RESUMEN
To investigate clonal hematopoiesis associated gene mutations in vitro and to unravel the direct impact on the human stem and progenitor cell (HSPC) compartment, we targeted healthy, young hematopoietic progenitor cells, derived from umbilical cord blood samples, with CRISPR/Cas9 technology. Site-specific mutations were introduced in defined regions of DNMT3A, TET2, and ASXL1 in CD34+ progenitor cells that were subsequently analyzed in short-term as well as long-term in vitro culture assays to assess self-renewal and differentiation capacities. Colony-forming unit (CFU) assays revealed enhanced self-renewal of TET2 mutated (TET2mut) cells, whereas ASXL1mut as well as DNMT3Amut cells did not reveal significant changes in short-term culture. Strikingly, enhanced colony formation could be detected in long-term culture experiments in all mutants, indicating increased self-renewal capacities. While we could also demonstrate preferential clonal expansion of distinct cell clones for all mutants, the clonal composition after long-term culture revealed a mutation-specific impact on HSPCs. Thus, by using primary umbilical cord blood cells, we were able to investigate epigenetic driver mutations without confounding factors like age or a complex mutational landscape, and our findings provide evidence for a direct impact of clonal hematopoiesis-associated mutations on self-renewal and clonal composition of human stem and progenitor cells.
Asunto(s)
Sistemas CRISPR-Cas , Sangre Fetal , Hematopoyesis Clonal , Hematopoyesis/genética , Células Madre Hematopoyéticas , HumanosRESUMEN
Evidence for umbilical cord blood (UCB) cell therapies as a potential intervention for neurological diseases is emerging. To date, most existing trials worked with allogenic cells, as the collection of autologous UCB from high-risk patients is challenging. In obstetric emergencies the collection cannot be planned. In preterm infants, late cord clamping and anatomic conditions may reduce the availability. The aim of the present study was to assess the feasibility of UCB collection in neonates at increased risk of brain damage. Infants from four high-risk groups were included: newborns with perinatal hypoxemia, gestational age (GA) ≤30 + 0 weeks and/or birthweight <1,500 g, intrauterine growth restriction (IUGR), or monochorionic twins with twin-to-twin transfusion syndrome (TTTS). Feasibility of collection, quantity and quality of obtained UCB [total nucleated cell count (TNC), volume, sterility, and cell viability], and neonatal outcome were assessed. UCB collection was successful in 141 of 177 enrolled patients (hypoxemia n = 10; GA ≤30 + 0 weeks n = 54; IUGR n = 71; TTTS n = 6). Twenty-six cases were missed. The amount of missed cases per month declined over the time. Volume of collected UCB ranged widely (median: 24.5 ml, range: 5.0-102 ml) and contained a median of 0.77 × 108 TNC (range: 0.01-13.0 × 108). TNC and UCB volume correlated significantly with GA. A total of 10.7% (19/177) of included neonates developed brain lesions. To conclude, collection of UCB in neonates at high risk of brain damage is feasible with a multidisciplinary approach and intensive training. High prevalence of brain damage makes UCB collection worthwhile. Collected autologous UCB from mature neonates harbors a sufficient cell count for potential therapy. However, quality and quantity of obtained UCB are critical for potential therapy in preterm infants. Therefore, for extremely preterm infants alternative cell sources such as UCB tissue should be investigated for autologous treatment options because of the low yield of UCB.
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Lesiones Encefálicas/fisiopatología , Encéfalo/patología , Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Trasplante Autólogo/métodos , Estudios de Factibilidad , Femenino , Humanos , Recién Nacido , Proyectos Piloto , Embarazo , Factores de RiesgoRESUMEN
BACKGROUND: Colorectal cancer is one of the most common malignancies worldwide; whilst approximately 20% of patients have hepatic disease at presentation. Hepatic resection remains the gold standard of care; however, it is associated with significant morbidity. We sought to establish whether the lymphocyte-to-monocyte ratio (LMR) could help predict post-operative complications, thus improving patient outcomes. METHODS: We performed a retrospective cohort study of patients undergoing hepatic resection at a single centre. Baseline demographics and complications within 30 days following surgery were recorded. White blood cell counts and C-reactive protein (CRP) were recorded pre-operatively, and until post-operative day 7. RESULTS: A total of 188 operations were included. About 47.3% of resections had a complicated recovery, of which 31.46% were major. The median LMR was 1.29 across the cohort, 1.60 for uncomplicated procedures, 1.14 for those with complications and 0.85 in major complications. For detecting major complications versus an uncomplicated recovery, median LMR was the best parameter (area under the curve 0.78), whilst it was the only parameter to accurately predict such complications within 48 hours of surgery (area under the curve 0.72 on day 1). It was consistently the most accurate parameter at detecting uncomplicated versus complicated recovery, minor versus major complications, and major complications versus an uncomplicated recovery, at numerous timepoints over the post-operative period. CONCLUSION: The LMR appears better at predicting complications following hepatic resection for colorectal liver metastases, as opposed to conventionally measured parameters.
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Adenocarcinoma/secundario , Neoplasias Colorrectales/patología , Técnicas de Apoyo para la Decisión , Hepatectomía , Leucocitos/metabolismo , Neoplasias Hepáticas/secundario , Complicaciones Posoperatorias/etiología , Adenocarcinoma/cirugía , Biomarcadores/sangre , Femenino , Estudios de Seguimiento , Humanos , Recuento de Leucocitos , Neoplasias Hepáticas/cirugía , Linfocitos/metabolismo , Masculino , Monocitos/metabolismo , Complicaciones Posoperatorias/sangre , Complicaciones Posoperatorias/diagnóstico , Curva ROC , Estudios Retrospectivos , Medición de Riesgo , Factores de RiesgoRESUMEN
BACKGROUND AND OBJECTIVES: Approximately 30-50% of patients with colorectal cancer develop liver metastasis for which liver resection is the only hope for potential cure. However, hepatic resection is associated with considerable morbidity. The aim was to detect early complications by utilising the neutrophil: lymphocyte ratio (NLR). METHODS: We performed a retrospective cohort study of patients undergoing hepatic resection at a single institution between 2008 and 2016. Baseline demographics and complications within 30 days following surgery were recorded, with blood tests measured until day 7. Statistical analysis was performed using Mann Whitney and ROC analysis. RESULTS: One hundred eighty-eight operations were included. 47.3% had an associated complication, of which 31.46% were major. The median NLR was 6.31 across the cohort, 5.44 for uncomplicated procedures, 7.0 for complications and 10.65 in major complications. Median NLR was the best parameter for detecting major complications versus minor complications (AUC 0.74) as opposed to lymphocytes (AUC 0.65), neutrophils (AUC 0.60), and CRP (AUC 0.60). The diagnostic ability of NLR increased further when predicting major complications versus an uncomplicated recovery (AUC 0.78), and it was the only significant parameter in the early post-operative period on days 2, 3, and 4 (AUC 0.70, 0.72, and 0.75). CONCLUSIONS: The NLR may have a role in predicting complications following hepatic resection for CLM, and with earlier detection, potentially improving outcomes.
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Neoplasias Colorrectales/patología , Hepatectomía/efectos adversos , Neoplasias Hepáticas/secundario , Linfocitos/patología , Neutrófilos/patología , Complicaciones Posoperatorias/diagnóstico , Anciano , Neoplasias Colorrectales/cirugía , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Hepáticas/cirugía , Metástasis Linfática , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Pronóstico , Estudios Prospectivos , Tasa de SupervivenciaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) has generally a poor prognosis, but recent data suggest that there are molecular subtypes differing in clinical outcome. This study examines the association between histopathologic heterogeneity, genetic profile, and survival. Tumor histology from 177 resected PDAC patients with follow-up data was subclassified according to predominant growth pattern, and four key genes were analyzed. PDACs were classified as conventional (51%), combined with a predominant component (41%), variants and special carcinomas (8%). Patients with combined PDACs and a dominant cribriform component survived longer than patients with conventional or other combined PDACs. Genetic alterations in at least two out of four genes were found in 95% of the patients (KRAS 93%, TP53 79%, CDKN2A/p16 75%, SMAD4 37%). Patients with less than four mutations survived significantly longer (p = 0.04) than those with alterations in all four genes. Patients with either wildtype KRAS or CDKN2A/p16 lived significantly longer than those with alterations in these genes (p = 0.018 and p = 0.006, respectively). Our data suggest that the number of altered genes, the mutational status of KRAS and certain morphological subtypes correlate with the outcome of patients with PDAC. Future pathology reporting of PDAC should therefore include the KRAS status and a detailed morphological description.
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Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/patología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/cirugía , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Estudios de Seguimiento , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mutación , Clasificación del Tumor , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/cirugía , Fenotipo , Pronóstico , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteína Smad4/genética , Análisis de Supervivencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Somatostatin receptor 2A expression is a feature of well-differentiated neuroendocrine neoplasms and is important for their diagnosis and therapy. Little is known about somatostatin receptor 2A expression in poorly differentiated neuroendocrine neoplasms in relation to TP53 and RB1 status and how these features may contribute to the separation of well from poorly differentiated neuroendocrine neoplasms with a proliferation index above 20%. This study investigates the expression of somatostatin receptors, p53 and Rb1, and TP53 alterations in pancreatic and extrapancreatic well and poorly differentiated neuroendocrine neoplasms (Ki67-index >20%). Thirty-seven poorly differentiated neuroendocrine neoplasms of pancreatic (n=12) and extrapancreatic origin (n=25) as well as 10 well-differentiated neuroendocrine neoplasms of the pancreas (n=9) and rectum (n=1) with a Ki67-index >20% were immunostained for synaptophysin, chromogranin A, Ki67, CD56, p53, Rb1, ATRX, DAXX, progesterone receptor, somatostatin receptor 2A, somatostatin receptor 5, and cytokeratin 20, and sequenced for TP53, exons 5-9. Somatostatin receptor 2A was positive in 6/37 of poorly differentiated and in 8/10 of well-differentiated neuroendocrine neoplasms. One well-differentiated and two poorly differentiated neuroendocrine neoplasms expressed somatostatin receptor 5. Abnormal nuclear p53 and Rb1 staining was found in 29/37 and 22/37 poorly differentiated neuroendocrine neoplasms, respectively, whereas all well-differentiated neuroendocrine neoplasms showed normal p53 and Rb1 expression. TP53 gene alterations were restricted to poorly differentiated neuroendocrine neoplasms (24/34) and correlated well with p53 expression. All cases were progesterone receptor negative. Somatostatin receptor 2A expression is not limited to well-differentiated neuroendocrine neoplasms but also occurs in 16% of poorly differentiated neuroendocrine neoplasms from various sites. Most poorly differentiated neuroendocrine neoplasms are characterized by TP53 alterations and Rb1 loss, usually in the absence of somatostatin receptor 2A expression. In the pancreas, these criteria contribute to separate well-differentiated neuroendocrine neoplasms with a Ki67-index above 20% from poorly differentiated neuroendocrine neoplasms.
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Tumores Neuroendocrinos/metabolismo , Páncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores de Somatostatina/metabolismo , Proteínas de Unión a Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Tumores Neuroendocrinos/patología , Páncreas/patología , Neoplasias Pancreáticas/patología , Adulto JovenRESUMEN
Here, we show CRISPR/Cas9-based targeted somatic multiplex-mutagenesis and its application for high-throughput analysis of gene function in mice. Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We observed Darwinian selection of target genes, which suppress tumorigenesis in the respective cellular/tissue context, such as Pten or Cdkn2a, and conversely found low frequency of Brca1/2 alterations, explaining mutational spectra in human ICC/HCC. Our studies show that multiplexed CRISPR/Cas9 can be used for recessive genetic screening or high-throughput cancer gene validation in mice. The analysis of CRISPR/Cas9-induced tumors provided support for a major role of chromatin modifiers in hepatobiliary tumorigenesis, including that of ARID family proteins, which have recently been reported to be mutated in ICC/HCC. We have also comprehensively characterized the frequency and size of chromosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISPR/Cas9 multiplexing, as well as opportunities for chromosome engineering in the context of hepatobiliary tumorigenesis. Our study describes novel approaches to model and study cancer in a high-throughput multiplexed format that will facilitate the functional annotation of cancer genomes.
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Sistemas CRISPR-Cas/genética , Carcinoma Hepatocelular/genética , Modelos Animales de Enfermedad , Genómica/métodos , Ensayos Analíticos de Alto Rendimiento , Neoplasias Hepáticas/genética , Mutagénesis/genética , Animales , Secuencia de Bases , Marcación de Gen , Técnicas Histológicas , Hígado/metabolismo , Ratones , Datos de Secuencia Molecular , Selección Genética/genéticaRESUMEN
Over the past decade, there have been substantial improvements in our knowledge of pancreatic neoplasms and their precursor lesions. Extensive genetic analyses, recently using high-throughput molecular techniques and next-generation sequencing methodologies, and the development of sophisticated genetically engineered mouse models closely recapitulating human disease, have improved our understanding of the genetic basis of pancreatic neoplasms. These advances are paving the way for refined, molecular-based classifications of pancreatic neoplasms with the potential to better predict prognosis and, possibly, response to therapy. Another major development resides in the identification of subsets of pancreatic exocrine and endocrine neoplasms which occur in the context of hereditary syndromes and whose genetic basis and tumor development have been at least partially defined. However, despite all molecular progress, correct and careful morphological characterization of tissue specimens both in the context of experimental and routine diagnostic pathology represents the basis for any further genetic investigation or clinical decision. This review focuses on the current and new concepts of classification and on the current models of tumor development, both in the field of exocrine and endocrine neoplasms, and underscores the importance of applying standardized terminology to allow adequate data interpretation and promote scientific exchange in the field of pancreas research.
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Neoplasias Experimentales/patología , Neoplasias Pancreáticas/patología , Animales , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Experimentales/clasificación , Neoplasias Experimentales/genética , Neoplasias Pancreáticas/clasificación , Neoplasias Pancreáticas/genéticaRESUMEN
BACKGROUND: Lenalidomide is a thalidomide analogue with immunomodulatory and anti-angiogenic properties that include altering cytokine production, activating T cells, and augmenting natural killer cell function. Lenalidomide is approved by the U.S. Food and Drug Administration (FDA) for single-agent treatment of myelodysplastic syndromes associated with a 5q deletion and as a combination therapy with dexamethasone for the treatment of multiple myeloma. METHODS: All prospective phase I-III clinical trials and preclinical data published until October 2011 and relevant literature were reviewed. RESULTS: In phase I and/or II studies of single-agent lenalidomide in patients with advanced cancer, responses were reported in patients with prostate, thyroid, hepatocellular, pancreatic, and renal cancer and melanoma. The most common toxicities were hematologic, and in the first clinical trials, thrombotic events were noted. When anticoagulation prophylaxis and exclusion of patients with a history of thrombosis were implemented, thrombotic complications became uncommon. CONCLUSION: Monitoring of blood counts and for evidence of thromboembolic events is essential for patients treated with lenalidomide. Ongoing trials of lenalidomide combination therapy offer a treatment option for patients with advanced cancer and will better define the role of lenalidomide in solid tumors.