RESUMO
Breast cancer is the most frequently diagnosed cancer in women worldwide. Although dramatically increased survival rates of early diagnosed cases have been observed, late diagnosed patients and metastatic cancer may still be considered fatal. The present study's main focus was on cancerassociated fibroblasts (CAFs) which is an active component of the tumor microenvironment (TME) regulating the breast cancer ecosystem. Transcriptomic profiling and analysis of CAFs isolated from breast cancer skin metastasis, cutaneous basal cell carcinoma, and squamous cell carcinoma unravelled major gene candidates such as IL6, VEGFA and MFGE8 that induced coexpression of keratins8/14 in the EMG3 cell line derived from infiltrating ductal breast carcinoma. Western blot analysis of selected keratins (keratin8, 14, 18, 19) and epithelialmesenchymal transitionassociated markers (SLUG, SNAIL, ZEB1, E/Ncadherin, vimentin) revealed specific responses pointing to certain heterogeneity of the studied CAF populations. Experimental in vitro treatment using neutralizing antibodies against IL-6, VEGFA and MFGE8 attenuated the modulatory effect of CAFs on EMG3 cells. The present study provided novel data in characterizing and understanding the interactions between CAFs and EMG3 cells in vitro. CAFs of different origins support the proinflammatory microenvironment and influence the biology of breast cancer cells. This observation potentially holds significant interest for the development of novel, clinically relevant approaches targeting the TME in breast cancer. Furthermore, its implications extend beyond breast cancer and have the potential to impact a wide range of other cancer types.
Assuntos
Neoplasias da Mama , Fibroblastos Associados a Câncer , Feminino , Humanos , Antígenos de Superfície , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Queratinas/genética , Queratinas/metabolismo , Células MCF-7 , Proteínas do Leite/genética , Proteínas do Leite/metabolismo , Prognóstico , Transcriptoma , Microambiente Tumoral/genética , Melanoma Maligno CutâneoRESUMO
Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL oncogene. Despite the high performance of treatment with tyrosine kinase inhibitors (TKI), about 30% of patients develop resistance to the therapy. To improve the outcomes, identification of new targets of treatment is needed. Here, we explored the Casein Kinase 2 (CK2) as a potential target for CML therapy. Previously, we detected increased phosphorylation of HSP90ß Serine 226 in patients non-responding to TKIs imatinib and dasatinib. This site is known to be phosphorylated by CK2, which was also linked to CML resistance to imatinib. In the present work, we established six novel imatinib- and dasatinib-resistant CML cell lines, all of which had increased CK2 activation. A CK2 inhibitor, CX-4945, induced cell death of CML cells in both parental and resistant cell lines. In some cases, CK2 inhibition also potentiated the effects of TKI on the cell metabolic activity. No effects of CK2 inhibition were observed in normal mononuclear blood cells from healthy donors and BCR-ABL negative HL60 cell line. Our data indicate that CK2 kinase supports CML cell viability even in cells with different mechanisms of resistance to TKI, and thus represents a potential target for treatment.
Assuntos
Caseína Quinase II , Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Mesilato de Imatinib/farmacologia , Dasatinibe/farmacologia , Proteínas de Fusão bcr-abl/metabolismo , Resistencia a Medicamentos Antineoplásicos , Apoptose , Inibidores de Proteínas Quinases/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Morte CelularRESUMO
BACKGROUND: Human papillomavirus (HPV) has been shown to adversely affect human reproduction. We aimed to evaluate the prevalence of human papillomavirus (HPV) infection in men and its correlation with semen parameters and reproductive outcomes. METHODS: Semen samples and penile swabs were collected from potential sperm donors (SD, n = 97) and male partners of infertile couples (IM, n = 328). The presence of HPV DNA in semen samples and penile swabs was analyzed. Associations between hrHPV positive status and fertility outcomes as well as socio-behavioral and health characteristics were evaluated using the R software package. RESULTS: High-risk HPV (hrHPV) genotypes were detected in 28.9% of SD and 35.1% of IM (P = 0.312). Penile swabs were more frequently positive for hrHPV genotypes than semen samples in both IM (32.3% vs. 11.9%, P < 0.001) and SD (26.8% vs. 6.2%, P = 0.006). Men with hrHPV positive semen samples had lower semen volume (median volume 2.5 ml vs. 3 ml, P = 0.009), sperm concentration (median concentration 16 × 106/ml vs. 31 × 106/ml, P = 0.009) and total sperm count (median count 46 × 106 vs. 82 × 106, P = 0.009) than men with hrHPV negative samples. No association was identified between penile hrHPV status and semen parameters. CONCLUSIONS: Our findings indicate that penile HPV infection is common in both potential sperm donors and men from infertile couples. Although HPV positivity is higher in penile swabs, only HPV infection in semen samples affects sperm parameters. However, there was no association between hrHPV positivity in semen and fertility outcomes including abortion rate.
Assuntos
Infertilidade/complicações , Infertilidade/diagnóstico , Infecções por Papillomavirus/complicações , Adulto , República Tcheca/epidemiologia , Características da Família , Feminino , Fertilização in vitro/estatística & dados numéricos , Humanos , Infertilidade/epidemiologia , Masculino , Pessoa de Meia-Idade , Papillomaviridae/fisiologia , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/epidemiologia , Gravidez , Resultado da Gravidez/epidemiologia , Prognóstico , Sêmen/fisiologia , Sêmen/virologia , Análise do Sêmen , Doadores de Tecidos/estatística & dados numéricos , Resultado do Tratamento , Adulto JovemRESUMO
Somatic mutations are a common molecular mechanism through which chronic myeloid leukemia (CML) cells acquire resistance to tyrosine kinase inhibitors (TKIs) therapy. While most of the mutations in the kinase domain of BCR-ABL1 can be successfully managed, the recurrent somatic mutations in other genes may be therapeutically challenging. Despite the major clinical relevance of mutation-associated resistance in CML, the mechanisms underlying mutation acquisition in TKI-treated leukemic cells are not well understood. This work demonstrated de novo acquisition of mutations on isolated single-cell sorted CML clones growing in the presence of imatinib. The acquisition of mutations was associated with the significantly increased expression of the LIG1 and PARP1 genes involved in the error-prone alternative nonhomologous end-joining pathway, leading to genomic instability, and increased expression of the UNG, FEN and POLD3 genes involved in the base-excision repair (long patch) pathway, allowing point mutagenesis. This work showed in vitro and in vivo that de novo acquisition of resistance-associated mutations in oncogenes is the prevalent method of somatic mutation development in CML under TKIs treatment.
RESUMO
BACKGROUND: Data about the genotype-specific human papillomavirus (HPV) prevalence in the Czech Republic is limited. We aimed to evaluate the prevalence and concordance of genotype-specific HPV infection detected in semen samples, penile swabs and cervical swabs from non-vaccinated heterosexual couples without HPV-associated disease. METHODS: Semen samples and penile swabs were collected from male partners and cervical swabs were collected from female partners of heterosexual couples treated for infertility (n = 195). Presence of HPV DNA in semen samples and cervical swabs was analyzed using the cobas® HPV Test and PapilloCheck®. Only the PapilloCheck® test was used to detect HPV in penile swabs. The genotype-specific prevalence and concordance of HPV infection not targeted by vaccine were evaluated using Fisher exact test. RESULTS: Both partners were infected with any HPV type in 13.8% (27/195) of couples and, of these couples, 55.6% (15/27) harbored at least one mutual genotype. High-risk HPV (hrHPV) genotypes were detected in 12.3% (24/195) of semen samples, 31.3% (61/195) of penile swabs, and 19.5% (38/195) of cervical swabs (P < 0.001). The most prevalent hrHPV genotype were HPV53 (2.56%; 5/195) in semen samples, HPV16 (6.67%, 13/195) in penile swabs and HPV39 (3.59%, 7/195) in cervical swabs. Low-risk (lrHPV) genotypes were detected in 5.13% (10/195) of semen samples, 15.9% (31/195) of penile swabs, and 4.10% (8/195) of cervical swabs (P < 0.001). Male sexual partners of HPV-positive women were more likely to be infected with at least one of the same HPV types than female sexual partners of HPV-positive men (34.9% vs. 17.9%, P = 0.055). CONCLUSIONS: This study showed that the detection of HPV infection differ by anatomic site and gender. Regardless the anatomic site, high prevalence of HPV genital infection was found in both Czech men and women.
Assuntos
Alphapapillomavirus , Infecções por Papillomavirus , Alphapapillomavirus/genética , República Tcheca/epidemiologia , Feminino , Genótipo , Heterossexualidade , Humanos , Masculino , Infecções por Papillomavirus/epidemiologia , PrevalênciaRESUMO
The karyotype of bone-marrow cells at the time of diagnosis is one of the most important prognostic factors in patients with myelodysplastic syndromes (MDS). In some cases, the acquisition of additional genetic aberrations (clonal evolution [CE]) associated with clinical progression may occur during the disease. We analyzed a cohort of 469 MDS patients using a combination of molecular cytogenomic methods to identify cryptic aberrations and to assess their potential role in CE. We confirmed CE in 36 (8%) patients. The analysis of bone-marrow samples with a combination of cytogenomic methods at diagnosis and after CE identified 214 chromosomal aberrations. The early genetic changes in the diagnostic samples were frequently MDS specific (17 MDS-specific/57 early changes). Most progression-related aberrations identified after CE were not MDS specific (131 non-MDS-specific/155 progression-related changes). Copy number neutral loss of heterozygosity (CN-LOH) was detected in 19% of patients. MDS-specific CN-LOH (4q, 17p) was identified in three patients, and probably pathogenic homozygous mutations were found in TET2 (4q24) and TP53 (17p13.1) genes. We observed a statistically significant difference in overall survival (OS) between the groups of patients divided according to their diagnostic cytogenomic findings, with worse OS in the group with complex karyotypes (P = .021). A combination of cytogenomic methods allowed us to detect many cryptic genomic changes and identify genes and genomic regions that may represent therapeutic targets in patients with progressive MDS.
Assuntos
Evolução Clonal , Síndromes Mielodisplásicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Aberrações Cromossômicas , Proteínas de Ligação a DNA/genética , Dioxigenases , Feminino , Humanos , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Mutação , Síndromes Mielodisplásicas/classificação , Síndromes Mielodisplásicas/patologia , Prognóstico , Proteínas Proto-Oncogênicas/genética , Análise de Sobrevida , Proteína Supressora de Tumor p53/genéticaRESUMO
OBJECTIVE: The aims of this study were to determine the prevalence of human papillomavirus (HPV) infection in women treated for infertility and oocyte donors, and to investigate the possible influence of HPV infection on reproductive outcomes. STUDY DESIGN: In this observational laboratory-based study, cervical swabs were collected from oocyte donors (n = 207), and women treated for infertility (n = 945) and analysed for the presence of high-risk HPV (hrHPV) genotypes using the cobas® 4800 HPV Test and PapilloCheck® HPV-Screening. Associations between hrHPV positive status and fertility outcome or socio-behavioral and health characteristics were evaluated using R statistical software. RESULTS: HrHPV prevalence was significantly higher in oocyte donors than in women treated for infertility (28.0% vs. 16.1%, P < 0.001). Women who became pregnant spontaneously (19.6%) and women not treated with in vitro fertilization (IVF, 18.1%) were more frequently hrHPV positive than women treated with IVF (12.7%, P = 0.077). Despite the high prevalence of hrHPV in both oocyte donors and infertile women, no associations between hrHPV positive status and pregnancy or abortion rates were found in IVF treated women or in oocyte recipients. Moreover, no associations between hrHPV positive status and abortion rates were found in spontaneously pregnant women. CONCLUSION: Despite the high prevalence of hrHPV in both oocyte donors and infertile women, HPV infection did not influence the outcomes of assisted reproductive technology.
RESUMO
In patients with hematological malignancies one of the most substantial findings is the karyotype of bone marrow cells at the time of diagnosis. The detection of clonal chromosome aberrations in diagnostic samples not only confirms a neoplastic or premalignant process but also provides important diagnostic and prognostic information essential for precise disease classification and choice of suitable therapy. Karyotype analysis during the disease course also allows monitoring of the treatment success reflected as well in the revised WHO classification where patients are often classified into the different diagnostic subtypes based on the finding of specific chromosome and/or genetic changes. Recently, also increases the number of advanced treatment approaches that directly or indirectly target the genetic aberrations present in tumor cells. Despite the large development of new sequencing technologies in recent years, cytogenetic analysis supplemented by the molecular cytogenetic methods still remains a very important part of diagnostics of hematological malignancies.
Assuntos
Aberrações Cromossômicas , Análise Citogenética , Neoplasias Hematológicas , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Humanos , Cariotipagem , PrognósticoRESUMO
High-risk human papillomavirus (hrHPV) infection is a cause of cervical cancer development. The addition of hrHPV testing to cervical cancer screening and monitoring of cervical intraepithelial neoplasia treatment improves the efficacy of screening and treatment, respectively. Self-sampling for hrHPV testing seems a promising tool for increasing patient participation in cervical cancer screening. In this project, 1198 cervical swabs obtained by physicians and 176 cervicovaginal swabs obtained by self-sampling (not collected in parallel) were analyzed for the presence of 14 hrHPV genotypes using three commercially available assays in comparison. HPV DNA was detected in 21.2% of all samples (21% of cervical swabs and 22.7% of cervicovaginal swabs). The cobas 4800 HPV Test was the most sensitive (0.983) and specific (0.992) for hrHPV detection overall. The PapilloCheck HPV-Screening and LMNX Genotyping Kit HPV GP had comparable specificity with that of the cobas (0.989 and 0.955, respectively), but lesser sensitivity (0.897 and 0.909, respectively). In physician-obtained cervical swabs, the cobas showed the highest sensitivity and specificity (0.980 and 0.994, respectively) for hrHPV detection, whereas in cervicovaginal swabs, the cobas had the highest sensitivity (1.00), but the PapilloCheck had the highest specificity (0.993). In conclusion, all of the detection methods evaluated were highly sensitive and specific for hrHPV detection from both clinician-collected cervical swabs and self-sampled cervicovaginal swabs.
Assuntos
DNA Viral/análise , Técnicas de Genotipagem/métodos , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Kit de Reagentes para Diagnóstico , Neoplasias do Colo do Útero/virologia , Vagina/virologia , Adolescente , Adulto , Idoso , DNA Viral/genética , Feminino , Humanos , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
The apical inflammatory cytokine TNF regulates numerous important biological processes including inflammation and cell death, and drives inflammatory diseases. TNF secretion requires TACE (also called ADAM17), which cleaves TNF from its transmembrane tether. The trafficking of TACE to the cell surface, and stimulation of its proteolytic activity, depends on membrane proteins, called iRhoms. To delineate how the TNF/TACE/iRhom axis is regulated, we performed an immunoprecipitation/mass spectrometry screen to identify iRhom-binding proteins. This identified a novel protein, that we name iTAP (iRhom Tail-Associated Protein) that binds to iRhoms, enhancing the cell surface stability of iRhoms and TACE, preventing their degradation in lysosomes. Depleting iTAP in primary human macrophages profoundly impaired TNF production and tissues from iTAP KO mice exhibit a pronounced depletion in active TACE levels. Our work identifies iTAP as a physiological regulator of TNF signalling and a novel target for the control of inflammation.
Assuntos
Proteína ADAM17/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Fator de Necrose Tumoral alfa/genética , Proteína ADAM17/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Linhagem Celular , Proteínas do Citoesqueleto/genética , Endossomos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/citologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Ligação Proteica , Proteólise , Células RAW 264.7 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Dicentric chromosomes (DCs) are considered markers of cancer in various malignancies. However, they can be overlooked when conventional analysis or multicolor fluorescence in situ hybridization (mFISH) is used to detect complex karyotypes. We analyzed the karyotypes of 114 patients with acute myeloid leukemia (AML) and complex karyotypes and verified the presence of monosomies by FISH using multi-centromeric probes. Monosomy was detected in 63% of patients by G-banding/mFISH and confirmed in 55% of patients by centromeric FISH. FISH analysis indicated a high frequency of DCs that were previously considered monosomies. In some cases, it was apparent that the derivative monocentric chromosome was a primary DC. DCs were formed mostly by chromosomes 17 and 20. In conclusion, chromosome loss and unbalanced translocation suggest the presence of a hidden DC or its previous existence. DCs undergo several stabilizing changes and can induce other chromosomal aberrations and/or the formation of new DCs. This can result in the clonal evolution of abnormal cells, which is considered an independent prognostic marker of an unfavorable disease course and short survival.
Assuntos
Centrômero , Aberrações Cromossômicas , Hibridização in Situ Fluorescente/métodos , Cariótipo , Leucemia Mieloide Aguda/genética , Idoso , Bandeamento Cromossômico , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 20 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monossomia , Prognóstico , Análise de SobrevidaRESUMO
Rhomboids are intramembrane serine proteases conserved in all kingdoms of life. They regulate epidermal growth factor receptor signalling in Drosophila by releasing signalling ligands from their transmembrane tethers. Their functions in mammals are poorly understood, in part because of the lack of endogenous substrates identified thus far. We used a quantitative proteomics approach to investigate the substrate repertoire of rhomboid protease RHBDL2 in human cells. We reveal a range of novel substrates that are specifically cleaved by RHBDL2, including the interleukin-6 receptor (IL6R), cell surface protease inhibitor Spint-1, the collagen receptor tyrosine kinase DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, BCAM and others. We further demonstrate that these substrates can be shed by endogenously expressed RHBDL2 and that a subset of them is resistant to shedding by cell surface metalloproteases. The expression profiles and identity of the substrates implicate RHBDL2 in physiological or pathological processes affecting epithelial homeostasis.
Assuntos
Epitélio/metabolismo , Homeostase , Proteoma , Proteômica , Serina Proteases/metabolismo , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Células Epiteliais/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteômica/métodos , Serina Endopeptidases , Serina Proteases/genética , Especificidade por SubstratoRESUMO
BACKGROUND: The high incidence of mutations and cytogenetic abnormalities in patients with myelodysplastic syndrome (MDS) suggests that defects in DNA repair mechanisms. We monitored DNA repair pathways in MDS and their alterations during disease progression. METHODS: Expression profiling of DNA repair genes was performed on CD34+ cells, and paired samples were used for monitoring of RAD51 and XRCC2 gene expression during disease progression. Immunohistochemical staining for RAD51 was done on histology samples. RESULTS: RAD51 and XRCC2 showed differential expression between low-risk and high-risk MDS (P<.0001), whereas RPA3 was generally decreased among the entire cohort (FC=-2.65, P<.0001). We demonstrated that RAD51 and XRCC2 expression gradually decreased during the progression of MDS. Down-regulation of XRCC2 and RAD51 expression was connected with abnormalities on chromosome 7 (P=.0858, P=.0457). Immunohistochemical staining revealed the presence of RAD51 only in the cytoplasm in low-risk MDS, while in both the cytoplasm and nucleus in high-risk MDS. The multivariate analysis identified RAD51 expression level (HR 0.49; P=.01) as significant prognostic factor for overall survival of patients with MDS. CONCLUSIONS: Our study demonstrates that the expression of DNA repair factors, primarily RAD51 and XRCC2, is deregulated in patients with MDS and presents a specific pattern with respect to prognostic categories.
Assuntos
Regulação da Expressão Gênica , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Reparo de DNA por Recombinação/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Medula Óssea/patologia , Aberrações Cromossômicas , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/mortalidade , Prognóstico , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Adulto JovemRESUMO
Rhomboid proteases are increasingly being explored as potential drug targets, but their potent and specific inhibitors are not available, and strategies for inhibitor development are hampered by the lack of widely usable and easily modifiable in vitro activity assays. Here we address this bottleneck and report on the development of new fluorogenic transmembrane peptide substrates, which are cleaved by several unrelated rhomboid proteases, can be used both in detergent micelles and in liposomes, and contain red-shifted fluorophores that are suitable for high-throughput screening of compound libraries. We show that nearly the entire transmembrane domain of the substrate is important for efficient cleavage, implying that it extensively interacts with the enzyme. Importantly, we demonstrate that in the detergent micelle system, commonly used for the enzymatic analyses of intramembrane proteolysis, the cleavage rate strongly depends on detergent concentration, because the reaction proceeds only in the micelles. Furthermore, we show that the catalytic efficiency and selectivity toward a rhomboid substrate can be dramatically improved by targeted modification of the sequence of its P5 to P1 region. The fluorogenic substrates that we describe and their sequence variants should find wide use in the detection of activity and development of inhibitors of rhomboid proteases.
Assuntos
Corantes Fluorescentes/química , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cinética , Lipossomos , Especificidade por SubstratoRESUMO
TP53 mutations are frequently detected in patients with higher-risk myelodysplastic syndromes (MDS); however, the clinical impact of these mutations on the disease course of patients with lower-risk MDS is unclear. In this study of 154 lower-risk MDS patients, TP53 mutations were identified in 13% of patients, with prevalence in patients with del(5q) (23.6%) compared to non-del(5q) (3.8%). Two-thirds of the mutations were detected at the time of diagnosis, and one-third were detected during the course of the disease. Multivariate analysis demonstrated that a TP53 mutation was the strongest independent prognostic factor for overall survival (OS) (HR: 4.39) and progression-free survival (PFS) (HR: 3.74). Evaluation of OS determined a TP53 variant allele frequency (VAF) threshold of 6% as an optimal cut-off for patient stratification. The median OS was 43.5 months in patients with mutations detected at the time of diagnosis and a mutational burden of > 6% VAF compared to 138 months (HR 12.2; p = 0.003) in patients without mutations; similarly, the median PFS was 20.2 months versus 116.6 months (HR 79.5; p < 0.0001). In contrast, patients with a mutational burden of < 6% VAF were stable for long periods without progression and had no significant impact on PFS or OS. Additionally, we found a high correlation in the mutational data from cells of the peripheral blood and those of the bone marrow, indicating that peripheral blood is a reliable source for mutation monitoring. Our results indicate that the clinical impact of TP53 mutations in lower-risk MDS patients depends on the level of mutational burden.
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Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Mutação , Síndromes Mielodisplásicas/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Feminino , Frequência do Gene , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Fatores de Risco , Adulto JovemRESUMO
Complex karyotypes are seen in approximately 20% of patients with myelodysplastic syndromes (MDS) and are associated with a high risk of transformation to acute myeloid leukemia and poor outcomes in patients. Copy number neutral loss of heterozygosity (CN-LOH, i.e., both copies of a chromosomal pair or their parts originate from one parent) might contribute to increased genomic instability in the bone-marrow cells of patients with MDS. The pathological potential of CN-LOH, which arises as a clonal aberration in a proportion of somatic cells, consists of tumor suppressor gene and oncogene homozygous mutations. The aim of our study was to evaluate the frequency of CN-LOH at 17p in bone-marrow cells of newly diagnosed MDS patients with complex chromosomal aberrations and to assess its correlation with mutations in the TP53 gene (17p13.1). CN-LOH was detected in 40 chromosomal regions in 21 (29%) of 72 patients analyzed. The changes in 27 of the 40 regions identified were sporadic. The most common finding was CN-LOH of the short arm of chromosome 17, which was detected in 13 (18%) of 72 patients. A mutational analysis confirmed the homozygous mutation of TP53 in all CN-LOH 17p patients, among which two frameshift mutations are not registered in the International Agency for Research on Cancer TP53 Database. CN-LOH 17p correlated with aggressive disease (median overall survival 4 months) and was strongly associated with a complex karyotype in the cohort studied, which might cause rapid disease progression in high-risk MDS. No other CN-LOH region previously recorded in MDS or AML patients (1p, 4q, 7q, 11q, 13q, 19q, 21q) was detected in our cohort of patients with complex karyotype examined at the diagnosis of MDS. The LOH region appeared to be balanced (i.e., with no DNA copy number change) when examined with conventional and molecular cytogenetic methods. Therefore, a microarray that detects single-nucleotide polymorphisms is an ideal method with which to identify and further characterize CN-LOH. Our data should specify the prognosis and should lead to the identification of potential targets for therapeutic interventions.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 17/genética , Dosagem de Genes , Síndromes Mielodisplásicas/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Perda de Heterozigosidade/genética , Masculino , Pessoa de Meia-Idade , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Estudos RetrospectivosRESUMO
Dicentric chromosomes (DCs) have been described in many hematological diseases, including acute myeloid leukemia (AML). They are markers of cancer and induce chromosomal instability, leading to the formation of other chromosomal aberrations and the clonal evolution of pathological cells. Our knowledge of the roles and behavior of human DCs is often derived from studies of induced DCs and cell lines. It is difficult to identify all the DCs in the karyotypes of patients because of the limitations of metaphase cytogenetic methods. The aim of this study was to revise the karyotypes of 20 AML patients in whom DCs were found with conventional G-banding or multicolor fluorescence in situ hybridization (mFISH) with (multi)centromeric probes and to characterize the DCs at the molecular cytogenetic level. FISH analyses confirmed 23 of the 29 expected DCs in 18 of 20 patients and identified 13 others that had not been detected cytogenetically. Fourteen DCs were altered by other chromosomal changes. In conclusion, karyotypes with DCs are usually very complex, and we have shown that they often contain more than one DC, which can be missed with conventional or mFISH methods. Our study indicates an association between number of DCs in karyotype and very short survival of patients.
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Cariótipo Anormal , Cromossomos Humanos/genética , Hibridização in Situ Fluorescente , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de SobrevidaRESUMO
OBJECTIVE: A new interleukin-6 (IL-6)-dependent plasma cell leukemia cell line UHKT-944 was established from bone marrow cells derived from a 55-yr-old man with plasma cell leukemia. RESULTS: The cell line possesses phenotypic characteristics of plasma cells including the production of a monoclonal immunoglobulin IgA1-kappa. VH3-9 region of IgVH genes was rearranged and somatically hypermutated. The UHKT-944 cells were found to be negative for most of tested B-cell, T-cell, and myeloid markers. According to cytogenetic analysis, the cells were classified as near tetraploid with several numerical and structural abnormalities including the t(14;20) involving IgH locus. CONCLUSION: The established permanent plasma cell leukemia cell line is a suitable model for the study of cellular and molecular mechanisms of pathogenesis of this rare malignant disease.
Assuntos
Leucemia Plasmocitária/metabolismo , Leucemia Plasmocitária/patologia , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células , Análise Citogenética , Humanos , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Imunofenotipagem , Leucemia Plasmocitária/diagnóstico , Masculino , Pessoa de Meia-IdadeRESUMO
Downregulation of cereblon (CRBN) gene expression is associated with resistance to the immunomodulatory drug lenalidomide and poor survival outcomes in multiple myeloma (MM) patients. However, the importance of CRBN gene expression in patients with myelodysplastic syndrome (MDS) and its impact on lenalidomide therapy are not clear. In this study, we evaluate cereblon expression in mononuclear cells isolated from bone marrow [23 lower risk MDS patients with isolated 5q deletion (5q-), 37 lower risk MDS patients with chromosome 5 without the deletion of long arms (non-5q-), and 24 healthy controls] and from peripheral blood (38 patients with 5q-, 52 non-5q- patients and 25 healthy controls) to gain insight into, firstly, the role of cereblon in lower risk MDS patients with or without 5q deletion and, secondly, into the mechanisms of lenalidomide action. Patients with 5q- lower risk MDS have the highest levels of CRBN mRNA in comparison with both lower risk MDS without the deletion of long arms of chromosome 5 and healthy controls. CRBN gene expression was measured using the quantitative TaqMan real-time PCR. High levels of CRBN mRNA were detected in all lenalidomide responders during the course of therapy. A significant decrease of the CRBN mRNA level during lenalidomide treatment is associated with loss of response to treatment and disease progression. These results suggest that, similar to the treatment of MM, high levels of full-length CRBN mRNA in lower risk 5q- patients are necessary for the efficacy of lenalidomide.
Assuntos
Anemia Macrocítica/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Fatores Imunológicos/uso terapêutico , Síndromes Mielodisplásicas/tratamento farmacológico , Peptídeo Hidrolases/genética , RNA Mensageiro/genética , Talidomida/análogos & derivados , Proteínas Adaptadoras de Transdução de Sinal , Anemia Macrocítica/genética , Anemia Macrocítica/metabolismo , Anemia Macrocítica/patologia , Estudos de Casos e Controles , Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Cromossomos Humanos Par 5/metabolismo , Humanos , Lenalidomida , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Peptídeo Hidrolases/metabolismo , Polimorfismo de Nucleotídeo Único , Splicing de RNA , RNA Mensageiro/metabolismo , Transdução de Sinais , Talidomida/uso terapêutico , Resultado do Tratamento , Ubiquitina-Proteína LigasesRESUMO
AIMS: Male infertility has been associated with imbalance in the polyunsaturated fatty acids arachidonic acid/docosahexaenoic acid (AA/DHA) ratio. This study aimed to assess whether use of the potent antioxidant, lycopene, would affect this imbalance in seminal plasma and to test whether modulation of the AA/DHA ratio correlates with male fertility. METHODS: The study included 44 males from infertile couples who were treated with lycopene for three months before scheduled IVF treatment. The control group was 13 fertile males who were also treated with lycopene for three months. AA and DHA levels were measured in seminal plasma before and after treatment. The outcome of IVF treatment after lycopene use in of males from infertile couples was recorded. RESULTS: The subjects were subdivided into 21 normozoospermic males from couples with idiopathic infertility and 23 males with semen abnormalities. Prior to the treatment with lycopene, the AA/DHA ratios in both subgroups of patients were significantly higher than in fertile controls and improved following treatment with lycopene. Improvement was more marked in the normospermic males. Seven spontaneous pregnancies (16%) occurred before the scheduled IVF treatment and 15 couples (42%) achieved pregnancy after IVF. CONCLUSIONS: Three months of treatment with lycopene led to a significant improvement in the AA/DHA ratio in seminal plasma of males from infertile couples and facilitated the spontaneous as well as IVF conception.