mRNA overexpression of the hypoxia inducible factor 1 alpha subunit gene (HIF1A): An independent predictor of poor overall survival in chronic lymphocytic leukemia

Highlights

• A cost-effective qPCR method for quantification of HIF1A mRNA levels was developed.

• HIF1A mRNA levels do not differ between normal blood samples and leukemic B cells.

• High HIF1A mRNA expression predicts poor overall survival in CLL patients.

• Prognostic significance of HIF1A mRNA is independent of clinicopathological factors.

• HIF1A mRNA is an unfavorable prognosticator in distinct subgroups of CLL patients.

Abstract

The hypoxia inducible factor 1 (HIF1) is a heterodimeric transcription factor that ultimately regulates cellular responses to changes in oxygen tension. In this study, we examined the potential diagnostic and prognostic potential of the mRNA expression of HIF1 regulatory α-subunit (HIF1A) in chronic lymphocytic leukemia (CLL). For this purpose, total RNA was isolated from peripheral blood mononuclear cells collected from 88 CLL patients and 33 non-leukemic blood donors, and poly(A)-RNA was reversely transcribed. HIF1A mRNA levels were quantified using real-time PCR. Kaplan-Meier survival analysis showed that high HIF1A mRNA expression predicts inferior overall survival for CLL patients (p = 0.001). Bootstrap univariate Cox regression analysis confirmed that HIF1A mRNA overexpression is a significant unfavorable prognosticator in CLL (hazard ratio = 3.75, bias-corrected and accelerated 95% confidence interval = 1.43–24.36, bootstrap p < 0.001), independent of other established prognostic factors, including CD38 expression, the mutational status of the immunoglobulin heavy chain variable region (IGHV), and the clinical stage (Binet or Rai stage) or risk group (p < 0.001 in all cases). Interestingly, HIF1A mRNA positivity retains its unfavorable prognostic value in distinct subgroups of patients, stratified according to established prognostic factors. Thus, HIF1A mRNA overexpression can be regarded as a promising, independent molecular biomarker of unfavorable prognosis in CLL.

Introduction

Chronic lymphocytic leukemia (CLL), the most common type of adult leukemia in the Western world, remains incurable despite recent improvements in prolonging patients’ survival [1], [2]. This hematological malignancy is characterized by the progressive accumulation of small mature CD5⁺ B lymphocytes in the peripheral blood, lymphoid organs, and bone marrow [3]. The clinical course of the disease is highly heterogeneous among different patients [2]. CLL mainly affects older adults. The average age at the time of diagnosis is around 71 years, whereas it is rarely encountered in people under the age of 40 and is extremely rare in children [1].

Two prognostic systems are currently used for the prediction of CLL patients’ outlook, namely the Rai staging [4] and the Binet staging [5] systems. Albeit useful for prognostic purposes, these staging systems cannot predict response to therapy nor distinguish prospectively patients with probably evolving disease from those retaining disease stability even for decades. Although the prognosis of CLL patients is traditionally based on clinicopathological parameters, several additional prognostic biomarkers in CLL have been proposed, validated, and introduced in clinical practice during the last decades [6]; these prognostic biomarkers include cytogenetics, mutational status of the immunoglobulin heavy chain variable region (IGHV), expression analysis of CD38, and zeta-chain–associated protein kinase 70 kDa (ZAP70) in leukemic B cells [7], [8]. Therefore, deletion of part of chromosome 13 (with no other chromosome abnormalities), low proportion of CLL cells containing ZAP70 (20% or less) or CD38 (30% or less), and a mutated IGHV status constitute favorable prognostic factors in CLL [9].

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors that ultimately regulate cellular responses to changes in oxygen tension during normal development or pathological processes, such as cancer or cardiovascular disease. These heterodimers consist of a regulatory α-subunit (HIF1A, HIF2A, or HIF3A) and a constitutively expressed β-subunit (HIF1B; also known as aryl hydrocarbon receptor nuclear translocator, ARNT). HIFs regulate a wide array of adaptive responses to hypoxia as they control the expression of many genes involved in apoptosis, invasion, angiogenesis, and metastasis [10], and are often activated in solid tumors and hematological malignancies, due to intratumoral hypoxia or activation of oncogenic pathways [11]. Importantly, HIF1A regulates the interaction of CLL cells with protective leukemia microenvironments and is also regulated by this interaction in a positive feedback loop promoting leukemic cell survival and propagation. Although it was previously shown that CLL cells express constitutively HIF1A under normal levels of oxygen [12], emerging evidence supports the notion that HIF1A mRNA levels present significant variation among CLL patients [13]. Overexpression of the oxygen-regulated component of HIF1, HIF1A, has also been observed in leukemic cells found in bone marrow of CLL patients [14]. Nevertheless, despite the fact that the relevance of HIF1A expression to CLL pathogenesis has already been highlighted, its putative role as a biomarker in CLL has not been elucidated yet.

The purpose of this study was to perform HIF1A mRNA expression analysis in patients with CLL and normal controls, using an accurate and cost-effective, in-house–developed, quantitative real-time PCR (qPCR) methodology based on the SYBR^® Green chemistry, and to examine its potential predictive and prognostic significance as a new molecular biomarker in CLL.