Contributions
Abstract: EP630
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of long-lived resting clonal B lymphocytes in blood, bone marrow, and secondary lymphoid organs. Oxidative stress is known to be one of the mechanisms important in CLL development and progression. Specifically, NRF2/KEAP1 axis plays a crucial role in redox balance, inducing the expression of target genes, namely those related to antioxidant defenses.
Aims
The goal of this study was to clarify the relevance of NRF2/KEAP1 axis in CLL development, particularly through the evaluation of its target genes expression levels and their correlation with clinical and laboratory data, in order to identify potential new CLL diagnostic biomarkers.
Methods
We evaluated the expression levels of NFE2L2, KEAP1, NFKB1, NQO1, HMOX1, GPX1, SQSTM1, TXNRD1, BCL2, GSTM1 and HPRT (endogenous control) genes in peripheral blood of 25 CLL patients and 30 healthy controls (CTL) using qPCR. Informed consent was obtained in accordance with the Helsinki Declarations. Mann-Whitney U and Kruskal-Wallis tests were used to assess the statistical significance between groups. Receiver operating characteristic (ROC) curves analysis was performed to analyze variables accuracy as diagnostic biomarkers. Patients were dichotomized according to the cut-off points obtained from the ROC curves constructed to predict death. All statistical analyses were two-sided and a p<0.05 was considered statistically significant.
Results
The 25 CLL patients studied were 13 males and 12 females with a median age of 73y, ranging between 48 and 83 years, and the 30 healthy controls, were 15 males and 15 females with a median age of 75 years, ranging between 54 and 99 years. At diagnosis, all CLL patients were Binet A (low risk). 24% of CLL patients presented low-risk cytogenetic abnormalities, 19% presented intermediate-risk, and 24% high-risk and 33% did not present any cytogenetic abnormalities. Our results showed, in CLL patients, an increase on gene expression levels of NQO1 [CLL: m=0.161; interquartile range (IR)=0.283; CTL: m=0.023; IR=0.098; p=0.005] and BCL2 [CLL: m=3,400; IR=6,353; CTL: m=0,434; IR=1,039; p<0,001) and a decrease of NFKB1 (CLL: m=0,900; IR=0,690; p=0,014; CTL: m=1,752; IR=5,220; p=0,014) and SQSTM1 (CLL: m=0,144; IR=0,395; CTL: m=0,880; IR=3,153; p<0,001), when compared to controls. None of the other evaluated genes showed significant differences between CLL patients and controls. ROC analysis for these genes suggested that their expression levels might be possible diagnostic biomarkers for CLL NQO1 (AUC:0,733; IC95%:0,593-0,873; cut-off>0,159; sensitivity=57%; specificity=85%; p=0,005); BCL2 (AUC:0,882; IC95%:0,786-0,978; cut-off>0,532; sensitivity=91%; specificity=74%; p<0,001); NFKB1 (AUC:0,695; IC95%:0,553-0,837; cut-off<1,030; sensitivity=64%; specificity=77%; p=0,014); SQSTM1 (AUC:0,799; IC95%:0,676-0,921; cut-off<0,443; sensitivity=68%; specificity=87%; p<0,001)]. No correlation was found between gene expression levels and CLL patients’ survival, as well as with other clinical and laboratory data.
Conclusion
The decrease of NFKB1 and SQSTM1 gene expression levels observed in our study confirms the importance of oxidative stress in CLL, and the increase of BCL2 and NQO1 reflect the ability of tumor cells to resist cell death. Furthermore, as these genes are target of the NRF2/KEAP1 axis, our study suggests the relevance of the NRF2/KEAP1 axis for CLL development, being the levels of NFKB1, SQSTM1, BCL2 and NQO1 genes potential new diagnostic biomarkers.
This work was supported by FMUC, CIMAGO and FCT (SFRH/BD/145531/2019).
Keyword(s): Chronic lymphocytic leukemia, Gene expression
Abstract: EP630
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of long-lived resting clonal B lymphocytes in blood, bone marrow, and secondary lymphoid organs. Oxidative stress is known to be one of the mechanisms important in CLL development and progression. Specifically, NRF2/KEAP1 axis plays a crucial role in redox balance, inducing the expression of target genes, namely those related to antioxidant defenses.
Aims
The goal of this study was to clarify the relevance of NRF2/KEAP1 axis in CLL development, particularly through the evaluation of its target genes expression levels and their correlation with clinical and laboratory data, in order to identify potential new CLL diagnostic biomarkers.
Methods
We evaluated the expression levels of NFE2L2, KEAP1, NFKB1, NQO1, HMOX1, GPX1, SQSTM1, TXNRD1, BCL2, GSTM1 and HPRT (endogenous control) genes in peripheral blood of 25 CLL patients and 30 healthy controls (CTL) using qPCR. Informed consent was obtained in accordance with the Helsinki Declarations. Mann-Whitney U and Kruskal-Wallis tests were used to assess the statistical significance between groups. Receiver operating characteristic (ROC) curves analysis was performed to analyze variables accuracy as diagnostic biomarkers. Patients were dichotomized according to the cut-off points obtained from the ROC curves constructed to predict death. All statistical analyses were two-sided and a p<0.05 was considered statistically significant.
Results
The 25 CLL patients studied were 13 males and 12 females with a median age of 73y, ranging between 48 and 83 years, and the 30 healthy controls, were 15 males and 15 females with a median age of 75 years, ranging between 54 and 99 years. At diagnosis, all CLL patients were Binet A (low risk). 24% of CLL patients presented low-risk cytogenetic abnormalities, 19% presented intermediate-risk, and 24% high-risk and 33% did not present any cytogenetic abnormalities. Our results showed, in CLL patients, an increase on gene expression levels of NQO1 [CLL: m=0.161; interquartile range (IR)=0.283; CTL: m=0.023; IR=0.098; p=0.005] and BCL2 [CLL: m=3,400; IR=6,353; CTL: m=0,434; IR=1,039; p<0,001) and a decrease of NFKB1 (CLL: m=0,900; IR=0,690; p=0,014; CTL: m=1,752; IR=5,220; p=0,014) and SQSTM1 (CLL: m=0,144; IR=0,395; CTL: m=0,880; IR=3,153; p<0,001), when compared to controls. None of the other evaluated genes showed significant differences between CLL patients and controls. ROC analysis for these genes suggested that their expression levels might be possible diagnostic biomarkers for CLL NQO1 (AUC:0,733; IC95%:0,593-0,873; cut-off>0,159; sensitivity=57%; specificity=85%; p=0,005); BCL2 (AUC:0,882; IC95%:0,786-0,978; cut-off>0,532; sensitivity=91%; specificity=74%; p<0,001); NFKB1 (AUC:0,695; IC95%:0,553-0,837; cut-off<1,030; sensitivity=64%; specificity=77%; p=0,014); SQSTM1 (AUC:0,799; IC95%:0,676-0,921; cut-off<0,443; sensitivity=68%; specificity=87%; p<0,001)]. No correlation was found between gene expression levels and CLL patients’ survival, as well as with other clinical and laboratory data.
Conclusion
The decrease of NFKB1 and SQSTM1 gene expression levels observed in our study confirms the importance of oxidative stress in CLL, and the increase of BCL2 and NQO1 reflect the ability of tumor cells to resist cell death. Furthermore, as these genes are target of the NRF2/KEAP1 axis, our study suggests the relevance of the NRF2/KEAP1 axis for CLL development, being the levels of NFKB1, SQSTM1, BCL2 and NQO1 genes potential new diagnostic biomarkers.
This work was supported by FMUC, CIMAGO and FCT (SFRH/BD/145531/2019).
Keyword(s): Chronic lymphocytic leukemia, Gene expression