Sex-dependent association of circulating sex steroids and pituitary hormones with treatment-free survival in chronic lymphocytic leukemia patients

Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia characterized by an accumulation and clonal proliferation of mature CD5⁺ B lymphocytes in peripheral blood, bone marrow, and lymphoid organs. A significant clinical and molecular heterogeneity characterizes CLL, leading to vast differences in disease progression, response to treatment, and risk of relapse, with clinical stage, cytogenetic abnormalities, and mutational status of IGHV being the most important prognostic markers [1]. To date, no curative therapy exists aside from allogeneic bone marrow transplantation, however, the development of targeting drugs and their introduction into treatment regimens in recent years portends the prolongation of overall survival (OS) which, depending on clinical stage, lies at a median of 6.5 years [1].

Sex is a risk factor with a significantly different male/female ratio (2:1) in the incidence of CLL that remains unexplained much like for other lymphoid malignancies [2, 3]. Furthermore, men develop progressive disease and resistance to treatment more frequently than women who have better prognosis independently of age and CLL stage, and respond better to therapy [2, 4]. CLL is not considered a hormone-regulated cancer but the sexual dimorphism associated with CLL incidence, prognosis, and response to therapy, in addition to what is known regarding the importance of sex hormones for development and function of the immune system, led to the speculation of a potential protective role for these molecules, most particularly estrogens, in hematologic malignancies [5, 6]. As second explanation, sex-specific somatic alterations in the non-pseudoautosomal and pseudoautosomal regions on chromosomes X and Y have been discussed, [4, 7] however, in-depth data are still lacking.

Sex hormones exert their influence via hormone-specific receptors. For estrogens, some studies support the expression of estrogen receptors (ERs) in CLL but with variable results [8‐10] with more recent data pointing towards a predominance of ERβ [11]. Hormone receptors like ERβ are ligand-regulated transcription factors activated by naturally produced steroid hormones that regulate transcription of genes controlling a wide variety of biological processes. The endogenous ligands of these hormone receptors comprise numerous steroids produced from cholesterol by the gonads and other organs such as adrenal glands (Fig. 1). Among C19 androgenic steroids, testosterone (Testo) and 5α-dihydrotestosterone (DHT) are the most potent ligands of the androgen receptor (AR), which has been sparingly studied in CLL or other B cell malignancies [12, 13]. For ERs comprised of ERα and ERβ isoforms, many endogenous ligands have been identified including parent C18 estrogens such as estradiol that binds both receptors [14]. Data also indicate that ERα is the major ER isoform expressed in T cells whereas a co-expression of ERβ splice isoforms ERβ1 and ERβ2 was reported in the majority of patients with CLL and normal lymphocytes [10, 11, 15]. Certain C19 steroids including androstenediol (5-diol) and androstanediol (3β-diol), both of which can be synthesized from dehydroepiandrosterone (DHEA), are also highly potent ERβ-ligands [16]. Parent estrogens (E₂) and estrone (E₁) may be further converted into diverse catechol estrogen (CE) metabolites that display specific biological activities (mitogenic, antiproliferative, antiangiogenic, pro-apoptotic, and genotoxic properties) [17]. A comprehensive evaluation of this array of hormones in CLL patients is still lacking.

In support of a potential effect of variable hormonal exposure on CLL progression, high expression of the androgen-inactivating UGT2B17 enzyme in peripheral blood mononuclear cells (PBMCs) of untreated CLL patients was associated with reduced treatment-free survival (TFS) and showed promise as a biomarker for IGHV-mutated CLL cases [18, 19]. The UGT2B17 gene encodes a uridine diphospho-glucuronosyltransferase (UGT) enzyme that conjugates small molecule substrates such as steroids to the polar sugar glucuronic acid, preventing receptor binding, leading to their inactivation and enhanced elimination through bile and urine. UGT2B17 substrates include the potent androgens Testo and DHT, and their metabolites androstane-3α, 17β-diol (3α-diol), and androsterone (ADT) [20]. The UGT2B17 gene is also highly polymorphic, with a complete gene deletion occurring at varying frequencies (14–92%) depending on ethnicity [21‐23]. In healthy donors and cancer patients, altered levels of circulating steroids were reported for individuals with the UGT2B17^null genotype (del/del) compared to individuals carrying one or two copies of the gene [24, 25].

To provide a comprehensive evaluation of the potential effect of circulating steroids on CLL prognosis, and most particularly ligands of ERβ, we established their plasma concentrations in a population of female and male CLL patients using mass spectrometry-validated assays, along with androgenic precursors and biologically active estrogen metabolites. We also measured levels of gonadotropins produced by the pituitary gland that act on the gonads including luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by immunoassay. Our primary objective was to examine their relationship to treatment-free survival (TFS) and after adjustment for established prognostic markers. We also studied the influence of UGT2B17 mRNA expression levels in peripheral mononuclear blood cells (PBMCs) and UGT2B17 common deletion polymorphism on circulating steroid levels of CLL patients, and their relationship to TFS.

Sex disparity in the development of hematological malignancies has been well documented. A genomic basis for these differences is in the occurrence of somatic mutations on the sex chromosomes [7]. Of the six genes for which a sex bias was found in different tumor entities [7], only DDX2X had been reported previously in the context of poor risk CLL and clonal evolution [30, 31], Considering, however, the importance of sex steroids and gonadotropins for development and function of the immune system [6], we decided to focus our study on hormones and hormone metabolites.

We report a comprehensive profiling of circulating sex steroids and pituitary hormones in men and women with CLL, revealing a sex-specific association of these signaling molecules to treatment-free survival. We observed that high levels of potent androgens and biologically active estrogen metabolites are linked to an improved survival of female CLL patients whereas higher LH levels are associated with shorter survival in male CLL patients. These observations support that CLL is a hormone-responsive disease and imply different biological mechanisms associated with progression of leukemia in men and women.

We observed a significant association of high levels of testosterone and DHT, and improved TFS in women suggesting that the AR signaling axis may confer beneficial effects, potentially delaying disease progression, whether directly or indirectly acting on leukemic cells. This relationship remained similar after adjustment for prognostic factors, suggesting that effect of androgens is not dependent on these molecular changes. Testosterone is one of very few steroids present at higher concentrations in circulation of women with CLL compared to healthy postmenopausal donors, despite lower levels of adrenal precursors, suggesting a potential influence of the disease on androgen synthesis. Little is known regarding the expression and function of the AR in CLL and this information would be essential to provide a better understanding of the potential impact of androgen signaling on CLL progression. While data denoted that androgens have immunosuppressive properties [5], some of these cellular processes may be relevant to CLL progression including the alteration of bone marrow stromal cell behavior and responses to small molecular weight mediators such as cytokines [32, 33]. In line, recent reports suggested potential therapeutic or prognostic applications to targeting the AR axis in other lymphoid malignancies such as mantle cell lymphoma and diffuse large B cell lymphoma [13, 34].

An alternative hypothesis to our observation in women with CLL may involve the aromatization of testosterone to estradiol, an efficient agonist for ERβ, which is the main ER expressed in lymphoid tissues also overexpressed in CLL [11, 15, 35]. Accordingly, the improved TFS in female cases may be, at least in part, the consequence of a greater local exposure of leukemic cells to estrogens, described as tumor-suppressive molecules acting via the ER axis in cells of lymphoid origin that express ERβ [36]. Concurring with this hypothesis, treatment with aromatase inhibitors blocking the conversion of androgen to estrogen was shown to enhance lymphoma growth in mice, but not androgens per se [37]. Also, activation of ERβ was shown to induce autophagy and impair cell proliferation of Hodgkin lymphoma [38] while ERβ1 nuclear expression was exposed as an independent prognostic factor for adverse progression-free survival in diffuse large B cell lymphoma (DLBCL) cases [39, 40].

We also observed that higher levels of methoxylated estrogen metabolites are associated with prolonged TFS in female CLL patients compared to those with lower levels. MeO-CE corresponds to abundant endogenous estrogen derivatives that cause growth arrest of hormone-dependent and hormone-independent tumors in vitro and in vivo, consistent with their protective effect observed in female CLL cases [41]. Several studies have shown that MeO-CE exerts anticarcinogenic, antiproliferative, antiangiogenic, pro-apoptotic, and anti-inflammatory properties [42, 43]. This is in opposition to their mitogenic hydroxylated counterparts (2/4OH-CE) that can be metabolized into quinones leading to the formation of quinone adducts and oxidative DNA damage [17]. These reactive estrogen metabolites are critical in the initiation of breast and prostate cancers as well as non-Hodgkin lymphoma, and have been found in abundance in leukemia cell lines of the NCI-60 human tumor cell lines panel [44, 45]. This may indicate that increased methylation of estrogens could be beneficial for CLL patients by preventing the formation and accumulation of damaging catechol estrogen metabolites and/or through their intrinsic beneficial effects. In support of the later, 2MeOE₂ was recently shown to abrogate preleukemic stem cell self-renewal when maintained in a niche-like environment, inducing apoptosis and exhibiting antileukemic activity in primary human T cell acute lymphoblastic leukemia blasts [46] concurring with the protective association for MeO-CE observed herein.

It may not be surprising to observe a more pronounced adverse effect associated with high mRNA expression of the steroid-inactivating UGT2B17 enzyme in leukemic cells of female patients, where a higher systemic exposure to its androgenic substrates is linked to beneficial effects on disease progression. Besides, we could verify the ability of the UGT2B17 enzyme to conjugate MeO-CE in vitro (not shown). This is supported by our observation of higher levels of MeO-CE in female CLL cases with the UGT2B17^null genotype compared to those carrying the gene, despite the small sample size. In turn, no differences were apparent in male CLL cases notwithstanding similar circulating concentrations of MeO-CE. This may reflect sex differences in the metabolic enzymes involved in the conversion of androgen to estrogen and their subsequent biotransformation to MeO-CE by catechol-O-methyltransferase (COMT). It is thus conceivable that UGT2B17 mRNA expression in CLL modifies exposure of leukemic cells and/or proliferation centers to these steroids with a resulting alteration of leukemic cell behavior or of the microenvironment.

For male CLL patients, we observed an association between higher levels of LH and shorter TFS. An earlier study reported higher levels of LH in male CLL patients compared to controls and an association to Rai stage [47], suggesting that men with higher levels of LH correspond to progressive cases. LH pituitary secretion is regulated by testosterone levels that decline with age with a corresponding increase in LH in aging men [48]. We did not observe a correlation between levels of these two circulating hormones in male CLL cases (r = 0.12; P = 0.30), denoting that LH may represent a biomarker in CLL. As for the underlying mechanism, a recent study presented evidence that pituitary gonadotropins may act on lymphocyte migration and that leukemia cells recognize these hormones as chemoattractants [49]. Hematopoietic stem cells have been shown to express nearly all hormone receptors, but it is unknown whether their expression is maintained through maturation and differentiation [50]. Evidence for the expression of LH receptor in mature B lymphocytes and CLL cells is still lacking.

Among the strengths of the study are the abundance of available clinical and molecular data as well as the wide range of hormones that are reported for the first time in CLL patients. TFS was retained as the primary endpoint since it is disease-specific and not limited by competing risks of death by other conditions. In addition, whereas previous studies used radioimmunoassays to measure sex steroids, we used validated MS assays that are more specific, sensitive, and accurate [27]. Our cohort of CLL patients was composed of aged men and postmenopausal women. We excluded premenopausal women to limit confounding factors such as menstrual cycle that largely affects circulating steroid levels. Some limitations of our study, exploratory in nature, also need to be considered. Body mass index was not recorded and may have affected the relationship between disease progression and steroid levels considering the association of body mass index with circulating steroids [51].

Sex differences in cytogenetic anomalies, as reported previously [4], were not statistically significant in this study cohort. We also explored potential differences in circulating hormone levels in CLL patients compared to those of healthy subjects revealing hormonal imbalances with the onset of CLL and raising the possibility that the disease may affect hormone synthesis and/or metabolism. The variations observed are unlikely to be linked to molecular events commonly associated with CLL, such as gene deletions or IGHV status, as no obvious association between these variables and hormone levels was noted (not shown).

This study reports a sex-specific hormonal imbalance and association between circulating sex steroids and pituitary hormones and treatment-free survival in CLL patients. Larger studies will need to be conducted to replicate our initial observations and to assess potential changes in hormone levels during the evolution of the disease and how drug treatment potentially affects their relationship with disease progression, both in men and women. Further work is also required to elucidate whether these effects are mediated by leukemic cells and/or affecting the dynamic interactions with the microenvironment in order to identify mediators and signaling pathways involved. Our work creates opportunities for additional studies on the role of sex steroids and pituitary hormones that may participate in autocrine/paracrine loops affecting the survival and proliferation of CLL cells.