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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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Aprendizado de Máquina , Redes Neurais de Computação , Humanos , Reprodutibilidade dos Testes , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
The current gold standard of response assessment in patients with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML) is morphologic complete remission (CR) and CR with incomplete count recovery (CRi), both of which require an invasive BM evaluation. Outside of clinical trials, BM evaluations are only performed in ~50% of patients during follow-up, pinpointing a clinical need for response endpoints that do not necessitate BM assessments. We define and validate a new response type termed "peripheral blood complete remission" (PB-CR) that can be determined from the differential blood count and clinical parameters without necessitating a BM assessment. We compared the predictive value of PB-CR with morphologic CR/CRi in 1441 non-selected, consecutive patients diagnosed with MDS (n = 522; 36.2%), CMML (n = 132; 9.2%), or AML (n = 787; 54.6%), included within the Austrian Myeloid Registry (aMYELOIDr; NCT04438889). Time-to-event analyses were adjusted for 17 covariates remaining in the final Cox proportional hazards (CPH) model. DeepSurv, a CPH neural network model, and permutation-based feature importance were used to validate results. 1441 patients were included. Adjusted median overall survival for patients achieving PB-CR was 22.8 months (95%CI 18.9-26.2) versus 10.4 months (95%CI 9.7-11.2) for those who did not; HR = 0.366 (95%CI 0.303-0.441; p < .0001). Among patients achieving CR, those additionally achieving PB-CR had a median adjusted OS of 32.6 months (95%CI 26.2-49.2) versus 21.7 months (95%CI 16.9-27.7; HR = 0.400 [95%CI 0.190-0.844; p = .0161]) for those who did not. Our deep neural network analysis-based findings from a large, prospective cohort study indicate that BM evaluations solely for the purpose of identifying CR/CRi can be omitted.
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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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Reparo do DNA por Junção de Extremidades , Proteína 1 com Domínio SAM e Domínio HD/fisiologia , Proteína 9 Associada à CRISPR/metabolismo , Quebra Cromossômica , Desoxirribonucleotídeos/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteína 1 com Domínio SAM e Domínio HD/metabolismoRESUMO
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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Bactérias/genética , Aberrações Cromossômicas , Transferência Genética Horizontal , Genoma Bacteriano , Genoma Humano , Leucemia Linfocítica Crônica de Células B/patologia , Microbiota , Bactérias/crescimento & desenvolvimento , Estudos de Casos e Controles , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/microbiologiaRESUMO
Chronic lymphocytic leukemia (CLL) is a heterogenous disease that is highly dependent on a cross talk of CLL cells with the microenvironment, in particular with T cells. T cells derived from CLL patients or murine CLL models are skewed to an antigen-experienced T-cell subset, indicating a certain degree of antitumor recognition, but they are also exhausted, preventing an effective antitumor immune response. Here we describe a novel mechanism of CLL tumor immune evasion that is independent of T-cell exhaustion, using B-cell-specific deletion of the transcription factor IRF4 (interferon regulatory factor 4) in Tcl-1 transgenic mice developing a murine CLL highly similar to the human disease. We show enhanced CLL disease progression in IRF4-deficient Tcl-1 tg mice, associated with a severe downregulation of genes involved in T-cell activation, including genes involved in antigen processing/presentation and T-cell costimulation, which massively reduced T-cell subset skewing and exhaustion. We found a strong analogy in the human disease, with inferior prognosis of CLL patients with low IRF4 expression in independent CLL patient cohorts, failed T-cell skewing to antigen-experienced subsets, decreased costimulation capacity, and downregulation of genes involved in T-cell activation. These results have therapeutic relevance because our findings on molecular mechanisms of immune privilege may be responsible for the failure of immune-therapeutic strategies in CLL and may lead to improved targeting in the future.
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Linfócitos B/imunologia , Deleção de Genes , Fatores Reguladores de Interferon/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Evasão Tumoral , Animais , Linfócitos B/metabolismo , Regulação Leucêmica da Expressão Gênica , Humanos , Fatores Reguladores de Interferon/imunologia , Leucemia Linfocítica Crônica de Células B/genética , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos TransgênicosRESUMO
Adhesive properties of leukemia cells shape the degree of organ infiltration and the extent of leukocytosis. CD44 and the integrin VLA-4, a CD49d/CD29 heterodimer, are important factors of progenitor cell adhesion in bone marrow (BM). Here, we report their cooperation in acute myeloid leukemia (AML) by a novel non-classical CD44-mediated way of inside-out VLA-4 activation. In primary AML BM samples from patients and the OCI-AML3 cell line, CD44 engagement by hyaluronan induced inside-out activation of VLA-4 resulting in enhanced leukemia cell adhesion on VCAM-1. This was independent from VLA-4 affinity regulation but based on ligand-induced integrin clustering on the cell surface. CD44-induced VLA-4 activation could be inhibited by the Src family kinase inhibitor PP2 and the multikinase inhibitor midostaurin. In further consequence, the increased adhesion on VCAM-1 allowed AML cells to strongly bind stromal cells. Thereby VLA-4/VCAM-1 interaction promoted activation of Akt, MAPK, NF-kB and mTOR signaling and decreased AML cell apoptosis. Collectively, our investigations provide a mechanistic description of an unusual CD44 function in regulating VLA-4 avidity in AML, supporting AML cell retention in the supportive BM microenvironment.
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Integrina alfa4beta1 , Leucemia Mieloide Aguda , Medula Óssea , Adesão Celular , Humanos , Receptores de Hialuronatos/genética , Microambiente Tumoral , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
BACKGROUND: Pancreatic carcinoma carries a devastating prognosis and is the 4th leading cause for cancer related death in the US and most European countries. Apart from imaging and CA 19-9, pancreatic carcinoma is still lacking reliable markers to assess tumor dynamics and to monitor treatment response over time. The aim of this study was to evaluate the feasibility of cell free tumor-DNA (cft-DNA), respectively KRAS mutation in peripheral blood, detection as a prognostic and predictive value for chemotherapy monitoring. METHODS: Serial plasma samples from 42 patients with KRAS mutated pancreatic cancer were prospectively collected and the ctKRAS Mutation Assay (Idylla™, Biocartis, Mechelen, Belgium) of cft-DNA was performed on 29 patients that did not receive curative surgery and went on to palliative chemotherapy. To monitor cft-DNA KRAS mutation levels during treatment quantitative assessment of cft-DNA was performed at baseline and during follow up at predetermined times. RESULTS: All 29 patients included in our analyses had a detected KRAS mutation in the tumor biopsy. In almost half (48.2%) of patients a KRAS mutation could also be detected in peripheral plasma. Patients with detectable KRAS mutations before treatment start in plasma had a significantly worse survival (16.8 months vs not reached, p < 0.031 and HR 3.303). Looking for a dynamic assessment of tumor response, we found a statistically significant association between the KRAS mutant ratio from first staging CT scan to basal levels with tumor response or progress (p = 0.014). CONCLUSION: Performing KRAS testing from peripheral blood for patients, who have no elevated tumor markers, might be a novel option for treatment monitoring complementing routine imaging techniques.
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DNA Tumoral Circulante , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras)/genética , Biomarcadores Tumorais/genética , Análise Mutacional de DNA , Humanos , Mutação , Neoplasias Pancreáticas/genética , Prognóstico , Neoplasias PancreáticasRESUMO
Chimeric antigen receptor (CAR) T-cells (CAR T-cells) are a promising therapeutic approach in treating hematological malignancies. CAR T-cells represent engineered autologous T-cells, expressing a synthetic CAR, targeting tumor-associated antigens (TAAs) independent of major histocompatibility complex (MHC) presentation. The most common target is CD19 on B-cells, predominantly used for the treatment of lymphoma and acute lymphocytic leukemia (ALL), leading to approval of five different CAR T-cell therapies for clinical application. Despite encouraging clinical results, treatment of other hematological malignancies such as acute myeloid leukemia (AML) remains difficult. In this review, we focus especially on CAR T-cell application in different hematological malignancies as well as strategies for overcoming CAR T-cell dysfunction and increasing their efficacy.
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Neoplasias Hematológicas/terapia , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/tendências , Antígenos de Neoplasias/imunologia , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Humanos , Imunoterapia/métodos , Leucemia/imunologia , Leucemia/terapia , Linfoma/imunologia , Linfoma/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologiaRESUMO
Chronic lymphocytic leukemia (CLL) is considered a clonal B cell malignancy. Sporadically, CLL cases with multiple productive heavy and light-chain rearrangements were detected, thus leading to a bi- or oligoclonal CLL disease with leukemic cells originating either from different B cells or otherwise descending from secondary immunoglobulin rearrangement events. This suggests a potential role of clonal hematopoiesis or germline predisposition in these cases. During the screening of 75 CLL cases for kappa and lambda light-chain rearrangements, we could detect a single case with CLL cells expressing two distinct kappa and lambda light chains paired with two separate immunoglobulin heavy-chain variable regions. Furthermore, this patient also developed a prostate carcinoma. Targeted genome sequencing of highly purified light-chain specific CLL clones from this patient and from the prostate carcinoma revealed the presence of a rare germline polymorphism in the POLE gene. Hence, our data suggest that the detected SNP may predispose for cancer, particularly for CLL.
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Processamento Alternativo , DNA Polimerase II/genética , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Polimorfismo de Nucleotídeo Único , Neoplasias da Próstata/patologia , Humanos , Leucemia Linfocítica Crônica de Células B/complicações , Leucemia Linfocítica Crônica de Células B/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias da Próstata/complicações , Neoplasias da Próstata/genéticaRESUMO
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 Crônica de Células B/fisiopatologia , Proteínas Proto-Oncogênicas/genética , Linfócitos T/imunologia , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/imunologia , Camundongos , Camundongos TransgênicosRESUMO
The therapeutic concept of unleashing a pre-existing immune response against the tumor by the application of immune-checkpoint inhibitors (ICI) has resulted in long-term survival in advanced cancer patient subgroups. However, the majority of patients do not benefit from single-agent ICI and therefore new combination strategies are eagerly necessitated. In addition to conventional chemotherapy, kinase inhibitors as well as tumor-specific vaccinations are extensively investigated in combination with ICI to augment therapy responses. An unprecedented clinical outcome with chimeric antigen receptor (CAR-)T cell therapy has led to the approval for relapsed/refractory diffuse large B cell lymphoma and B cell acute lymphoblastic leukemia whereas response rates in solid tumors are unsatisfactory. Immune-checkpoints negatively impact CAR-T cell therapy in hematologic and solid malignancies and as a consequence provide a therapeutic target to overcome resistance. Established biomarkers such as programmed death ligand 1 (PD-L1) and tumor mutational burden (TMB) help to select patients who will benefit most from ICI, however, biomarker negativity does not exclude responses. Investigating alterations in the antigen presenting pathway as well as radiomics have the potential to determine tumor immunogenicity and response to ICI. Within this review we summarize the literature about specific combination partners for ICI and the applicability of artificial intelligence to predict ICI therapy responses.
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Antineoplásicos Imunológicos/farmacologia , Inteligência Artificial , Inibidores de Checkpoint Imunológico/farmacologia , Animais , Antineoplásicos Imunológicos/uso terapêutico , Biomarcadores Tumorais , Vacinas Anticâncer/efeitos adversos , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Terapia Combinada , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/efeitos adversos , Imunoterapia/métodos , Terapia de Alvo Molecular , Neoplasias/etiologia , Neoplasias/metabolismo , Neoplasias/terapia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Resultado do TratamentoRESUMO
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 terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Quimioterapia de Consolidação , Intervalo Livre de Doença , Relação Dose-Resposta a Droga , Esquema de Medicação , Toxidermias/etiologia , Feminino , Doenças Hematológicas/induzido quimicamente , Humanos , Imunidade Celular/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Imunoterapia , Estimativa de Kaplan-Meier , Lenalidomida , Leucemia Linfocítica Crônica de Células B/genética , Contagem de Linfócitos , Quimioterapia de Manutenção , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Indução de Remissão , Rituximab/administração & dosagem , Rituximab/efeitos adversos , Subpopulações de Linfócitos T/efeitos dos fármacos , Talidomida/administração & dosagem , Talidomida/efeitos adversos , Talidomida/análogos & derivados , Talidomida/farmacologia , Resultado do Tratamento , Vidarabina/administração & dosagem , Vidarabina/efeitos adversos , Vidarabina/análogos & derivadosRESUMO
Although chronic lymphocytic leukaemia (CLL) is a B cell malignancy, earlier studies have indicated a role of T cells in tumour growth and disease progression. In particular, the functional silencing of antigen-experienced T cells, called T cell exhaustion, has become implicated in immune evasion in CLL. In this study, we tested whether T cell exhaustion is recapitulated in the TCL1(tg) mouse model for CLL. We show that T cells express high levels of the inhibitory exhaustion markers programmed cell death 1 (PDCD1, also termed PD-1) and lymphocyte-activation gene 3 (LAG3), whereas CLL cells express high levels of CD274 (also termed PD-ligand 1). In addition, the fraction of exhausted T cells increases with CLL progression. Finally, we demonstrate that exhausted T cells are reinvigorated towards CLL cytotoxicity by inhibition of PDCD1/CD274 interaction in vivo. These results suggest that T cell exhaustion contributes to CLL pathogenesis and that interference with PDCD1/CD274 signalling holds high potential for therapeutic approaches.
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Regulação Leucêmica da Expressão Gênica/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Regulação Leucêmica da Expressão Gênica/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Transdução de Sinais/genética , Linfócitos T/patologiaRESUMO
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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Processamento Alternativo , Citidina Desaminase/genética , Leucemia Linfocítica Crônica de Células B/enzimologia , Animais , Citidina Desaminase/análise , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The activation-induced cytidine deaminase (AID) mediates somatic hypermutation and class switch recombination of the Ig genes by directly deaminating cytosines to uracils. As AID causes a substantial amount of off-target mutations, its activity has been associated with lymphomagenesis and clonal evolution of B-cell malignancies. Although it has been shown that AID is expressed in B-cell chronic lymphocytic leukemia (CLL), a clear analysis of in vivo AID activity in this B-cell malignancy remained elusive. In this study performed on primary human CLL samples, we report that, despite the presence of a dominant VDJ heavy chain region, a substantial intraclonal diversity was observed at VDJ as well as at IgM switch regions (Sµ), showing ongoing AID activity in vivo during disease progression. This AID-mediated heterogeneity was higher in CLL subclones expressing CD86, which we identified as the proliferative CLL fraction. Finally, CD86 expression correlated with shortened time to first treatment and increased γ-H2AX focus formation. Our data demonstrate that AID is active in CLL in vivo and thus, AID likely contributes to clonal evolution of CLL.
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Antígeno B7-2/imunologia , Proliferação de Células , Citidina Desaminase/imunologia , Dano ao DNA/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Proteínas de Neoplasias/imunologia , Feminino , Regulação Leucêmica da Expressão Gênica/imunologia , Genes de Cadeia Pesada de Imunoglobulina/imunologia , Histonas/imunologia , Humanos , Região de Troca de Imunoglobulinas/imunologia , Cadeias mu de Imunoglobulina/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , MasculinoRESUMO
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 Tumorais , Fezes , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Neoplasias Retais , Humanos , Fezes/química , Neoplasias Retais/genética , Neoplasias Retais/patologia , Neoplasias Retais/sangue , Biomarcadores Tumorais/genética , Biópsia Líquida/métodos , Feminino , Masculino , DNA Tumoral Circulante/genética , DNA Tumoral Circulante/sangue , Pessoa de Meia-Idade , Idoso , Análise Mutacional de DNA , Heterogeneidade Genética , DNA de Neoplasias/sangue , DNA de Neoplasias/genéticaRESUMO
Cyclooxygenase-2 (COX-2) is a key regulator of inflammation. High constitutive COX-2 expression enhances survival and proliferation of cancer cells, and adversely impacts antitumor immunity. The expression of COX-2 is modulated by various signaling pathways. Recently, we identified the melastatin-like transient-receptor-potential-7 (TRPM7) channel-kinase as modulator of immune homeostasis. TRPM7 protein is essential for leukocyte proliferation and differentiation, and upregulated in several cancers. It comprises of a cation channel and an atypical α-kinase, linked to inflammatory cell signals and associated with hallmarks of tumor progression. A role in leukemia has not been established, and signaling pathways are yet to be deciphered. We show that inhibiting TRPM7 channel-kinase in chronic myeloid leukemia (CML) cells results in reduced constitutive COX-2 expression. By utilizing a CML-derived cell line, HAP1, harboring CRISPR/Cas9-mediated TRPM7 knockout, or a point mutation inactivating TRPM7 kinase, we could link this to reduced activation of AKT serine/threonine kinase and mothers against decapentaplegic homolog 2 (SMAD2). We identified AKT as a direct in vitro substrate of TRPM7 kinase. Pharmacologic blockade of TRPM7 in wildtype HAP1 cells confirmed the effect on COX-2 via altered AKT signaling. Addition of an AKT activator on TRPM7 kinase-dead cells reconstituted the wildtype phenotype. Inhibition of TRPM7 resulted in reduced phosphorylation of AKT and diminished COX-2 expression in peripheral blood mononuclear cells derived from CML patients, and reduced proliferation in patient-derived CD34+ cells. These results highlight a role of TRPM7 kinase in AKT-driven COX-2 expression and suggest a beneficial potential of TRPM7 blockade in COX-2-related inflammation and malignancy.
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Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide , Canais de Cátion TRPM , Humanos , Proteínas Proto-Oncogênicas c-akt/genética , Ciclo-Oxigenase 2/genética , Canais de Cátion TRPM/genética , Leucócitos Mononucleares/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Inflamação , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Background: Next generation sequencing (NGS) has become indispensable for diagnosis, risk stratification, prognostication, and monitoring of response in patients with myeloid neoplasias. Guidelines require bone marrow evaluations for the above, which are often not performed outside of clinical trials, indicating a need for surrogate samples. Methods: Myeloid NGS analyses (40 genes and 29 fusion drivers) of 240 consecutive, non-selected, prospectively collected, paired bone marrow/peripheral blood samples were compared. Findings: Very strong correlation (r = 0.91, p < 0.0001), high concordance (99.6%), sensitivity (98.8%), specificity (99.9%), positive predictive value (99.8%), and negative predictive value (99.6%) between NGS analyses of paired samples was observed. A total of 9/1321 (0.68%) detected mutations were discordant, 8 of which had a variant allele frequency (VAF) ≤ 3.7%. VAFs between peripheral blood and bone marrow samples were very strongly correlated in the total cohort (r = 0.93, p = 0.0001) and in subgroups without circulating blasts (r = 0.92, p < 0.0001) or with neutropenia (r = 0.88, p < 0.0001). There was a weak correlation between the VAF of a detected mutation and the blast count in either the peripheral blood (r = 0.19) or the bone marrow (r = 0.11). Interpretation: Peripheral blood samples can be used to molecularly classify and monitor myeloid neoplasms via NGS without loss of sensitivity/specificity, even in the absence of circulating blasts or in neutropenic patients.