Sickle cell anemia: hierarchical cluster analysis and clinical profile in a cohort in Brazil

Abstract Introduction Sickle cell anemia is a monogenic disorder caused by a mutation in the β-hemoglobin gene, resulting in sickle hemoglobin that can polymerize. Presentation and clinical course have significant inter-individual variability and classifying these patients for severity is a challenge. Methods We applied hierarchical clusters with 10 routine laboratory tests to understand if this grouping could be associated with clinical manifestations. We included 145 adult homozygous patients (SS) at an outpatient clinic in a retrospective study. Results We found five clusters by counting those that had been differentiated by unconjugated bilirubin, reticulocytes, LDH, leukocytes, lymphocytes and monocytes. When comparing groups to clinical findings, the clusters were different only for liver abnormality. Cluster 3 had the lower median of reticulocytes, LDH, leukocytes, lymphocytes and monocytes and a higher percentage of patients under treatment. Clusters 4 and 5 had higher frequencies of liver impairment and higher medians of reticulocytes, LDH, leukocytes, lymphocytes and monocytes. Hemolysis and inflammation seemed to influence the grouping. Conclusion In our study, cluster analysis showed five groups that exhibited different degrees of inflammation and hemolysis. When comparing clinical data, the result was different only for the criteria of liver abnormality.

Sickle cell anemia: hierarchical cluster analysis and clinical profile in a cohort in Brazil.

INTRODUCTION: Sickle cell anemia is a monogenic disorder caused by a mutation in the ß-hemoglobin gene, resulting in sickle hemoglobin that can polymerize. Presentation and clinical course have significant inter-individual variability and classifying these patients for severity is a challenge. METHODS: We applied hierarchical clusters with 10 routine laboratory tests to understand if this grouping could be associated with clinical manifestations. We included 145 adult homozygous patients (SS) at an outpatient clinic in a retrospective study. RESULTS: We found five clusters by counting those that had been differentiated by unconjugated bilirubin, reticulocytes, LDH, leukocytes, lymphocytes and monocytes. When comparing groups to clinical findings, the clusters were different only for liver abnormality. Cluster 3 had the lower median of reticulocytes, LDH, leukocytes, lymphocytes and monocytes and a higher percentage of patients under treatment. Clusters 4 and 5 had higher frequencies of liver impairment and higher medians of reticulocytes, LDH, leukocytes, lymphocytes and monocytes. Hemolysis and inflammation seemed to influence the grouping. CONCLUSION: In our study, cluster analysis showed five groups that exhibited different degrees of inflammation and hemolysis. When comparing clinical data, the result was different only for the criteria of liver abnormality.

miRNA profile and disease severity in patients with sickle cell anemia.

Identification of biomarkers associated with severity in sickle cell anemia is desirable. Circulating serum microRNAs (miRNA) are targets studied as diagnostic or prognostic markers, but few studies have been conducted in sickle cell anemia. The purpose of this study is to identify specific signatures of miRNAs in plasma samples from sickle cell anemia patients according to severity indexes. Screening of the miRNAs expression was performed in 8 patients, classified by tricuspid regurgitation velocity (TRV) measure: 4 with TRV ≥ 2.5 m/s and 4 with TRV < 2.5 m/s. The samples were analyzed by real-time PCR using Megaplex RT Human Pool A and Pool B comprising 667 distinct miRNAs. Seventeen miRNAs were differentially expressed between the two groups (p < 0.05). Five differentially expressed miRNAs (miR15b, miR502, miR510, miR544, miR629) were selected for validation in a cohort of 52 patient samples, 26 with TRV ≥ 2.5 m/s. Another two severity scores were also used: organ injury score (OIS) and Bayesian score (BS). Univariate binary logistic regressions were performed to analyze the data. Five out of 17 differentially expressed miRNAs were selected for validation in 52 patient samples: miR15b, miR502, miR510, miR544, and miR629. Two miRNAs (miR510 and miR629) were significantly decreased in cases of greater severity. Whereas miR510 expression discriminated the patients according to TRV and OIS, miR629 expression did it according to BS. This is the first study investigating plasma miRNAs as possible biomarkers for SCA severity. Our data suggest that low levels of miR510 and miR629 expression are associated with greater SCA disease severity. Further studies are still necessary to elucidate mechanism of these miRNAs and their related proteins.

Downregulation of DCC sensitizes multiple myeloma cells to bortezomib treatment.

Multiple myeloma (MM) is an incurable disease; a better understanding of the molecular aspects of this hematological malignancy could contribute to the development of new treatment strategies and help to improve the survival rates of patients with MM. Previously, the methylation status of the deleted in colorectal cancer (DCC) gene was correlated with the survival rate of patients with MM, thus the main goal of this study was to understand DCC contribution to MM tumorigenesis, and to assess the impact of DCC inhibition in the MM response to treatment with bortezomib. Our results demonstrated that hypermethylation of the DCC promoter inhibits gene expression, and DCC silencing is significantly correlated with a reduction in cell viability and an increase in cell death induced by bortezomib. In conclusion, our results suggested that hypermethylation is an important mechanism of DCC expression regulation in MM and that the absence of DCC contributes to the enhanced sensitivity to treatment with bortezomib.

OIP5 Expression Sensitize Glioblastoma Cells to Lomustine Treatment.

Glioblastoma (GBM) is an incurable disease ranked among the deadliest solid cancers worldwide. A better understanding on the molecular aspects of this malignancy could contribute to the development of new treatment strategies and help to improve survival rates. Previously, our group had shown that GBM patients expressing the cancer/testis antigen Opa Interacting Protein 5 (OIP5) present a longer survival period than the OIP5-negative group. The main goal of this study was to evaluate the OIP5 contribution to GBM tumorigenesis and assess the role of OIP5 in GBM cell response to lomustine, an alkylating agent used in the treatment of this malignancy. So, the effect of OIP5 knockdown was evaluated in A172 and T98G GBM cell lines. Our results demonstrated that downregulation of the OIP5 stimulates glioma cell viability and inhibits cell death-induced necrosis prompted by lomustine. In conclusion, our data shows that OIP5 expression in GBM cells seems to be able to enhance lomustine cytotoxic effects, reinforcing that this gene is a potential therapeutic target and putative molecular biomarker for treatment response in GBM.

Expression of cancer/testis antigens is correlated with improved survival in glioblastoma.

BACKGROUND: Glioblastoma (GBM) confers a dismal prognosis despite advances in current therapy. Cancer-testis antigens (CTA) comprise families of tumor-associated antigens that are immunogenic in different cancers. The aim of this study was to determine the expression profile of a large number of CTA genes in GBM. METHODS: We selected, from 153 CTA genes, those genes potentially expressed in GBM. The expression pattern of 30 CTA was then evaluated by RT-PCR in a series of 48 GBM and 5 normal brain samples. The presence of CTCFL protein was also evaluated by immunohistochemical staining. RESULTS: Among the genes with no expression in normal brain, ACTL8 (57%), OIP5 (54%), XAGE3 (44%) and CTCFL (15%) were frequently expressed in GBM, while over 85% of the tumors expressed at least 1 of these four CTA. Coexpression of two or more CTA occurred in 49% of cases. CTCFL protein expression was detected in 13% of the GBM and was negative in normal brain samples. GBM expressing 3-4 CTA was associated with significantly better overall survival (OS) rates (P = 0.017). By multivariate analysis, mRNA positivity for 3-4 CTA (P = 0.044), radiotherapy (P = 0.010) and chemotherapy (P = 0.001) were independent prognostic factors for OS. CONCLUSIONS: GBM frequently express ACTL8, OIP5, XAGE3 and CTCFL. A relatively high percentage of tumors expressed at least one of these four CTA, opening the perspective for their utility in antigen-specific immunotherapy. Furthermore, mRNA positivity for 3-4 CTA is an independent predictor of better OS for GBM patients.