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BACKGROUND: Shallow whole genome sequencing (Shallow-seq) is used to determine the copy number aberrations (CNA) in tissue samples and circulating tumor DNA. However, costs of NGS and challenges of small biopsies ask for an alternative to the untargeted NGS approaches. The mFAST-SeqS approach, relying on LINE-1 repeat amplification, showed a good correlation with Shallow-seq to detect CNA in blood samples. In the present study, we evaluated whether mFAST-SeqS is suitable to assess CNA in small formalin-fixed paraffin-embedded (FFPE) tissue specimens, using vulva and anal HPV-related lesions. METHODS: Seventy-two FFPE samples, including 36 control samples (19 vulva;17 anal) for threshold setting and 36 samples (24 vulva; 12 anal) for clinical evaluation, were analyzed by mFAST-SeqS. CNA in vulva and anal lesions were determined by calculating genome-wide and chromosome arm-specific z-scores in comparison with the respective control samples. Sixteen samples were also analyzed with the conventional Shallow-seq approach. RESULTS: Genome-wide z-scores increased with the severity of disease, with highest values being found in cancers. In vulva samples median and inter quartile ranges [IQR] were 1[0-2] in normal tissues (n = 4), 3[1-7] in premalignant lesions (n = 9) and 21[13-48] in cancers (n = 10). In anal samples, median [IQR] were 0[0-1] in normal tissues (n = 4), 14[6-38] in premalignant lesions (n = 4) and 18[9-31] in cancers (n = 4). At threshold 4, all controls were CNA negative, while 8/13 premalignant lesions and 12/14 cancers were CNA positive. CNA captured by mFAST-SeqS were mostly also found by Shallow-seq. CONCLUSION: mFAST-SeqS is easy to perform, requires less DNA and less sequencing reads reducing costs, thereby providing a good alternative for Shallow-seq to determine CNA in small FFPE samples.
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Variações do Número de Cópias de DNA , Inclusão em Parafina , Humanos , Feminino , Variações do Número de Cópias de DNA/genética , Inclusão em Parafina/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Formaldeído , Fixação de Tecidos/métodos , Sequenciamento Completo do Genoma/métodos , Neoplasias Vulvares/genética , Neoplasias Vulvares/patologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/diagnóstico , Neoplasias do Ânus/genética , Neoplasias do Ânus/diagnósticoRESUMO
Large-scale chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage. Suitable functional genomics approaches to study the oncogenic driving capacity of large-scale deletions are limited. Here, we present an effective technique to engineer large-scale deletions by CRISPR-Cas9 and create isogenic cell line models. We simultaneously induce double-strand breaks (DSBs) at two ends of a chromosomal arm and select the cells that have lost the intermittent region. Using this technique, we induced large-scale deletions on chromosome 11q (65 Mb) and chromosome 6q (53 Mb) in neuroblastoma cell lines. A high frequency of successful deletions (up to 30% of selected clones) and increased colony forming capacity in the 11q deleted lines suggest an oncogenic advantage of these deletions. Such isogenic models enable further research on the role of large-scale deletions in tumor development and growth, and their possible therapeutic potential.
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Sistemas CRISPR-Cas , DNA/metabolismo , Neuroblastoma/genética , Linhagem Celular Tumoral , Aberrações Cromossômicas , Deleção Cromossômica , HumanosRESUMO
Detection of DNA copy number aberrations by shallow whole-genome sequencing (WGS) faces many challenges, including lack of completion and errors in the human reference genome, repetitive sequences, polymorphisms, variable sample quality, and biases in the sequencing procedures. Formalin-fixed paraffin-embedded (FFPE) archival material, the analysis of which is important for studies of cancer, presents particular analytical difficulties due to degradation of the DNA and frequent lack of matched reference samples. We present a robust, cost-effective WGS method for DNA copy number analysis that addresses these challenges more successfully than currently available procedures. In practice, very useful profiles can be obtained with â¼0.1× genome coverage. We improve on previous methods by first implementing a combined correction for sequence mappability and GC content, and second, by applying this procedure to sequence data from the 1000 Genomes Project in order to develop a blacklist of problematic genome regions. A small subset of these blacklisted regions was previously identified by ENCODE, but the vast majority are novel unappreciated problematic regions. Our procedures are implemented in a pipeline called QDNAseq. We have analyzed over 1000 samples, most of which were obtained from the fixed tissue archives of more than 25 institutions. We demonstrate that for most samples our sequencing and analysis procedures yield genome profiles with noise levels near the statistical limit imposed by read counting. The described procedures also provide better correction of artifacts introduced by low DNA quality than prior approaches and better copy number data than high-resolution microarrays at a substantially lower cost.
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Biologia Computacional , Variações do Número de Cópias de DNA , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Algoritmos , Composição de Bases , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Biologia Computacional/métodos , Genômica/métodos , Humanos , Neoplasias/genética , SoftwareRESUMO
Pulmonary carcinoids comprise a well-differentiated subset of neuroendocrine tumors usually associated with a favorable prognosis, but mechanisms underlying disease progression are poorly understood. In an explorative approach to identify pathways associated with progression, we compared gene expression profiles of tumors from five patients with a favorable and five with a poor disease outcome. Differentially expressed genes were validated using quantitative real-time PCR on 65 carcinoid tumors, in combination with survival analysis. One of the identified pathways was further examined using immunohistochemistry. As compared with other chromosomal locations, a significantly higher number of genes downregulated in carcinoids with a poor prognosis were located at chromosome 11q (P = 0.00017), a region known to be frequently lost in carcinoids. In addition, a number of upregulated genes were found involved in the mitotic spindle checkpoint, the chromosomal passenger complex (CPC), mitotic kinase CDC2 activity and the BRCA-Fanconi anemia pathway. At the individual gene level, BIRC5 (survivin), BUB1, CD44, IL20RA, KLK12 and OTP were independent predictors of patient outcome. For survivin, the number of positive nuclei was also related to poor prognosis within the group of carcinoids. Aurora B kinase and survivin, major components of the CPC, were particularly upregulated in high-grade carcinomas and may therefore comprise therapeutic targets for these tumors. To our knowledge, this is the first expression profiling study focusing specifically on pulmonary carcinoids and progression. We have identified novel pathways underlying malignant progression and validated several genes as being strong prognostic indicators, some of which could serve as putative therapeutic targets.
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Tumor Carcinoide/genética , Tumor Carcinoide/patologia , Regulação Neoplásica da Expressão Gênica/genética , Transdução de Sinais/genética , Transcriptoma/genética , Adulto , Idoso , Tumor Carcinoide/mortalidade , Instabilidade Cromossômica/genética , Cromossomos Humanos Par 11/genética , Progressão da Doença , Regulação para Baixo/genética , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mitose/genética , Prognóstico , Análise de Sobrevida , Regulação para Cima/genética , Adulto JovemRESUMO
Formalin-fixed, paraffin-embedded (FFPE) archival tissue is an important source of DNA material. The most commonly used technique to identify copy number aberrations from chromosomal DNA in tumorigenesis is array comparative genomic hybridization (aCGH). Although copy number analysis using DNA from FFPE archival tissue is challenging, several research groups have reported high quality and reproducible DNA copy number results using aCGH. Aim of this study is to compare the commercially available aCGH platforms suitable for high-resolution copy number analysis using FFPE-derived DNA. Two dual channel aCGH platforms (Agilent and NimbleGen) and a single channel SNP-based platform (Affymetrix) were evaluated using seven FFPE colon cancer samples, and median absolute deviation (MAD), deflection, signal-to-noise ratio, and DNA input requirements were used as quality criteria. Large differences were observed between platforms; Agilent and NimbleGen showed better MAD values (0.13 for both) compared with Affymetrix (0.22). On the contrary, Affymetrix showed a better deflection of 0.94, followed by 0.71 for Agilent and 0.51 for NimbleGen. This resulted in signal-to-nose ratios that were comparable between the three commercially available platforms. Interestingly, DNA input amounts from FFPE material lower than recommended still yielded high quality profiles on all platforms. Copy number analysis using DNA derived from FFPE archival material is feasible using all three high-resolution copy number platforms and shows reproducible results, also with DNA input amounts lower than recommended.
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Neoplasias do Colo/genética , Hibridização Genômica Comparativa/métodos , Variações do Número de Cópias de DNA , DNA/genética , Preservação de Tecido/métodos , DNA/isolamento & purificação , DNA de Neoplasias/genética , Formaldeído , Humanos , Inclusão em Parafina , Razão Sinal-RuídoRESUMO
Somatic copy number alterations can be detected in cell-free DNA (cfDNA) by shallow whole genome sequencing (sWGS). PCR is typically included in library preparations, but a PCR-free method could serve as a high-throughput alternative. To evaluate a PCR-free method for research and diagnostics, archival peripheral blood or bone marrow plasma samples, collected in EDTA- or lithium-heparin-containing tubes, were collected from patients with non-small-cell lung cancer (n = 10 longitudinal samples; 4 patients), B-cell lymphoma (n = 31), and acute myeloid leukemia (n = 15), or from healthy donors (n = 14). sWGS was performed on PCR-free and PCR library preparations, and the mapping quality, percentage of unique reads, genome coverage, fragment lengths, and copy number profiles were compared. The percentage of unique reads was significantly higher for PCR-free method compared with PCR method, independent of the type of collection tube: EDTA PCR-free method, 96.4% (n = 35); EDTA PCR method, 85.1% (n = 32); heparin PCR-free method, 94.5% (n = 25); and heparin PCR method, 89.4% (n = 10). All other evaluated metrics were highly comparable for PCR-free and PCR library preparations. These results demonstrate the feasibility of somatic copy number alteration detection by PCR-free sWGS using cfDNA from plasma collected in EDTA- or lithium-heparin-containing tubes and pave the way for an automated cfDNA analysis workflow for samples from cancer patients.
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Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , Variações do Número de Cópias de DNA , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/genética , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Linfoma de Células B/sangue , Linfoma de Células B/genética , Reação em Cadeia da Polimerase/métodos , Sequenciamento Completo do Genoma/métodos , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Coleta de Amostras Sanguíneas/métodos , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Estudos de Casos e Controles , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Estudos de Viabilidade , Humanos , Leucemia Mieloide Aguda/diagnóstico , Limite de Detecção , Biópsia Líquida , Estudos Longitudinais , Neoplasias Pulmonares/diagnóstico , Linfoma de Células B/diagnósticoRESUMO
PURPOSE: Bone marrow is a common homing organ for early disseminated tumor cells (DTC) and their presence can predict the subsequent occurrence of overt metastasis and survival in lung cancer. It is still unclear whether the shedding of DTC from the primary tumor is a random process or a selective release driven by a specific genomic pattern. EXPERIMENTAL DESIGN: DTCs were identified in bone marrow from lung cancer patients by an immunocytochemical cytokeratin assay. Genomic aberrations and expression profiles of the respective primary tumors were assessed by microarrays and fluorescence in situ hybridization analyses. The most significant results were validated on an independent set of primary lung tumors and brain metastases. RESULTS: Combination of DNA copy number profiles (array comparative genomic hybridization) with gene expression profiles identified five chromosomal regions differentiating bone marrow-negative from bone marrow-positive patients (4q12-q32, 10p12-p11, 10q21-q22, 17q21, and 20q11-q13). Copy number changes of 4q12-q32 were the most prominent finding, containing the highest number of differentially expressed genes irrespective of chromosomal size (P=0.018). Fluorescence in situ hybridization analyses on further primary lung tumor samples confirmed the association between loss of 4q and bone marrow-positive status. In bone marrow-positive patients, 4q was frequently lost (37% versus 7%), whereas gains could be commonly found among bone marrow-negative patients (7% versus 17%). The same loss was also found to be common in brain metastases from both small and non-small cell lung cancer patients (39%). CONCLUSIONS: Thus, our data indicate, for the first time, that early hematogenous dissemination of tumor cells might be driven by a specific pattern of genomic changes.
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Neoplasias Encefálicas/genética , Deleção Cromossômica , Cromossomos Humanos Par 4/genética , Perfilação da Expressão Gênica , Neoplasias Pulmonares/genética , Adenocarcinoma/genética , Adenocarcinoma/secundário , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/secundário , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/secundário , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/secundário , Hibridização Genômica Comparativa , Feminino , Dosagem de Genes , Genoma Humano , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/secundárioRESUMO
OBJECTIVES: The majority of patients with locally advanced larynx or hypopharynx squamous cell carcinoma are treated with organ-preserving chemoradiotherapy (CRT). Clinical outcome following CRT varies greatly. We hypothesized that tumor microRNA (miRNA) expression is predictive for outcome following CRT. METHODS: Next-generation sequencing (NGS) miRNA profiling was performed on 37 formalin-fixed paraffin-embedded (FFPE) tumor samples. Patients with a recurrence-free survival (RFS) of less than 2 years and patients with late/no recurrence within 2 years were compared by differential expression analysis. Tumor-specific miRNAs were selected based on normal mucosa miRNA expression data from The Cancer Genome Atlas database. A model was constructed to predict outcome using group-regularized penalized logistic ridge regression. Candidate miRNAs were validated by RT-qPCR in the initial sample set as well as in 46 additional samples. RESULTS: Thirteen miRNAs were differentially expressed (p < 0.05, FDR < 0.1) according to outcome group. Initial class prediction in the NGS cohort (n = 37) resulted in a model combining five miRNAs and disease stage, able to predict CRT outcome with an area under the curve (AUC) of 0.82. In the RT-qPCR cohort (n = 83), 25 patients (30%) experienced early recurrence (median RFS 8 months; median follow-up 42 months). Class prediction resulted in a model combining let-7i-5p, miR-192-5p and disease stage, able to discriminate patients with good versus poor clinical outcome (AUC:0.80). CONCLUSION: The combined miRNA expression and disease stage prediction model for CRT outcome is superior to using either factor alone. This study indicates NGS miRNA profiling using FFPE specimens is feasible, resulting in clinically relevant biomarkers.
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Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy (CRS-HIPEC) may be curative for colorectal cancer patients with peritoneal metastases (PMs) but it has a high rate of morbidity. Accurate preoperative patient selection is therefore imperative, but is constrained by the limitations of current imaging techniques. In this pilot study, we explored the feasibility of circulating tumor (ct) DNA analysis to select patients for CRS-HIPEC. Thirty patients eligible for CRS-HIPEC provided blood samples preoperatively and during follow-up if the procedure was completed. Targeted Next-Generation Sequencing (NGS) of DNA from PMs was used to identify bespoke mutations that were subsequently tested in corresponding plasma cell-free (cf) DNA samples using droplet digital (dd) PCR. CtDNA was detected preoperatively in cfDNA samples from 33% of patients and was associated with a reduced disease-free survival (DFS) after CRS-HIPEC (median 6.0 months vs median not reached, p = 0.016). This association could indicate the presence of undiagnosed systemic metastases or an increased metastatic potential of the tumors. We demonstrate the feasibility of ctDNA to serve as a preoperative marker of recurrence in patients with PMs of colorectal cancer using a highly sensitive technique. A more appropriate treatment for patients with preoperative ctDNA detection may be systemic chemotherapy in addition to, or instead of, CRS-HIPEC.
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BACKGROUND: The active form of Vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), has strong anti-proliferative effects, yet the molecular mechanisms underneath this effect remain unclear. In contrast, the molecular mechanism of 1,25D for the regulation of calcium homeostasis has principally been resolved, demonstrating a pivotal role for the vitamin D receptor (VDR). RESULTS: We first addressed the question whether the anti-proliferative effects of 1,25D are influenced by VDR. Knockdown of VDR by siRNA did not affect the anti-proliferative effects of 1,25D in MCF7 breast cancer cells. This unanticipated finding led us to take an alternative approach using genome wide screens to study the molecular mechanisms of 1,25D in proliferation. For that purpose, four independently developed and stable 1,25D resistant MCF7 cell lines were analyzed. Array CGH identified a copy number alteration in a region of 13.5 Mb at chromosome 11q13.4-14.1 common to all four 1,25D resistant cell lines. Expression arrays revealed that no single gene was differentially expressed between the sensitive and resistant cells, but multiple membrane receptor signaling pathways were altered in the 1,25D resistant cell lines. Importantly, in the genome wide experiments neither VDR, CYP24A1 nor other known vitamin D signaling pathway genes were associated with 1,25D resistance. CONCLUSION: In conclusion, siRNA and genome wide studies both suggest that the anti-proliferative effects of 1,25D in MCF7 breast tumor cell lines do not rely on classical Vitamin D pathway per se.
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RNA Interferente Pequeno/genética , Receptores de Calcitriol/deficiência , Receptores de Calcitriol/genética , Vitamina D/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromossomos Humanos Par 11/genética , Resistencia a Medicamentos Antineoplásicos/genética , Dosagem de Genes/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Receptores de Calcitriol/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0223827.].
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Multiple tumors in patients are frequently diagnosed, either synchronous or metachronous. The distinction between a second primary and a metastasis is important for treatment. Chromosomal DNA copy number aberrations (CNA) patterns are highly unique to specific tumors. The aim of this study was to assess genome-wide CNA-patterns as method to identify clonally related tumors in a prospective cohort of patients with synchronous or metachronous tumors, with at least one intrapulmonary tumor. In total, 139 tumor pairs from 90 patients were examined: 35 synchronous and 104 metachronous pairs. Results of CNA were compared to histological type, clinicopathological methods (Martini-Melamed-classification (MM) and ACCP-2013-criteria), and, if available, EGFR- and KRAS-mutation analysis. CNA-results were clonal in 74 pairs (53%), non-clonal in 33 pairs (24%), and inconclusive in 32 pairs (23%). Histological similarity was found in 130 pairs (94%). Concordance between histology and conclusive CNA-results was 69% (74 of 107 pairs: 72 clonal and two non-clonal). In 31 of 103 pairs with similar histology, genetics revealed non-clonality. In two out of four pairs with non-matching histology, genetics revealed clonality. The subgroups of synchronous and metachronous pairs showed similar outcome for the comparison of histological versus CNA-results. MM-classification and ACCP-2013-criteria, applicable on 34 pairs, and CNA-results were concordant in 50% and 62% respectively. Concordance between mutation matching and conclusive CNA-results was 89% (8 of 9 pairs: six clonal and two non-clonal). Interestingly, in one patient both tumors had the same KRAS mutation, but the CNA result was non-clonal. In conclusion, although some concordance between histological comparison and CNA profiling is present, arguments exist to prefer extensive molecular testing to determine whether a second tumor is a metastasis or a second primary.
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Hibridização Genômica Comparativa/métodos , Variações do Número de Cópias de DNA , Neoplasias Pulmonares/diagnóstico , Segunda Neoplasia Primária/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Clonais/química , Células Clonais/patologia , Diagnóstico Diferencial , Receptores ErbB/genética , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Masculino , Pessoa de Meia-Idade , Mutação , Segunda Neoplasia Primária/genética , Estudos Prospectivos , Proteínas Proto-Oncogênicas p21(ras)/genética , Estudos Retrospectivos , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: First line chemotherapy is effective in 75 to 80% of patients with metastatic colorectal cancer (mCRC). We studied whether microRNA (miR) expression profiles can predict treatment outcome for first line fluoropyrimidine containing systemic therapy in patients with mCRC. METHODS: MiR expression levels were determined by next generation sequencing from snap frozen tumor samples of 88 patients with mCRC. Predictive miRs were selected with penalized logistic regression and posterior forward selection. The prediction co-efficients of the miRs were re-estimated and validated by real-time quantitative PCR in an independent cohort of 81 patients with mCRC. RESULTS: Expression levels of miR-17-5p, miR-20a-5p, miR-30a-5p, miR-92a-3p, miR-92b-3p and miR-98-5p in combination with age, tumor differentiation, adjuvant therapy and type of systemic treatment, were predictive for clinical benefit in the training cohort with an AUC of 0.78. In the validation cohort the addition of the six miR signature to the four clinicopathological factors demonstrated a significant increased AUC for predicting treatment response versus those with stable disease (SD) from 0.79 to 0.90. The increase for predicting treatment response versus progressive disease (PD) and for patients with SD versus those with PD was not significant. in the validation cohort. MiR-17-5p, miR-20a-5p and miR-92a-3p were significantly upregulated in patients with treatment response in both the training and validation cohorts. CONCLUSION: A six miR expression signature was identified that predicted treatment response to fluoropyrimidine containing first line systemic treatment in patients with mCRC when combined with four clinicopathological factors. Independent validation demonstrated added predictive value of this miR-signature for predicting treatment response versus SD. However, added predicted value for separating patients with PD could not be validated. The clinical relevance of the identified miRs for predicting treatment response has to be further explored.
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Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/terapia , MicroRNAs/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Área Sob a Curva , Biomarcadores Tumorais/metabolismo , Estudos de Coortes , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Curva ROCRESUMO
Purpose Patients with metastatic colorectal cancer (mCRC) have limited benefit from the addition of bevacizumab to standard chemotherapy. However, a subset probably benefits substantially, highlighting an unmet clinical need for a biomarker of response to bevacizumab. Previously, we demonstrated that losses of chromosomes 5q34, 17q12, and 18q11.2-q12.1 had a significant correlation with progression-free survival (PFS) in patients with mCRC treated with bevacizumab in the CAIRO2 clinical trial but not in patients who did not receive bevacizumab in the CAIRO trial. This study was designed to validate these findings. Materials and Methods Primary mCRC samples were analyzed from two cohorts of patients who received bevacizumab as first-line treatment; 96 samples from the European multicenter study Angiopredict (APD) and 81 samples from the Italian multicenter study, MOMA. A third cohort of 90 samples from patients with mCRC who did not receive bevacizumab was analyzed. Copy number aberrations of tumor biopsy specimens were measured by shallow whole-genome sequencing and were correlated with PFS, overall survival (OS), and response. Results Loss of chromosome 18q11.2-q12.1 was associated with prolonged PFS most significantly in both the cohorts that received bevacizumab (APD: hazard ratio, 0.54; P = .01; PFS difference, 65 days; MOMA: hazard ratio, 0.55; P = .019; PFS difference, 49 days). A similar association was found for OS and overall response rate in these two cohorts, which became significant when combined with the CAIRO2 cohort. Median PFS in the cohort of patients with mCRC who did not receive bevacizumab and in the CAIRO cohort was similar to that of the APD, MOMA, and CAIRO2 patients without an 18q11.2-q12.1 loss. Conclusion We conclude that the loss of chromosome 18q11.2-q12.1 is consistently predictive for prolonged PFS in patients receiving bevacizumab. The predictive value of this loss is substantiated by a significant gain in OS and overall response rate.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Deleção Cromossômica , Cromossomos Humanos Par 18 , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Bevacizumab/administração & dosagem , Capecitabina/administração & dosagem , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 5 , Ensaios Clínicos Fase III como Assunto , Estudos de Coortes , Neoplasias Colorretais/patologia , Hibridização Genômica Comparativa , Feminino , Testes Genéticos , Humanos , Irinotecano/administração & dosagem , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Oxaliplatina/administração & dosagem , Intervalo Livre de Progressão , Reprodutibilidade dos TestesRESUMO
Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase. Gemcitabine can be inactivated by the enzyme dCyd deaminase (dCDA). In most in vitro models, resistance to gemcitabine was associated with a decreased dCK activity. In all these models, resistance was established using continuous exposure to gemcitabine with increasing concentrations; however, these in vitro models have limited clinical relevance. To develop in vivo resistance to gemcitabine, we treated mice bearing a moderately sensitive tumor Colon 26-A (T/C = 0.25) with a clinically relevant schedule (120 mg/kg every 3 days). By repeated transplant of the most resistant tumor and continuation of gemcitabine treatment for >1 year, the completely resistant tumor Colon 26-G (T/C = 0.96) was created. Initial studies focused on resistance mechanisms known from in vitro studies. In Colon 26-G, dCK activity was 1.7-fold decreased; dCDA and DNA polymerase were not changed; and Colon 26-G accumulated 1.5-fold less dFdCTP, 6 hours after a gemcitabine injection, than the parental tumor. Based on in vitro studies, these relative minor changes were considered insufficient to explain the completely resistant phenotype. Therefore, an expression microarray was done with Colon 26-A versus Colon 26-G. Using independently grown nonresistant and resistant tumors, a striking increase in expression of the RRM1 subunit gene was found in Colon 26-G. The expression of RRM1 mRNA was 25-fold increased in the resistant tumor, as measured by real-time PCR, which was confirmed by Western blotting. In contrast, RRM2 mRNA was 2-fold decreased. However, ribonucleotide reductase enzyme activity was only moderately increased in Colon 26-G. In conclusion, this is the first model with in vivo induced resistance to gemcitabine. In contrast to most in vitro studies, dCK activity was not the most important determinant of gemcitabine resistance. Expression microarray identified RRM1 as the gene with the highest increase in expression in the Colon 26-G, which might clarify its complete gemcitabine-resistant phenotype. This study is the first in vivo evidence for a key role for RRM1 in acquired gemcitabine resistance.
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Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Desoxicitidina/análogos & derivados , Ribonucleotídeo Redutases/biossíntese , Animais , Western Blotting , Neoplasias do Colo/genética , Citidina Desaminase , Desoxicitidina/farmacologia , Desoxicitidina Quinase/biossíntese , Desoxicitidina Quinase/genética , Nucleotídeos de Desoxicitosina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Nucleosídeo Desaminases/biossíntese , Nucleosídeo Desaminases/genética , Análise de Sequência com Séries de Oligonucleotídeos , Subunidades Proteicas , Ribonucleotídeo Redutases/genética , GencitabinaRESUMO
Myocardial right ventricular (RV) hypertrophy due to pulmonary hypertension is aimed at normalizing ventricular wall stress. Depending on the degree of pressure overload, RV hypertrophy may progress to a state of impaired contractile function and heart failure, but this cannot be discerned during the early stages of ventricular remodeling. We tested whether critical differences in gene expression profiles exist between ventricles before the ultimate development of either a compensated or decompensated hypertrophic phenotype. Both phenotypes were selectively induced in Wistar rats by a single subcutaneous injection of either a low or a high dose of the pyrrolizidine alkaloid monocrotaline (MCT). Spotted oligonucleotide microarrays were used to investigate pressure-dependent cardiac gene expression profiles at 2 wk after the MCT injections, between control rats and rats that would ultimately develop either compensated or decompensated hypertrophy. Clustering of significantly regulated genes revealed specific expression profiles for each group, although the degree of hypertrophy was still similar in both. The ventricles destined to progress to failure showed activation of pro-apoptotic pathways, particularly related to mitochondria, whereas the group developing compensated hypertrophy showed blocked pro-death effector signaling via p38-MAPK, through upregulation of MAPK phosphatase-1. In summary, we show that, already at an early time point, pivotal differences in gene expression exist between ventricles that will ultimately develop either a compensated or a decompensated phenotype, depending on the degree of pressure overload. These data reveal genes that may provide markers for the early prediction of clinical outcome as well as potential targets for early intervention.
Assuntos
Insuficiência Cardíaca/genética , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Direita/genética , RNA Mensageiro/genética , Animais , Fator Natriurético Atrial/genética , ATPases Transportadoras de Cálcio/genética , Primers do DNA , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Hipertensão/genética , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/genética , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificação , Ratos , Ratos Wistar , ATPases Transportadoras de Cálcio do Retículo SarcoplasmáticoRESUMO
Diagnostic potential of the Chlamydia trachomatis ligase chain reaction system (LCx) to assess the presence of C. trachomatis in urine and semen specimens was evaluated. Paired urine and semen specimens from 153 asymptomatic male partners of subfertile couples attending our Center for Reproductive Medicine were examined by LCx. As controls, 19 semen samples from four donors who were participating in the programme for artificial insemination were used. Of these, 12 samples had previously been shown to be C. trachomatis-positive by an in-house PCR. C. trachomatis was detected by LCx in seven of 153 (5 %) urine samples. None of the 153 semen samples tested positive by LCx. Also, none of the 12 C. trachomatis-containing control semen samples were positive by LCx. By in-house PCR, seven urine specimens and two of 153 (1 %) semen samples tested positive. The corresponding urine samples of these male partners were also C. trachomatis-positive, as well as the 12 C. trachomatis-containing samples from donors. In conclusion, LCx is not sensitive enough to assess the presence of C. trachomatis in semen specimens; therefore, this method is not recommended to routinely screen semen specimens from donors who participate in programmes for artificial insemination or male partners of subfertile couples for C. trachomatis.
Assuntos
Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/isolamento & purificação , Reação em Cadeia da Ligase/métodos , Sêmen/microbiologia , Chlamydia trachomatis/genética , DNA Bacteriano/análise , Humanos , Inseminação Artificial Heteróloga , Masculino , Reação em Cadeia da Polimerase , Urina/microbiologiaRESUMO
PURPOSE: Next generation DNA sequencing (NGS) holds promise for diagnostic applications, yet implementation in routine molecular pathology practice requires performance evaluation on DNA derived from routine formalin-fixed paraffin-embedded (FFPE) tissue specimens. The current study presents a comprehensive analysis of TruSeq Amplicon Cancer Panel-based NGS using a MiSeq Personal sequencer (TSACP-MiSeq-NGS) for somatic mutation profiling. METHODS: TSACP-MiSeq-NGS (testing 212 hotspot mutation amplicons of 48 genes) and a data analysis pipeline were evaluated in a retrospective learning/test set approach (n = 58/n = 45 FFPE-tumor DNA samples) against 'gold standard' high-resolution-melting (HRM)-sequencing for the genes KRAS, EGFR, BRAF and PIK3CA. Next, the performance of the validated test algorithm was assessed in an independent, prospective cohort of FFPE-tumor DNA samples (n = 75). RESULTS: In the learning set, a number of minimum parameter settings was defined to decide whether a FFPE-DNA sample is qualified for TSACP-MiSeq-NGS and for calling mutations. The resulting test algorithm revealed 82% (37/45) compliance to the quality criteria and 95% (35/37) concordant assay findings for KRAS, EGFR, BRAF and PIK3CA with HRM-sequencing (kappa = 0.92; 95% CI = 0.81-1.03) in the test set. Subsequent application of the validated test algorithm to the prospective cohort yielded a success rate of 84% (63/75), and a high concordance with HRM-sequencing (95% (60/63); kappa = 0.92; 95% CI = 0.84-1.01). TSACP-MiSeq-NGS detected 77 mutations in 29 additional genes. CONCLUSION: TSACP-MiSeq-NGS is suitable for diagnostic gene mutation profiling in oncopathology.
Assuntos
Análise Mutacional de DNA/métodos , DNA de Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Neoplasias/genética , Biomarcadores Tumorais/genética , Classe I de Fosfatidilinositol 3-Quinases , DNA de Neoplasias/química , Receptores ErbB/genética , Formaldeído/química , Células HCT116 , Humanos , Neoplasias/diagnóstico , Inclusão em Parafina/métodos , Fosfatidilinositol 3-Quinases/genética , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fixação de Tecidos/métodos , Proteínas ras/genéticaRESUMO
Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA. This enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes, including BRCA1 and BRCA2, and enables haplotyping. We show that TLA can also be used to uncover insertion sites and sequences of integrated transgenes and viruses. TLA therefore promises to be a useful method in genetic research and diagnostics when comprehensive or allele-specific genetic information is needed.
Assuntos
Genômica/métodos , Haplótipos/genética , Modelos Genéticos , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência de DNA/métodos , Fusão Gênica/genética , Genes BRCA1 , Genes BRCA2 , Loci Gênicos/genética , Humanos , Neoplasias/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling, we used biotinylated trimethylpsoralen as a DNA structure probe to show that the human genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF insulator protein-binding sites. Underwound domains are transcriptionally active and enriched in topoisomerase I, 'open' chromatin fibers and DNase I sites, but they are depleted of topoisomerase II. Furthermore, DNA supercoiling affects additional levels of chromatin compaction as underwound domains are cytologically decondensed, topologically constrained and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation, providing an evolutionary purpose for clustering genes along chromosomes.