencing with droplet microfluidics of individual nuclei isolated from frozen tissue samples, providing the opportunity to determine the repertoire of somatic mutations at single cell resolution and to infer cancer clonal architecture and dynamics utilizing banked solid cancer tissue samples. Citation Format: Arnaud Da Cruz Paula, Yingjie Zhu, Shirin Issa Bhaloo, Fresia Pareja, Timothy Hoang, Pier Selenica, Etta Hanlon, Thais Basili, Higinio Dopeso, Nadeem Abu-Rustum, Jorge Sergio Reis-Filho, Britta Weigelt. Single-cell DNA sequencing from frozen endometrial tumors to address clonal evolution of somatic mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1692.

Clonal Hematopoiesis in Erdheim-Chester Disease

Rationale: In patients with Erdheim-Chester disease (ECD), accumulation of foamy histiocytes leads to multi-systemic disease with various organs involvement. During the past few years, we showed that Langerhans-cell histiocytosis (LCH) and other hemopathies were frequently associated with ECD. These findings, as well as the fact that BRAFV600Emutation is found in as much as 70% of ECD tissues, led to the reclassification of ECD as a myeloid neoplasm.Methods: We conducted a systematic next-generation sequencing (NGS) using bone marrow samples obtained from 69 patients with ECD (49 males, median age at the time of NGS 62.68 years SD 13.33, median time of evolution since ECD diagnosis 4.2 years). We performed molecular analysis using high-throughput sequencing focused on 36 genes recurrently mutated in myeloid malignancies. Libraries were prepared using the Ampliseq System according to the manufacter's instructions and run on Ion Proton (Thermofisher). Raw data were analyzed with both Torrent Browser (Thermofisher) and SeqNext (JSI Medical System).Results: Overall, 75 somatic mutations other than BRAFV600E in myeloid cancer candidate genes were present in 29 patients (42%), in a limited number of genes. Seventeen of these 29 patients (59%) had multiple mutations (range: 2-7 mutations). The most frequently mutated genes were TET2 , ASXL1 , DNMT3A and NRAS (in 16, 7, 5 and 4 patients respectively). CBL , KRAS , JAK2 and SRSF2 were mutated in 3 patients each. These genes accounted for 77% of all mutation-positive patients. Both the presence and the number of mutations per patients positively correlated with age (p=0.0055 and p=0.0069 respectively). Some patients harbored multiple mutations in the same genes, including TET2 , ASXL1 and NRAS . Six out of 41 patients (15%) carried karyotypic abnormalities. Twelve patients (17%) had a definite myeloproliferative neoplasm other than ECD (mainly CMML in 7 patients). Additionally, BRAFV600E was present in tissues histiocytes of 40/61 patients (not determined in 8 others) and in 13/60 patients in bone marrow (not determined in 9), some of the latter being treated with BRAF inhibitors. Two patients had a BRAFV600E mutation detectable in bone marrow but not in tissues. The presence of at least one TET2 mutation was associated with the presence of the BRAFV600E mutation in tissues (p=0.04). Among the 40 patients with the absence of somatic mutation in the panel of myeloid genes, only 17 were BRAFV600E Wild-Type and therefore showed no evidence of clonal hematopoiesis. These patients were characterized by less associated LCH (0 versus 23%; p=0.03), less vascular (41 versus 73%; p=0.02) and less cardiac (29 versus 69%; p=0.005) involvements.Conclusions: A subset of genes that are mutated in patients with myeloid neoplams is frequently mutated in ECD. These mutations may represent early events in the development of hematologic malignancies. DisclosuresEmile:Merck Serono: Honoraria.

ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile

AbstractThe presence or absence of somatic mutations in the expressed immunoglobulin heavy chain variable regions (IgVH) of chronic lymphocytic leukemia (CLL) cells provides prognostic information. Patients whose leukemic cells express unmutated IgVH regions (Ig-unmutated CLL) often have progressive disease, whereas patients whose leukemic cells express mutated IgVH regions (Ig-mutated CLL) more often have an indolent disease. Given the difficulty in performing IgVH sequencing in a routine diagnostic laboratory, this prognostic distinction is currently unavailable to most patients. Pilot gene expression profiling studies in patients with CLL identified genes that were differentially expressed between the Ig-unmutated and Ig-mutated CLL subtypes. Here, we have profiled an expanded cohort of 107 patients and show that ZAP-70 is the gene that best distinguishes the CLL subtypes. Ig-unmutated CLL expressed ZAP-70 5.54-fold more highly than Ig-mutated CLL (P < 10-21). ZAP-70 expression correctly predicted IgVH mutation status in 93% of patients. ZAP-70 expression and IgVH mutation status were comparable in their ability to predict time to treatment requirement following diagnosis. In 7 patients, ZAP-70 expression and IgVH mutation status were discordant: 4 Ig-mutated CLLs had high ZAP-70 expression and 3 Ig-unmutated CLLs had low ZAP-70 expression. Among these ZAP-70 “outliers,” those with Ig-mutated CLL had clinical features that are uncharacteristic of this CLL subtype: 2 required early treatment and 2 used a mutated VH3-21 gene, an IgVH gene that has been associated with progressive disease. We developed reverse transcriptase–polymerase chain reaction and immunohistochemical assays for ZAP-70 expression that can be applied clinically and would yield important prognostic information for patients with CLL.

Diagnosis and prognosis are supported by integrated assessment of next-generation sequencing in chronic myeloid malignancies. A real-life study.

Next-generation sequencing (NGS) is used to investigate the presence of somatic mutations. The utility of incorporating routine sequencing to guide diagnosis and therapeutic decisions remains unclear. We report the findings of an observational, multicenter study that aimed to assess the impact of somatic mutation testing by NGS in a reallife setting of chronic myeloid malignancies. A total of 177 patients were enrolled, partitioned into two overlapping groups. In group A (n=94), the indication was to search for clonal hematopoiesis, in a context of suspected myelodysplastic syndrome or myeloproliferative neoplasia. In group B (n=95), the theranostic impact of somatic mutations was studied. A panel of 34 genes was used on DNA extracted from blood or bone marrow samples. Within group A, the detection of clonal hematopoiesis supported the diagnosis of chronic myeloid malignancies for 31 patients while the absence of clonal hematopoiesis ruled out the suspected diagnosis in 47 patients. Within group B, NGS identified prognostically relevant somatic mutations in 32 patients, which had a therapeutic impact in 18 cases. By determining the presence or absence of somatic mutations, the application of NGS in daily practice was found to be useful for an integrated final diagnosis in 83% of the patients. Moreover, the search for somatic mutations had a prognostic impact that led to treatment modification in 19% of the cases. This study outlines the fact that adequate implementation of new investigations may have a significant positive medico-economic impact by enabling appropriate management of patients.

Identification and clinical impact of potentially actionable somatic oncogenic mutations in solid tumor samples

BackgroundAn increasing number of anti-cancer therapeutic agents target specific mutant proteins that are expressed by many different tumor types. Successful use of these therapies is dependent on the presence or absence of somatic mutations within the patient’s tumor that can confer clinical efficacy or drug resistance.MethodsThe aim of our study was to determine the type, frequency, overlap and functional proteomic effects of potentially targetable recurrent somatic hotspot mutations in 47 cancer-related genes in multiple disease sites that could be potential therapeutic targets using currently available agents or agents in clinical development.ResultsUsing MassArray technology, of the 1300 patient tumors analysed 571 (43.9%) had at least one somatic mutation. Mutations were identified in 30 different genes. KRAS (16.5%), PIK3CA (13.6%) and BRAF (3.8%) were the most frequently mutated genes. Prostate (10.8%) had the lowest number of somatic mutations identified, while no mutations were identified in sarcoma. Ocular melanoma (90.6%), endometrial (72.4%) and colorectal (66.4%) tumors had the highest number of mutations. We noted high concordance between mutations in different parts of the tumor (94%) and matched primary and metastatic samples (90%). KRAS and BRAF mutations were mutually exclusive. Mutation co-occurrence involved mainly PIK3CA and PTPN11, and PTPN11 and APC. Reverse Phase Protein Array (RPPA) analysis demonstrated that PI3K and MAPK signalling pathways were more altered in tumors with mutations compared to wild type tumors.ConclusionsHotspot mutational profiling is a sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular based therapeutics for treatment of cancer, and could potentially be of use in identifying novel opportunities for genotype-driven clinical trials.

Phylogenetic Tree Inference: A Top-Down Approach to Track Tumor Evolution.

Recently, an increasing number of studies sequence multiple biopsies of primary tumors, and even paired metastatic tumors to understand heterogeneity and the evolutionary trajectory of cancer progression. Although several algorithms are available to infer the phylogeny, most tools rely on accurate measurements of mutation allele frequencies from deep sequencing, which is often hard to achieve for clinical samples (especially FFPE samples). In this study, we present a novel and easy-to-use method, PTI (Phylogenetic Tree Inference), which use an iterative top-down approach to infer the phylogenetic tree structure of multiple tumor biopsies from same patient using just the presence or absence of somatic mutations without their allele frequencies. Therefore PTI can be used in a wide range of cases even when allele frequency data is not available. Comparison with existing state-of-the-art methods, such as LICHeE, Treeomics, and BAMSE, shows that PTI achieves similar or slightly better performance within a short run time. Moreover, this method is generally applicable to infer phylogeny for any other data sets (such as epigenetics) with a similar zero and one feature-by-sample matrix.

Multicentric Real Life Evaluation of the Impact of Next-Generation Sequencing on the Clinical Management of Chronic Myeloid Malignancies

Introduction Next generation sequencing (NGS) has allowed to improve knowledge about the genomic landscape of hematological malignancies. Somatic mutations (SM) are valuable new biomarkers but the utility of incorporating routine sequencing to guide diagnosis and therapeutic decisions remains challenging. We report here an observational multicentric study aimed at assessing the impact of SM testing by NGS in a real-life setting on the diagnosis and treatment of chronic myeloid malignancies (CMM). Patients and Method All patients who benefited from molecular assessment, between 10/2014 and 03/2019 in our University Hospital were included. All provided informed consent for data collection. All NGS requests were validated during a regional multidisciplinary concertation meeting. A custom targeted panel of 34 genes (145kbp i.e. ASXL1,BCOR, BCORL1, CBL, CSF3R, DNMT3A, ETV6, EZH2, GATA2, IDH1, IDH2, JAK2, KDM6A, KIT, KRAS, MPL, NPM1, NRAS, PIGA, PTEN, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET2, TNFAIP3, TP53, U2AF1, ZRSR2) was applied on DNA extracted from peripheral blood or bone marrow samples. DNA libraries, built with the Haloplex® target enrichment protocol (Agilent Technologies, Santa Clara, CA), were paired-end sequenced (150bp reads) with a MiSeq® Instrument (Illumina, San Diego, CA). Data analysis used an in-house pipeline including three variant callings (GATK HaplotypeCaller, VarScan and SAMTools). In a first group (A), NGS indication was to search for clonal hematopoiesis (CH), defined by the presence of at least one SM, in order to confirm or rule out a diagnosis of Idiopathic Cytopenia of Undetermined Significance (ICUS), Clonal Cytopenia of Undetermined Significance (CCUS), myelodysplastic syndrome (MDS), mixed myelodysplastic/myeloproliferative neoplasm (MDS/MPN), aplastic anemia (AA)/hypoplastic myelodysplasia (hMDS) or myeloproliferative neoplasm (MPN), based on recommendations of the WHO classification. In a second group (B), the theranostic impact of SM was studied. Prognostic SMs according to Bejar (2011) were used for MDS and MDS/MPN excluding chronic myelomonocytic leukemia that were analyzed with Itzykson score (2013) and/or CPSS-Mol score (Elena 2016). Prognostic SMs according to Vannucchi (2013) were used for myelofibrosis. Results The median age of the cohort was 60 years old (range: 10-87) with a median follow up of 1.1 years from molecular assessment to last follow-up. Within group A (94 patients), the most frequent blood count anomalies were cytopenia (68%), thrombocytosis (16%), and monocytosis (13%). The karyotype was normal in 77% and failed in 5% of the cases. Non-specific abnormalities (i.e. loss of chr Y, del 20q), were found in 8% of the cases. Before molecular assessment, the diagnoses proposed were ICUS (n=37), suspicion of MDS/MPN (n=16), AA/hMDS (n=16), or MPN (n=25). CH was detected in 31 patients comforting the diagnosis of CMM for 33% of group A (8 CCUS, 3 MDS, 7 MDS/MPN, 6 medullary hypoplasia, 7 MPN) patients. Considering the patients for whom no CH was detected (n=63), the initial suspected diagnosis of CMM was ruled out in 47 patients (i.e. 50% of group A). For the 16 remaining (i.e. 17% of group A), no firm diagnosis could be retained. Within group B (95 patients), NGS identified prognosis SM in 33% of the patients, i.e. poor prognosis SM in 24, including 8/40 MDS, 10/29 MDS/MPN and 6/17 myelofibrosis and good prognosis SM(SF3B1) in 7 of them, respectively 6/40 MDS and 1/29 MDS/MPN. Prognostic SMs had a therapeutic impact in 18/95 pts (19%). Indeed 13 patients with poor prognosis SM had a therapeutic change including 12 allogeneic stem-cell transplantation and 1 hypomethylating agent. Conversely, 5 patients with a good prognosis SM or absence of poor prognosis SM had a de-escalation of treatment intensity. Conclusion The use of NGS in daily practice had a clinical impact in both diagnostic and therapeutic decisions provided that the prescription is made in a critically explored context and not as a systematic test. In this "real life" cohort, the presence or absence of SM was a useful complement for integrated diagnoses in 83% of the patients, allowing to confirm (33%), or exclude (50%) a suspected condition. Moreover, in this cohort 34% of the patients had a SM with a reported prognostic impact and the treatment was modified in 19% of the cases. Yet, it remains necessary to integrate these results with other diagnostic criteria. Disclosures Peterlin: AbbVie Inc: Consultancy; Jazz Pharma: Consultancy; Astellas: Consultancy; Daiichi-Sankyo: Consultancy. Moreau:Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Le Gouill:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche-Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support. Chevallier:Daiichi Sankyo: Honoraria; Incyte: Consultancy, Honoraria; Jazz Pharmaceuticals: Honoraria.

Expression of the Tigit/CD226/CD155 Receptors/Ligand System in Chronic Lymphocytic Leukemia

Introduction: T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a surface receptor mainly expressed by CD8+, regulatory T lymphocytes and natural killer (NK) cells, but not by normal B cells. It performs as an inhibitory immune checkpoint, activated through binding of CD155. TIGIT competes with CD226 for CD155 binding, resulting in opposite outcomes: while CD226 enhances cytotoxicity of T lymphocytes and NK cells, TIGIT exerts immunosuppressive effects. Whether TIGIT engagement triggers an alternative signaling cascade, or whether it simply prevents CD226 activation, remains an open point. Tumor-infiltrating T lymphocytes generally express high levels of the molecule, together with the other checkpoint inhibitor PD-1. On this basis, antagonist antibodies targeting TIGIT are under evaluation to restore immunity and treat cancer patients, alone or in various combinations. Chronic lymphocytic leukemia (CLL), the most common adult leukemia, is characterized by a highly heterogeneous clinical outcome. Several molecular markers can help in stratifying patients, including the presence or absence of somatic mutations in B cell receptor, cytogenetic aberrations and single gene mutations. Interestingly, CLL cells express several T cell specific antigens, including CD5. A previous report indicates that, in CLL, TIGIT is expressed by circulating CD4+T cells, increasing during disease progression, while nothing is known about its expression on CLL cells. Aim:This work was undertaken with the aim of studying expression of the TIGIT/CD226/CD155 axis in CLL. Methods:We assembled a cohort of 101 primary CLL samples (40% females, mean age of 61). All patients were either untreated or had not received treatment in the 6 months prior to analysis. PBMC samples were tested for expression of TIGIT, CD155 and CD226 in both T and B subsets. A multiparametric flow cytometry strategy was designed, combining anti-TIGIT, anti-CD155 and anti-CD226 antibodies with a panel of B- (anti-CD19, anti-CD5, anti-CD38, anti-CD49d and anti-CD73) and T-mono/NK specific (anti-CD3, anti-CD8, anti-CD4, anti-CD14 and anti-CD56) markers. The number of TIGIT molecules on leukemic cells was estimated by interpolating values of mean fluorescence intensity (MFI) of each sample with that of PE-Quantibrite beads. Results:CLL cells heterogeneously express surface TIGIT, ranging from 0.2 to 81% (mean value 20%, median 10%, SEM ±2.145). The estimated number of molecules per cell was in the range of 32.5-3571 (mean 1140, median 841.1, SEM ±83.6). Expression of TIGIT was independent of gender or age at diagnosis and there was no correlation between TIGIT levels and lymphocyte counts in peripheral blood. In contrast, in this cohort of untreated patients, we observed a significantly lower TIGIT expression in samples with advanced disease (RAI III-IV) compared to early stages (RAI 0-I). Accordingly, low TIGIT associated with unmutated (UM) IGHVgenes and with an unfavorable FISH profile (trisomy 12, deletion 17 and deletion 11 vs. deletion 13 or normal karyotype). Lower, although not significant, TIGIT levels were observed in NOTCH1-mutated CLL samples (n=11) compared to counterpart (n=89). Looking at the T cell population, we observed overall higher TIGIT levels in the CD8+vs CD4+subset (mean %TIGIT+cells in CD8+56.7±1.8 vs 27.2±1.3 in CD4+). In line with reported observations, we found a modest but significant increase of TIGIT+T cells in advanced stage CLLs, at variance with what observed on the leukemic B cell side. Accordingly, we observed higher percentages of TIGIT+/CD4+cells in CLL samples carrying UM IGHVgenes. CD226 and CD155 were more homogeneously expressed in all subsets without significant differences, both in CLL and T cell components. Conclusions: This work shows that CLL cells express the immunomodulatory molecule TIGIT, particularly in the early stages of the disease in untreated patients. While further studies are needed to characterize its functional implications as well as treatment effect on TIGIT expression, it is tempting to speculate that TIGIT expression by CLL cells may serve to trigger an immunosuppressive behavior in these cells, which is no longer needed when the disease becomes advanced. This observation represents a starting point for future studies investigating the role of TIGIT in CLL and hints to a possible use of anti-TIGIT antibodies to target different cellular components of the disease. Disclosures Hoofd: iTeos Therapeutics: Employment. Coscia:Abbvie: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm Therapeutics: Research Funding. Gaidano:Sunesys: Consultancy, Honoraria; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra-Zeneca: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Furman:Acerta Pharma: Consultancy; Beigene: Consultancy; Incyte: Consultancy; Janssen: Consultancy; Oncotracker: Consultancy; Pharmacyclics: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy; Genentech: Consultancy; Abbvie: Consultancy; AstraZeneca: Consultancy. Deaglio:VelosBio Inc.: Research Funding; Verastem Inc: Research Funding; iTeos Therapeutics: Research Funding.

IgA Function in Relation to the Intestinal Microbiota.

IgA is the dominant immunoglobulin isotype produced in mammals, largely secreted across the intestinal mucosal surface. Although induction of IgA has been a hallmark feature of microbiota colonization following colonization in germ-free animals, until recently appreciation of the function of IgA in host-microbial mutualism has depended mainly on indirect evidence of alterations in microbiota composition or penetration of microbes in the absence of somatic mutations in IgA (or compensatory IgM). Highly parallel sequencing techniques that enable high-resolution analysis of either microbial consortia or IgA sequence diversity are now giving us new perspectives on selective targeting of microbial taxa and the trajectory of IgA diversification according to induction mechanisms, between different individuals and over time. The prospects are to link the range of diversified IgA clonotypes to specific antigenic functions in modulating the microbiota composition, position and metabolism to ensure host mutualism.

Stem Cell Evolutionary Dynamics of Differentiation and Plasticity

The role of tissue-specific stem cells, differentiation, and the resulting phenotypic heterogeneity in evolution of new mutations has attracted the interest of theoreticians and experimentalists. In the current paper, we first summarize the literature on population dynamics of stem cell and differentiated populations, in the absence of somatic mutations, with focus on applications to cancer therapeutics. We then review recent experimental and theoretical works on the evolutionary dynamics of stem cells and their progenitors in the presence of rare somatic mutation. The recent experiments show that the selection process among intestinal stem cells closely follows the previously proposed Moran model in finite populations. We also overview recent theoretical developments in the literature that generalizes selection processes inside the tissue with variable level differentiation and de-differentiation. This review tries to give a mathematical description on how phenotypic heterogeneity due to stem cell hierarchy influences growth dynamics and natural selection, inside an epithelial tissue or hematopoietic system.

El estado mutacional de las inmunoglobulinas en pacientes con leucemia linfocítica crónica: significado y pronóstico

Chronic lymphocytic leukaemia is a malignant disease characterised by proliferation and accumulation of mature B lymphocytes in bone marrow, blood, lymph nodes, and spleen. As part of its process of differentiation, immunoglobulin molecules of B cells, in addition to the characteristic rearrangements in their heavy and light chains, present a set of somatic mutations that greatly increase their diversity. The presence or absence of somatic mutations in the variable regions of the immunoglobulin gene in the malignant B-cells defines the aggressiveness and prognosis of the disease in individuals with chronic lymphocytic leukaemia.

Book review: The Epigenetics Revolution

Epigenetics is a rapidly evolving science that is often only described in scientific literature or textbooks. In “The Epigenetics Revolution”, Nessa Carey eloquently bridges the spheres of academia and scientific journalism (Carey, 2012). The phrasing “revolution” is a dramatic use of English that effectively portrays a momentous shift in biological thinking. For the novice, it is a good introduction to epigenetics whilst it provides professional readers with a concise summary of historically significant experiments and translational context. The text is divided into an introduction and 16 chapters which handle various concepts pertaining to epigenetic theory, key experimental studies and the potential for future therapeutic application. Carey starts with some historical context; she illustrates the successes and limitations of the human genome project and quotes some of the arguably overdramatic claims made by various organizations of the time. The first two chapters approach the issue of cellular differentiation by explaining the work of John Gurdon and the concept of a genetic “switch” rather than a loss of unneeded material. The analogy that DNA is a script to be interpreted is a particularly articulate explanation for novice readers. From this, Carey explains the epigenetic underpinnings of pluripotency and explores the potential application of stem cell therapies in regenerative medicine. Chapters 3 and 4 give an overview of genetic and epigenetic molecular biology, including the genetic code, DNA replication, transcription, translation, chromatin, DNA methylation, and histone modifications. These biological concepts are supplemented with an explanation of twin studies to illustrate the profound effect of environmental experience on genetically identical individuals. She makes reference to specific biological signals that induce epigenetic modification e.g. the hormone receptor. In the absence of somatic mutation, Carey also explores how epigenetic modifications can induce cellular abnormalities. Overall, this section provides an excellent narrative that uses famous scientific work to illustrate basic epigenetic concepts which is perfectly suited to novice readers or undergraduate students. Chapter 6 focuses on the heritability of epigenetic modifications. Environmental events such as the Dutch famine have clear effects on the health outcomes of offspring. Carey explains that this only occurs in susceptible regions of the genome and that epigenetic reprogramming during early embryogenesis circumvents the passage of many undesirable or dangerous modifications. Generation X is a very interesting chapter that explains the history of sex determination; an epigenetic process that depends upon tight regulation of X chromosome genes, termed “X chromosome dosage compensation.” The last quarter of the book focuses on epigenetic pathology. Carey illustrates this section with two very different disease states; psychiatric illness and cancer. This is purposefully designed to portray the universal influence of epigenetics. Carey makes an intelligent case for the role of childhood experience in shaping the epigenetic landscape; specifically, the association between abuse and the development of mental illness. Another interesting section explores the current contention surrounding neuro-epigenetics. This implies that epigenetic theory isn't sacrosanct and provokes a good debate for the reader. Disappointingly, neurodegeneration is only briefly mentioned. On the contrary, the concept of “epigenetic” aging is thorough and chapter 13 eloquently explains the global decrease in methylation and changes to the telomere system that are associated with the development of cancer. The final chapter elegantly draws together the biological principles, historical events, and translational examples that were given throughout the book. Carey concludes by exploring the potential for future drug discovery and the limitations and dangers that such agents may present. There is a description of recent research that is beginning to answer many elusive questions, such as the mechanisms underpinning the circadian rhythm. This section builds upon basic concepts with more advanced theory which might become challenging for some inexperienced readers. With that said, it is brilliantly written and provides a comprehensive review of many historically significant feats of epigenetic biology. “The Epigenetics Revolution” is a concise text that is accessible to a wide audience. However, such a compromise presents drawbacks; for the absolute beginner, gene nomenclature and complex scientific concepts may be confusing at times. If one is familiar with the field, many of the examples are unoriginal e.g. the agouti mouse experiments, the classic Dutch famine and its correlation with obesity, the royal jelly etc. Surprisingly, there is no mention of the early work by nobel laureates Barbara McClintock or Paul Berg. Despite these relatively minor criticisms, “The Epigenetics Revolution” is an excellent scientific biography that charts the past, present, and optimistic future of the field. It is especially suitable for informed non-specialists and students of biological sciences. Carey enthusiastically portrays a revolutionary system that could unlock the answers to many of nature's best kept secrets.

Abstract CN06-02: Insight into colorectal cancer etiology through MPE: Unmasking the links with obesity and smoking

Abstract Colorectal cancer is a leading cause of cancer morbidity and mortality worldwide. Epidemiologic studies have identified a host of genetic, lifestyle and demographic factors associated with colorectal cancer incidence, including high body mass index (BMI) and, perhaps less convincingly, cigarette smoking. Epidemiologic studies have often considered colorectal cancer as a singular disease outcome, ignoring potential disease heterogeneity; more recently, however, studies have further investigated whether risk and prognostic factors for colorectal cancer differ by tumor molecular phenotype, including microsatellite instability (MSI) status. MSI is the result of defective DNA mismatch repair capacity and it is a distinct tumor phenotype whereby short repetitive DNA sequences undergo a change in repeat length. Approximately 15% of colorectal tumors display MSI, usually the result of epigenetic modification of MLH1, a gene that encodes a mismatch repair protein, or less often, because the affected individual has a germline mutation in a DNA mismatch repair gene, consistent with Lynch syndrome. The majority of colorectal tumors develop despite having competent mismatch repair; these tumors, known as microsatellite (MS)-stable, arise through chromosomal instability and do not display marked gains or losses in MS regions. Other important molecular phenotypes for colorectal cancer include CpG island methylator phenotype (CIMP), and presence or absence of somatic mutations in the KRAS and BRAF genes. While BMI is a consistent and convincing risk factor for colorectal cancer overall (odds ratios and relative risks in the range of 1.3 to 1.5 are common for an obese BMI relative to a normal BMI), recent studies have suggested the associations are higher, or even restricted to, tumors that are MS-stable. Using the Colon Cancer Family Registry, our group showed that an obese BMI (&amp;gt;30 kg/m^2) relative to a normal BMI (BMI: 18.5-24.99 kg/m^2) was associated with an approximate 2-fold increased risk of MS-stable colorectal cancer while the association with MSI-high colorectal cancer was null. Other recent studies have shown a similar pattern, and other groups have shown that these results might be corroborated with FASN. Current work in the Colon Cancer Family Registry is seeking to address whether the prognostic (i.e., survival) benefit of MSI-high compared to MS-stable tumors further differs by level of obesity. The link between colorectal cancer and cigarette smoking has been more equivocal, with studies generally showing null associations or only modestly higher risks among smokers compared to non-smokers. Herein, tumor molecular phenotype data have been particularly revealing. Data from the Colon Cancer Family Registry showed a null association between ever smoking and colorectal cancer risk overall; however, when stratified by MSI status, risk of MSI-high cancer was about 2-fold higher among long-duration smokers relative to never smokers. Similar findings were reported in the prospective Iowa Women's Health Study where doubling in risks of MSI-high, CIMP-positive and BRAF mutation-positive colorectal cancers were reported for ever smoking while null associations were shown for tumors that lacked these molecular phenotypes. Additional work from one study center involved in the Colon Cancer Family Registry has suggested that the adverse impact of smoking on colorectal cancer survival is limited to patients diagnosed with MSI-high tumors. In addition to presenting published and unpublished results on associations of BMI and smoking with colorectal cancer incidence and survival, this presentation will also share recent experiences with collecting tumor blocks from a prospective cohort study, the American Cancer Society's Cancer Prevention Study-II. Citation Format: Peter T. Campbell. Insight into colorectal cancer etiology through MPE: Unmasking the links with obesity and smoking. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr CN06-02.

A reciprocal role of prostate cancer on stromal DNA damage

DNA damage found in prostate cancer-associated fibroblasts (CAF) promotes tumor progression. In the absence of somatic mutations in CAF, epigenetic changes dictate how stromal co-evolution is mediated in tumors. Seventy percent of prostate cancer patients lose expression of transforming growth factor-beta type II receptor (TGFBR2) in the stromal compartment (n = 77, p value = 0.0001), similar to the rate of glutathione S-transferase P1 (GSTP1) silencing. Xenografting of human prostate cancer epithelia, LNCaP, resulted in the epigenetic Tgfbr2 silencing of host mouse prostatic fibroblasts. Stromal Tgfbr2 promoter hypermethylation initiated by LNCaP cells was found to be dependent on IL-6 expression, based on neutralizing antibody studies. We further found that pharmacologic and transgenic knockout of TGF-β responsiveness in prostatic fibroblasts induced Gstp1 promoter methylation. It is known that TGF-β promotes DNA stability, however the mechanism is not well understood. Both prostatic human CAF and mouse transgenic knockout of Tgbr2 had elevated DNA methyltransferase I (DNMT1) activity and histone H3 lysine 9 trimethylation (H3K9me3) to suggest greater promoter methylation. Interestingly, the conditional knockout of Tgfbr2 in mouse prostatic fibroblasts, in modeling epigenetic silencing of Tgfbr2, had greater epigenetic gene silencing of multiple DNA damage repair and oxidative stress response genes, based on promoter methylation array analysis. Homologous gene silencing was validated by RT-PCR in mouse and human prostatic CAF. Not surprisingly, DNA damage repair gene silencing in the prostatic stromal cells corresponded with the presence of DNA damage. Restoring the expression of the epigenetically silenced genes in wild type fibroblasts with radiation-induced DNA damage reduced tumor progression. Tumor progression was inhibited even when epigenetic silencing was reversed in the Tgfbr2 knockout prostatic fibroblasts. Thus, fibroblastic epigenetic changes causative of DNA damage, initiated by association with cancer epithelia, is a dominant mediator of tumor progression over TGF-β responsiveness.

Mitochondria, obesity and aging

Mitochondria, obesity and aging

Genomic Analysis of Serial Chronic Lymphocytic Leukemia Samples Suggests That Epigenetic Changes, Rather Than Clonal Evolution, May Drive the Progression of This Leukemia

AbstractAbstract 4563The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous, with some patients requiring treatment relatively soon after diagnosis and others having indolent disease for many years. Some patients with indolent disease, however may develop more aggressive disease over time that requires therapy. To identify genetic and epigenetic changes that associate with the transition from indolent to aggressive disease, we used genomic methods to analyze sequential samples obtained from 19 CLL patients evaluated at the UC San Diego Moores Cancer Center who ultimately required treatment, as per iwCLL guidelines.For all patients, the first time point sample collection (SC1) was obtained within 1 year post-diagnosis and the second time point sample collection (SC2) was obtained within 1 year before treatment. We performed whole-exome sequencing (Agilent 50Mb capture, 100×) and methylation (450K) array analyses on leukemia cells and germline DNA. Somatic allele frequencies ranged from < 10% to 50%, suggesting heterogeneity within the tumor. When comparing SC1 versus SC2, we observed changes in somatic allele frequency for 6 (32%) of 19 patients, however 13 (68%) of 19 patients did not have evidence for clonal evolution at the somatic level, suggesting that the acquisition of additional somatic mutations did not drive CLL progression and that the clonal population structure remains stable throughout disease progression with multiple clones per patient. Using 450K CpG methylation arrays, we identified 52,409 sites (FDR=0.05) that changed consistently between SC1 and SC2 across 19 patients, suggesting that epigenetic changes were widespread, even without detectable somatic mutations.In summary, our results imply that CLL progression can occur in the absence of somatic mutations, but rather may reflect non-stochastic alterations in the epigenome altering RNA expression. Disclosures:No relevant conflicts of interest to declare.

Signaling pathways activated by the B-cell receptor in chronic lymphocytic leukemia

Over the past decade, several features of the B-cell receptor (BCR) complex have emerged as major markers for prognostic classification of B-cell chronic lymphocytic leukemia (B-CLL). In particular, the absence of somatic mutations within the immunoglobulin variable heavy chain genes (IGHV), the presence of ZAP-70 and a higher ability of the BCR to translate signals within the cell have been associated with an aggressive clinical course. Indeed, the stratification of patients with B-CLL based on BCR features suggests that heterogeneity of B-CLL clinical courses may reflect BCR signaling differences that have arisen during the evolution of leukemia. Therefore, characterizing BCR signaling profiles may help to identify signaling markers useful for patient stratification, disease monitoring and therapeutic targeting in B-CLL.

35 BCR-signaling profiles associated with prognosis and progression in B-CLL

Background. B-cell chronic lymphocytic leukemia (B-CLL) patients exhibit a variable clinical course. Several biological parameters have been shown to be associated with clinical outcome in CLL. Among them, the most reliable markers are represented by the absence of somatic mutations within the immunoglobulin variable heavy chain genes (IGHV), the expression of CD38 antigen, or the presence of the ZAP-70 tyrosine kinase. These parameters of poor clinical outcome are structurally and/or functionally linked to B-cell Receptor (BCR) expressed by CLL cells, thereby strengthening the hypothesis that antigenic stimulation mediated by the BCR represents a driving event in the onset and progression of the malignant B cells. Methods. We applied a “network level” analysis of BCR signaling by measuring single-cell profiles of phosphoprotein networks by flow cytometry. We evaluated the response to BCR engagement in primary cells isolated from 27 CLL patients by analyzing the phosphorylation states of 5 phosphoproteins on the route of BCR signaling, including p-Syk, p-NF-kappaB, p-Erk1/2, p-p38 and p-JNK. BCR was cross-linked by incubating cells with anti-IgM antibodies. Results. Unsupervised clustering analysis distinguished BCR response profiles of phosphoproteins that differentiated cases of CLL with mutated IGHV from those with unmutated IGHV (P=0.0003), cases with low levels of CD38 expression from those with high levels (P=0.0004) and cases with ZAP-70negative leukemic cells from cases that were ZAP-70-positive (P=0.001). Furthermore, the same BCR response profiles were also associated with time to progression (P=0.0014) and with overall survival (P=0.049), as assessed by Kaplan–Meier curves and the log-rank test. Independent survival analysis of time to progression via fitting Cox proportional hazards models comprising clinical covariates and/or BCR network response to modulation demonstrated that measuring modulated BCR network signaling can yield improved prognostic information compared to CD38 status alone (likelihood ratio test 5.8 for CD38 versus 10.6 for signaling) and enhance prognostic assessment using IGHV status (likelihood ratio test for IGHV = 14.8 versus for IGHV + signaling = 17.9).

Low frequency of somatic mutations in uterine sarcomas: A molecular analysis and review of the literature

The rarity of uterine sarcomas along with their pathological and molecular heterogeneities render their study particularly challenging. We evaluated a panel of somatic mutations principally centering on the tyrosine kinase gene family and their downstream signaling cascades in an attempt to identify potential candidate markers that may assist in diagnostic or therapeutic decisions in these tumors. We performed mutational analysis of 20 exons from 9 genes (EGFR, CDKN2A, MET, KIT, RAS, BRAF, PI3KCA, HER-2 and PDGFR-alpha) on biopsy material from 25 patients who underwent primary surgery for uterine sarcoma between October 1995 and October 2003. Due to the limited number of studies conducted we have also undertaken a literature review of somatic mutations in uterine sarcomas. A total of 3 different somatic mutations were identified: one KRAS (codon G12D) in a carcinosarcoma and two exon 20 PI3KCA mutations (H1047R and H1047Y) both in carcinosarcomas. Mutational status of all mutations was confirmed using germline DNA extracted from peripheral blood. Consistent with the literature data, no other mutations regarding the rest of the genes of the panel were identified. Due to the low number of somatic mutations in our series, we did not perform further clinicopathological correlations. The absence of somatic mutations in the majority of genes that are considered critical in neoplastic transformation hampers the identification of potential therapeutic targets in patients with uterine sarcoma.