Contributions
Abstract: PB2081
Type: Publication Only
Background
Hif-1α expression has been previously associated with an adverse prognosis in patients with solid tumors and some hematological malignancies. Moreover, it was recently correlated with poor overall survival and disease progression in MDS. Hif-1α is also known to regulate the expression of human equilibrative nucleoside transporters (ENTs) and ribonucleotide reductase (RR), both of which are involved in the metabolism of hypomethylating agents, such as azacitidine. AZA is the standard of care for patients with high-risk myelodysplastic syndromes (MDS) and AML patients not candidate for treatment with intensive chemotherapy. Nevertheless, approximately 40% of patients fail to respond, whereas even responders will inevitably relapse.
Aims
This study aims at investigating the expression level and clinical significance of Hif-1α in both MDS AZA responders and non-responders, before and after treatment.
Methods
Bone marrow samples from 39 de novo MDS patients, 5 CMML patients and 12 AML [43 M/13 F, median age 75 (52-89)] and 10 healthy donors were collected. MDS patients were classified according to WHO as RCMD (4/39), RAEB I (11/39) and RAEB II (24/39) and according to IPSS as low (3/39), intermediate-1 (7/39), intermediate-2 (13/39) and high (16/39). All patients received AZA treatment at the dose of 75mg/m2 x7 days SC. Low and inter-1 MDS patients received azacitidine as off label therapy. BM samples from 29 MDS [RCMD (1/29), RAEB I (9/29), RAEB II (19/29)], 2 CMML and 2 AML of the aforementioned patients were also collected 6 months after AZA therapy [26 M/7 F, median age 75 (52-89)]. After BM mononuclear cells’ isolation (Ficoll-paque method), RNA extraction (TRIzol method) and cDNA preparation (Superscript II reverse transcriptase), Hif-1α expression was estimated by Rt-PCR TaqMan gene expression assay. Relative gene expression was calculated by comparative threshold cycle (ΔΔCt) method. β-actin was used as a housekeeping gene. Statistical analyses were performed through Kruskal Wallis, Mann-Whitney U and Wilcoxon signed rank tests.
Results
Out of the 56 patients used in our study, 32 responded to azacitidine-treatment (including CR, PR and HI) while 24 failed to respond. AZA responders presented with a statistically significantly increased 2-ΔΔCt ratio of Hif-1α/β-Actin median expression compared to both control samples and non responders (1.377 vs 0.698 and 0.73, p=0.004 and 0.008, respectively). AZA non-responders presented no statistical significance compared to control samples. Hb levels were not significantly different between R and NR patients’ groups. Moreover, an increase in Hif-1α expression was observed comparing Inter-I and Inter-II IPSS risk groups (0.730 vs 1.672, p=0.037) in all MDS patients examined, while a positive correlation between Hif-1α expression and IPSS-R risk scores (CC=0.371, p=0.024) was also found. Finally, the 2-ΔΔCt ratio of Hif-1α/β-Actin median expression after 6-months of therapy presented a decreasing trend for both responders [from 1.377 to 1.269 (7.85% decrease)] and non-responders [from 0.730 to 0.598 (18% decrease)].
Conclusion
Our data indicate that patients with increased pretreatment Hif-1α expression seem to better respond to AZA therapy. Hif-1α expression gradually increases across IPSS and IPSS-R subgroups, suggesting an association of Hif-1α with a more aggressive disease. Moreover, AZA treatment seems to downregulate Hif-1α expression in both responders and non responders, indicating a potential role of hypoxia signaling in azacitidine resistance
Session topic: 9. Myelodysplastic syndromes – Biology & Translational Research
Keyword(s): Azacitidine, MDS
Abstract: PB2081
Type: Publication Only
Background
Hif-1α expression has been previously associated with an adverse prognosis in patients with solid tumors and some hematological malignancies. Moreover, it was recently correlated with poor overall survival and disease progression in MDS. Hif-1α is also known to regulate the expression of human equilibrative nucleoside transporters (ENTs) and ribonucleotide reductase (RR), both of which are involved in the metabolism of hypomethylating agents, such as azacitidine. AZA is the standard of care for patients with high-risk myelodysplastic syndromes (MDS) and AML patients not candidate for treatment with intensive chemotherapy. Nevertheless, approximately 40% of patients fail to respond, whereas even responders will inevitably relapse.
Aims
This study aims at investigating the expression level and clinical significance of Hif-1α in both MDS AZA responders and non-responders, before and after treatment.
Methods
Bone marrow samples from 39 de novo MDS patients, 5 CMML patients and 12 AML [43 M/13 F, median age 75 (52-89)] and 10 healthy donors were collected. MDS patients were classified according to WHO as RCMD (4/39), RAEB I (11/39) and RAEB II (24/39) and according to IPSS as low (3/39), intermediate-1 (7/39), intermediate-2 (13/39) and high (16/39). All patients received AZA treatment at the dose of 75mg/m2 x7 days SC. Low and inter-1 MDS patients received azacitidine as off label therapy. BM samples from 29 MDS [RCMD (1/29), RAEB I (9/29), RAEB II (19/29)], 2 CMML and 2 AML of the aforementioned patients were also collected 6 months after AZA therapy [26 M/7 F, median age 75 (52-89)]. After BM mononuclear cells’ isolation (Ficoll-paque method), RNA extraction (TRIzol method) and cDNA preparation (Superscript II reverse transcriptase), Hif-1α expression was estimated by Rt-PCR TaqMan gene expression assay. Relative gene expression was calculated by comparative threshold cycle (ΔΔCt) method. β-actin was used as a housekeeping gene. Statistical analyses were performed through Kruskal Wallis, Mann-Whitney U and Wilcoxon signed rank tests.
Results
Out of the 56 patients used in our study, 32 responded to azacitidine-treatment (including CR, PR and HI) while 24 failed to respond. AZA responders presented with a statistically significantly increased 2-ΔΔCt ratio of Hif-1α/β-Actin median expression compared to both control samples and non responders (1.377 vs 0.698 and 0.73, p=0.004 and 0.008, respectively). AZA non-responders presented no statistical significance compared to control samples. Hb levels were not significantly different between R and NR patients’ groups. Moreover, an increase in Hif-1α expression was observed comparing Inter-I and Inter-II IPSS risk groups (0.730 vs 1.672, p=0.037) in all MDS patients examined, while a positive correlation between Hif-1α expression and IPSS-R risk scores (CC=0.371, p=0.024) was also found. Finally, the 2-ΔΔCt ratio of Hif-1α/β-Actin median expression after 6-months of therapy presented a decreasing trend for both responders [from 1.377 to 1.269 (7.85% decrease)] and non-responders [from 0.730 to 0.598 (18% decrease)].
Conclusion
Our data indicate that patients with increased pretreatment Hif-1α expression seem to better respond to AZA therapy. Hif-1α expression gradually increases across IPSS and IPSS-R subgroups, suggesting an association of Hif-1α with a more aggressive disease. Moreover, AZA treatment seems to downregulate Hif-1α expression in both responders and non responders, indicating a potential role of hypoxia signaling in azacitidine resistance
Session topic: 9. Myelodysplastic syndromes – Biology & Translational Research
Keyword(s): Azacitidine, MDS