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Objectives: In this study, stool samples were evaluated for tumor mutation analysis via a targeted next generation sequencing (NGS) approach in a small patient cohort suffering from localized rectal cancer. Introduction: Colorectal cancer (CRC) causes the second highest cancer-related death rate worldwide. Thus, improvements in disease assessment and monitoring that may facilitate treatment allocation and allow organ-sparing "watch-and-wait" treatment strategies are highly relevant for a significant number of CRC patients. Methods: Stool-based results were compared with mutation profiles derived from liquid biopsies and the gold standard procedure of tumor biopsy from the same patients. A workflow was established that enables the detection of de-novo tumor mutations in stool samples of CRC patients via ultra-sensitive cell-free tumor DNA target enrichment. Results: Notably, only a 19% overall concordance was found in mutational profiles across the compared sample specimens of stool, tumor, and liquid biopsies. Conclusion: Based on these results, the analysis of stool and liquid biopsy samples can provide important additional information on tumor heterogeneity and potentially on the assessment of minimal residual disease and clonal tumor evolution.
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Biomarcadores de Tumor , Heces , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Neoplasias del Recto , Humanos , Heces/química , Neoplasias del Recto/genética , Neoplasias del Recto/patología , Neoplasias del Recto/sangre , Biomarcadores de Tumor/genética , Biopsia Líquida/métodos , Femenino , Masculino , ADN Tumoral Circulante/genética , ADN Tumoral Circulante/sangre , Persona de Mediana Edad , Anciano , Análisis Mutacional de ADN , Heterogeneidad Genética , ADN de Neoplasias/sangre , ADN de Neoplasias/genéticaRESUMEN
INTRODUCTION: Targeting the B cell receptor (BCR) pathway via ibrutinib, a specific inhibitor of Bruton's tyrosine kinase, has shown marked clinical efficacy in treatment of patients with chronic lymphocytic leukemia (CLL), thus becoming a preferred first line option independent of risk factors. However, acquired resistance to ibrutinib poses a major clinical problem and requires the development of novel treatment combinations to increase efficacy and counteract resistance development and clinical relapse rates. CASE PRESENTATION: In this study, we performed exome and transcriptome analyses of an ibrutinib resistant CLL patient in order to investigate genes and expression patterns associated with ibrutinib resistance. Here we provide evidence that ibrutinib resistance can be attributed to aberrant mammalian target of rapamycin (MTOR) signaling. CONCLUSION: Thus, our study proposes that combined use of MTOR inhibitors with ibrutinib could be a possible option to overcome therapy resistance in ibrutinib treated patients.
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Accurate somatic variant calling from next-generation sequencing data is one most important tasks in personalised cancer therapy. The sophistication of the available technologies is ever-increasing, yet, manual candidate refinement is still a necessary step in state-of-the-art processing pipelines. This limits reproducibility and introduces a bottleneck with respect to scalability. We demonstrate that the validation of genetic variants can be improved using a machine learning approach resting on a Convolutional Neural Network, trained using existing human annotation. In contrast to existing approaches, we introduce a way in which contextual data from sequencing tracks can be included into the automated assessment. A rigorous evaluation shows that the resulting model is robust and performs on par with trained researchers following published standard operating procedure.
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Aprendizaje Automático , Redes Neurales de la Computación , Humanos , Reproducibilidad de los Resultados , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
Chronic lymphocytic leukemia (CLL) is a very common and mostly incurable B-cell malignancy. Recent studies revealed high interpatient mutational heterogeneity and worsened therapy response and survival of patients with complex genomic aberrations. In line with this, a better understanding of the underlying mechanisms of specific genetic aberrations would reveal new prognostic factors and possible therapeutic targets. It is known that chromosomal rearrangements including DNA insertions often play a role during carcinogenesis. Recently it was reported that bacteria (microbiome)-human lateral gene transfer occurs in somatic cells and is enriched in cancer samples. To further investigate this mechanism in CLL, we analyzed paired-end RNA sequencing data of 45 CLL patients and 9 healthy donors, in which we particularly searched for bacterial DNA integrations into the human somatic genome. Applying the Burrows-Wheeler aligner (BWA) first on a human genome and then on bacterial genome references, we differentiated between sequencing reads mapping to the human genome, to the microbiome or to bacterial integrations into the human genome. Our results indicate that CLL samples featured bacterial DNA integrations more frequently (approx. two-fold) compared to normal samples, which corroborates the latest findings in other cancer entities. Moreover, we determined common integration sites and recurrent integrated bacterial transcripts. Finally, we investigated the contribution of bacterial integrations to oncogenesis and disease progression.
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Bacterias/genética , Aberraciones Cromosómicas , Transferencia de Gen Horizontal , Genoma Bacteriano , Genoma Humano , Leucemia Linfocítica Crónica de Células B/patología , Microbiota , Bacterias/crecimiento & desarrollo , Estudios de Casos y Controles , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/microbiologíaRESUMEN
The reinvigoration of anti-cancer immunity by immune checkpoint therapies has greatly improved cancer treatment. In chronic lymphocytic leukemia (CLL), patients as well as in the Tcl1 mouse model for CLL, PD1-expressing, exhausted T cells significantly expand alongside CLL development; nevertheless, PD1 inhibition has no clinical benefit. Hence, exhausted T cells are either not activatable by simple PD1 blocking in CLL and/or only an insufficient number of exhausted T cells are CLL-specific. In this study, we examined the latter hypothesis by exploiting the Tcl1 transgenic CLL mouse model in combination with TCR transgene expression specific for a non-cancer antigen. Following CLL tumor development, increased PD1 levels were detected on non-CLL specific T cells that seem dependent on the presence of (tumor-) antigen-specific T cells. Transcriptome analysis confirmed a similar exhaustion phenotype of non-CLL specific and endogenous PD1pos T cells. Our results indicate that in the CLL mouse model, a substantial fraction of non-CLL specific T cells becomes exhausted during disease progression in a bystander effect. These findings have important implications for the general efficacy assessment of immune checkpoint therapies in CLL.
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Leucemia Linfocítica Crónica de Células B/fisiopatología , Proteínas Proto-Oncogénicas/genética , Linfocitos T/inmunología , Animales , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/inmunología , Ratones , Ratones TransgénicosRESUMEN
Adaptive somatic mutations conferring treatment resistance and accelerated disease progression is still a major problem in cancer therapy. Additionally in CLL, patients receiving novel, efficient drugs frequently become treatment refractory and eventually relapse. Activation-induced deaminase (AID) is a cytosine deaminase that catalyzes somatic hypermutation of genomic DNA at the immunoglobulin locus in activated B cells. As considerable off-target mutations by AID have been discerned in chronic lymphocytic leukemia, it is essential to investigate to which extent these mutations contribute to disease progression to estimate whether AID inhibition could counteract drug resistance mechanisms. In this study, we examined the TCL1 mouse model for CLL on an AID pro- and deficient background by comparing disease development and mutational landscapes. We provide evidence that AID contributes to the acquisition of somatic cancer-specific mutations also in the TCL1 model and accelerates CLL development particularly in the transplant setting. We conclude that AID is directly determining the fitness of the CLL clone, which prompts further studies to assess the effect of AID inhibition on the occurrence of drug resistance.
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Aberrant end joining of DNA double strand breaks leads to chromosomal rearrangements and to insertion of nuclear or mitochondrial DNA into breakpoints, which is commonly observed in cancer cells and constitutes a major threat to genome integrity. However, the mechanisms that are causative for these insertions are largely unknown. By monitoring end joining of different linear DNA substrates introduced into HEK293 cells, as well as by examining end joining of CRISPR/Cas9 induced DNA breaks in HEK293 and HeLa cells, we provide evidence that the dNTPase activity of SAMHD1 impedes aberrant DNA resynthesis at DNA breaks during DNA end joining. Hence, SAMHD1 expression or low intracellular dNTP levels lead to shorter repair joints and impede insertion of distant DNA regions prior end repair. Our results reveal a novel role for SAMHD1 in DNA end joining and provide new insights into how loss of SAMHD1 may contribute to genome instability and cancer development.
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Reparación del ADN por Unión de Extremidades , Proteína 1 que Contiene Dominios SAM y HD/fisiología , Proteína 9 Asociada a CRISPR/metabolismo , Rotura Cromosómica , Desoxirribonucleótidos/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteína 1 que Contiene Dominios SAM y HD/metabolismoRESUMEN
Activation induced deaminase (AID) has two distinct and well defined roles, both relying on its deoxycytidine (dC) deaminating function: one as a DNA mutator and another in DNA demethylation. In chronic lymphocytic leukemia (CLL), AID was previously shown to be an independent negative prognostic factor. While there is substantial impact on DNA mutations, effects of AID on gene expression by promoter demethylation of disease related target genes in leukemia has not been addressed. To shed light on this question, we aimed at determining genome wide methylation changes as well as gene expression changes in response to AID expression in CLL. Although we found minor differences in individual methylation variable positions following AID expression, we could not find recurrent methylation changes of specific target sites or changes in global methylation.
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Epigénesis Genética , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/genética , Porfiria Intermitente Aguda/metabolismo , Biología Computacional , Metilación de ADN/fisiología , Regulación Neoplásica de la Expresión Génica , Humanos , Leucocitos Mononucleares/enzimologíaRESUMEN
The original version of this article contained a mistake. The name of Tanja Nicole Hartman should have been Tanja Nicole Hartmann. The original article has been corrected.
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Despite recent advances, chemoimmunotherapy remains a standard for fit previously untreated chronic lymphocytic leukaemia patients. Lenalidomide had activity in early monotherapy trials, but tumour lysis and flare proved major obstacles in its development. We combined lenalidomide in increasing doses with six cycles of fludarabine and rituximab (FR), followed by lenalidomide/rituximab maintenance. In 45 chemo-naive patients, included in this trial, individual tolerability of the combination was highly divergent and no systematic toxicity determining a maximum tolerated dose was found. Grade 3/4 neutropenia (71%) was high, but only 7% experienced grade 3 infections. No tumour lysis or flare > grade 2 was observed, but skin toxicity proved dose-limiting in nine patients (20%). Overall and complete response rates after induction were 89 and 44% by intention-to-treat, respectively. At a median follow-up of 78.7 months, median progression-free survival (PFS) was 60.3 months. Minimal residual disease and immunoglobulin variable region heavy chain mutation state predicted PFS and TP53 mutation most strongly predicted OS. Baseline clinical factors did not predict tolerance to the immunomodulatory drug lenalidomide, but pretreatment immunophenotypes of T cells showed exhausted memory CD4 cells to predict early dose-limiting non-haematologic events. Overall, combining lenalidomide with FR was feasible and effective, but individual changes in the immune system seemed associated with limiting side effects. clinicaltrials.gov (NCT00738829) and EU Clinical Trials Register ( www.clinicaltrialsregister.eu , 2008-001430-27).
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Quimioterapia de Consolidación , Supervivencia sin Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Erupciones por Medicamentos/etiología , Femenino , Enfermedades Hematológicas/inducido químicamente , Humanos , Inmunidad Celular/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Inmunoterapia , Estimación de Kaplan-Meier , Lenalidomida , Leucemia Linfocítica Crónica de Células B/genética , Recuento de Linfocitos , Quimioterapia de Mantención , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Inducción de Remisión , Rituximab/administración & dosificación , Rituximab/efectos adversos , Subgrupos de Linfocitos T/efectos de los fármacos , Talidomida/administración & dosificación , Talidomida/efectos adversos , Talidomida/análogos & derivados , Talidomida/farmacología , Resultado del Tratamiento , Vidarabina/administración & dosificación , Vidarabina/efectos adversos , Vidarabina/análogos & derivadosRESUMEN
Cancer is a genetic disease caused by mutations and chromosomal abnormalities that contribute to uncontrolled cell growth. In addition, cancer cells can rapidly respond to conventional and targeted therapies by accumulating novel and often specific genetic lesions leading to acquired drug resistance and relapsing disease. In chronic lymphocytic leukemia (CLL), however, diverse chromosomal aberrations often occur. In many cases, improper repair of DNA double-strand breaks (DSB) is a major source for genomic abnormalities. Therefore, this study examined the repair of DNA DSBs by nonhomologous end joining (NHEJ) in CLL by performing plasmid-based repair assays in primary CLL cells and normal B cells, isolated from patients, as well as TALEN/Cas9-induced chromosomal deletions in the CLL cell line Mec1. It is demonstrated that DNA repair is aberrant in CLL cells, featuring perturbed DNA break structure preference with efficient joining of noncohesive ends and more deletions at repair junctions. In addition, increased microhomology-mediated end joining (MMEJ) of DNA substrates was observed in CLL together with increased expression of MMEJ-specific repair factors. In summary, these data identify major differences in DNA repair efficiency between CLL cells and normal B cells isolated from patients.Implications: This study suggests inherently aberrant DNA DSB repair in the acquisition of subclonal genomic structural variations important for clonal evolution and treatment resistance in CLL. Mol Cancer Res; 16(3); 428-38. ©2017 AACR.
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Reparación del ADN por Unión de Extremidades , Leucemia Linfocítica Crónica de Células B/genética , Línea Celular Tumoral , ADN Ligasa (ATP)/biosíntesis , ADN Ligasa (ATP)/genética , ADN Ligasa (ATP)/metabolismo , Expresión Génica , Células HEK293 , Humanos , Transfección , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/biosíntesis , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismoRESUMEN
While research on T cell exhaustion in context of cancer particularly focuses on CD8+ cytotoxic T cells, the role of inhibitory receptors on CD4+ T-helper cells have remained largely unexplored. TIGIT is a recently identified inhibitory receptor on T cells and natural killer (NK) cells. In this study, we examined TIGIT expression on T cell subsets from CLL patients. While we did not observe any differences in TIGIT expression in CD8+ T cells of healthy controls and CLL cells, we found an enrichment of TIGIT+ T cells in the CD4+ T cell compartment in CLL. Intriguingly, CLL patients with an advanced disease stage displayed elevated numbers of CD4+ TIGIT+ T cells compared to low risk patients. Autologous CLL-T cell co-culture assays revealed that depleting CD4+ TIGIT+ expressing T cells from co-cultures significantly decreased CLL viability. Accordingly, a supportive effect of TIGIT+CD4+ T cells on CLL cells in vitro could be recapitulated by blocking the interaction of TIGIT with its ligands using TIGIT-Fc molecules, which also impeded the T cell specific production of CLL-prosurvival cytokines. Our data reveal that TIGIT+CD4+T cells provide a supportive microenvironment for CLL cells, representing a potential therapeutic target for CLL treatment.
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Chronic lymphocytic leukemia develops within a complex network driven by genetic mutations and microenvironmental interactions. Among the latter a complex interplay with the immune system is established by the clone. Next to a proposed recruitment of support from T and myeloid cells, potential anti-CLL immune reactions need to be subverted. By using TCL1 mice as a CLL model, we show that TCR-Vß7+ NK1.1+ T cells are overrepresented in this disease model and constitute a main subset of peripheral CD3+ cells with biased TCR usage, showing that these cells account for a major part for T cell skewing in TCL1 mice. Moreover, we show that overrepresentation is dependent on CD1d expression in TCL1 mice, implicating that these cells belong to a NKT-like cell fraction which are restricted to antigen presented by the MHC-like surface marker CD1d. Accordingly, we observed a high fraction of CD161+ cells within overrepresented T cells in CLL patients and we found downregulation of CD1d on the surface of CLL cells, both in TCL1 mice and patients. Finally, we show that in TCL1 mice, CD1d deficiency resulted in shortened overall survival. Our results point to an interaction between CLL and CD161+ T cells that may represent a novel therapeutic target for immune modulation.
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Antígenos CD1d/metabolismo , Linfocitos T CD8-positivos/citología , Leucemia Linfocítica Crónica de Células B/metabolismo , Células T Asesinas Naturales/citología , Anciano , Anciano de 80 o más Años , Animales , Complejo CD3/metabolismo , Progresión de la Enfermedad , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Mutación , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismoAsunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Antígenos CD40/inmunología , Evolución Clonal , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Evolución Clonal/genética , Evolución Clonal/inmunología , Regiones Determinantes de Complementariedad/genética , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Receptores de Antígenos de Linfocitos B/genéticaRESUMEN
Activation-induced deaminase (AID) is a DNA-mutating enzyme that mediates class-switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off-target activity, AID is implicated in lymphoma development by introducing genome-wide DNA damage and initiating chromosomal translocations such as c-myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein levels of AID splice isoforms by introducing an AID splice reporter construct into cell lines and primary CLL cells from patients as well as from WT and TCL1(tg) C57BL/6 mice (where TCL1 is T-cell leukemia/lymphoma 1). The splice construct is 5'-fused to a GFP-tag, which is preserved in all splice isoforms and allows detection of translated protein. Summarizing, we show a thorough quantification of alternatively spliced AID transcripts and demonstrate that the corresponding protein abundances, especially those of splice variants AID-ivs3 and AID-ΔE4, are not stoichiometrically equivalent. Our data suggest that enhanced proteasomal degradation of low-abundance proteins might be causative for this discrepancy.
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Empalme Alternativo , Citidina Desaminasa/genética , Leucemia Linfocítica Crónica de Células B/enzimología , Animales , Citidina Desaminasa/análisis , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The combination of cytotoxic treatment with strategies for immune activation represents an attractive strategy for tumour therapy. Following reduction of high tumour burden by effective cytotoxic agents, two major immune-stimulating approaches are being pursued. First, innate immunity can be activated by monoclonal antibodies triggering antibody-dependent cellular cytotoxicity. Second, tumour-specific T cell responses can be generated by immunization of patients with peptides derived from tumour antigens and infused in soluble form or loaded onto dendritic cells. The choice of cytotoxic agents for such combinatory regimens is crucial since most substances such as fludarabine are considered immunosuppressive while others such as cyclophosphamide can have immunostimulatory activity. We tested in this study whether fludarabine and/or cyclophosphamide, which represent a very effective treatment regimen for chronic lymphocytic leukaemia, would interfere with a therapeutic strategy of T cell activation. Analysis of peripheral blood samples from patients prior and during fludarabine/cyclophosphamide therapy revealed rapid and sustained reduction of tumour cells but also of CD4(+) and CD8(+) T cells. This correlated with a significant cytotoxic activity of fludarabine/cyclophosphamide on T cells in vitro. Unexpectedly, T cells surviving fludarabine/cyclophosphamide treatment in vitro had a more mature phenotype, while fludarabine-treated T cells were significantly more responsive to mitogenic stimulation than their untreated counterparts and showed a shift towards T(H)1 cytokine secretion. In conclusion, fludarabine/cyclophosphamide therapy though inducing significant and relevant T cell depletion seems to generate a micromilieu suitable for subsequent T cell activation.