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Inherited gene variants play an important role in malignant diseases. The transcriptional repressor growth factor independence 1 (GFI1) regulates hematopoietic stem cell (HSC) self-renewal and differentiation. A single-nucleotide polymorphism of GFI1 (rs34631763) generates a protein with an asparagine (N) instead of a serine (S) at position 36 (GFI136N) and has a prevalence of 3%–5% among Caucasians. Because GFI1 regulates myeloid development, we examined the role of GFI136N on the course of MDS disease. To this end, we determined allele frequencies of GFI136N in four independent MDS cohorts from the Netherlands and Belgium, Germany, the ICGC consortium, and the United States. The GFI136N allele frequency in the 723 MDS patients genotyped ranged between 9% and 12%. GFI136N was an independent adverse prognostic factor for overall survival, acute myeloid leukemia-free survival, and event-free survival in a univariate analysis. After adjustment for age, bone marrow blast percentage, IPSS score, mutational status, and cytogenetic findings, GFI136N remained an independent adverse prognostic marker. GFI136S homozygous patients exhibited a sustained response to treatment with hypomethylating agents, whereas GFI136N patients had a poor sustained response to this therapy. Because allele status of GFI136N is readily determined using basic molecular techniques, we propose inclusion of GFI136N status in future prospective studies for MDS patients to better predict prognosis and guide therapeutic decisions.
GFI1 is a zinc finger transcriptional repressor that recruits histone-modifying enzymes, such as histone deacetylases, to the loci of its target genes 1, 2. GFI1 regulates the functions of hematopoietic stem cells (HSCs) 1, 3 as well as myeloid–lymphoid lineage decisions 4, 5. A variant form of the GFI1 gene (denominated GFI136N) is associated with a predisposition to develop de novo acute myeloid leukemia (AML) , but it has also been reported to be involved in a case of neutropenia . Taking into consideration the predisposing role of GFI136N to de novo AML and its role in myeloid development, we investigated the role of GFI136N in myelodysplastic syndrome (MDS).
All patient samples (peripheral blood [PB] or bone marrow [BM] aspirates) were obtained with informed consent according to the Declaration of Helsinki. The respective local ethics committees approved the use of all patient samples.
The clinical characteristics of patients with a confirmed diagnosis of MDS used in this study have been previously described 8, 9, 10, 11, 12, 13. Events in “event-free survival” were defined as death from any cause or progression of MDS to AML with blast counts higher than 20%. Overall survival events are defined as death from any cause.
Bone marrow morphology studies were performed at individual centers. MDS was classified based on the World Health Organization (WHO) definition .
Genotyping was performed according to published procedures .
Significance of differences in percentages was determined using the two-sided, two-sample t test. Survival of the different human cohorts is based on the presence of GFI136N univariate analysis using Kaplan–Meier survival methods. Differences were assessed using the log-rank (Mantel–Cox) test. We used Cox proportional-hazards regression modeling to determine the influence of different factors with respect to survival. Factors taken into account were International Prognostic Scoring System (IPSS) risk group, BM blast count, age, sex, cytogenetic findings (based on IPSS classification), and in a last step, presence of GFI136N. Analyses were performed either separately (with each factor analyzed independently) or with the presence or absence of GFI136N. All p values reported are two-sided. Because of the exploratory nature of this study, no adjustment for multiple testing was done. All analyses presented were performed using GraphPad Prism 6 software (GraphPad Software, La Jolla, CA) or SPSS Version 19 (IBM, Armonk, NY).
Between 9% and 12% of all adult MDS patients in four different Caucasian cohorts from Europe and the United States 8, 9, 10, 11, 12 were heterozygous for GFI136N, and only two patients from the European cohorts (and none of the U.S. cohort) were homozygous for GFI136N (Table 1). GFI136N allele frequency was higher among MDS patients than among the control cohort (3%–5%) as reported in our previous study regarding the role of GFI136N in de novo AML . Although we did not determine the frequency of GFI136N among sex- and age-matched control subjects from every region, it is possible that GFI136N predisposes to MDS, similar to its predisposing role in de novo AML .
We analyzed the effect of GFI136N on MDS disease course in two independent cohorts. Patients were recruited and treated either in the United States or in Europe. The European cohorts consisted of patients recruited and treated at different centers in Germany, Belgium, and The Netherlands 8, 9, 10, 11, 12. The U.S. cohorts were referred to the Cleveland Clinic.
In the two MDS cohorts from Europe and the United States, presence of GFI136N was associated with an inferior event-free survival rate (Fig. 1A, B). GFI136N also had a negative impact on overall survival (Fig. 1C, D) and on AML-free survival (data not shown).
Next, we examined the association between GFI136N and established prognostic factors. GFI136N carriers were older, exhibited a tendency toward higher BM blast counts at diagnosis, were diagnosed with a more advanced stage of the disease according to histologic parameters, and had more adverse cytogenetic findings (Table 1). With respect to key blood parameters, no other differences between GFI136N and GFI136S carriers were observed (Table 1).
American and European GFI136S homozygous patients had median follow-ups of 1,100 and 975 days, respectively. American and European GFI136N carriers had median follow-ups of 540 and 350 days, respectively. To gain more statistical power and to perform more specific analysis, we combined the U.S. and European cohorts. One approach used to predict outcome of MDS patients is based on IPSS 16, 17. A recently introduced, revised version of IPSS (denominated IPSS-R) distinguishes between more subclasses based on cytogenetic findings and cytopenic lineages . However, not all of the specific data for determining IPSS-R status were present in our databases. Therefore, we focused our examination on the nonrevised version of IPSS.
As reported previously , IPSS scoring predicted event-free outcome of GFI136S homozygous patients (Fig. 1E). On the basis of the same scoring system, GFI136N carriers had a significantly shorter event-free survival (Fig. 1F). Especially among MDS patients in the low-risk groups (groups low and intermediate 1 based on IPSS), GFI136N carriers had a much shorter event-free (Fig. 1G) and AML-free survival (data not shown) than GFI136S homozygous patients. We also examined the association between allele status, cytogenetic findings and event-free survival. Similarly, presence of GFI136N was associated with an inferior outcome, independent of the cytogenetic finding (Table 1). After stratification for cytogenetic risk groups (“low” as one group and “intermediate and high” as a second group), the presence of a GFI136N allele was again linked to inferior event-free survival (Fig. 1H, I) in both comparisons.
MDS patients with somatic mutations within EZH2 or ASXL1 have an inferior prognosis 18, 19 (Fig. 2A, B). Presence of GFI136N in the absence of ASXL1 or EZH2 mutations had a similar effect on event-free survival as the presence of ASXL1 or EZH2 mutations (Fig. 2C, D). Furthermore, the combined presence of a mutated form of either ASXL1 or EZH2 and a GFI136N allele had an additional adverse effect on event-free survival (Fig. 2C, D). Similar analyses were not possible for mutations of P53 or RUNX1 18, 19, because of the small number of cases with P53 or RUNX1 mutation and GFI136N.
Because MDS patients who are hetero- or homozygous for GFI136N tend to be older at diagnosis, have more frequent adverse cytogenetic findings, and have a higher blast cell count at diagnosis, we examined whether the presence of GFI136N represents an adverse prognostic factor after adjusting for these findings. We found that GFI136N was an independent marker after adjusting for the variables IPSS score, cytogenetic findings, and age, either alone or in combination (Supplementary Table E1, Supplementary Table E2, online only, available at www.exphem.org).
One possible explanation why GFI136N accelerates AML development in MDS patients could be based on our findings using murine models. We previously generated mice expressing GFI136N or GFI136S instead of murine Gfi1 . We reported that GFI136N is not able to bind to its target genes to the same extent as the more common “wild-type” GFI136S variant. Hence, presence of one allele of GFI136N led to higher genomewide levels of acetylated histone 3 at lysine 9 (H3K9) at Gfi1 target genes. This led to active expression of genes favoring development of myeloid malignancies , which could explain how GFI136N accelerates both AML development and MDS–AML progression. We recently reported that a low level of GFI1 expression, which might mimic the presence of GFI136N on a functional level, accelerates AML progression in different murine models of AML, including one model of MDS . It is not yet clear why altering one amino acid changes the function of GFI1, and initial experiments regarding expression level, stability, ability to induce apoptosis, or interaction with histone deacetylases (HDACs) or lysine-specific demethylase 1 (LSD1) did not reveal any significant differences between GFI136N and GFI136S  (unpublished data).
We also examined whether GFI136N could predict response to therapy. To explore this in more detail, we focused on patients that were treated with 5-azacitidine. This treatment is used for patients who are otherwise not fit for allogeneic bone marrow transplantation or as a bridging to a definitive therapy 21, 22, 23. There was no difference between GFI136N heterozygous carriers and GFI136S patients achieving response to treatment with 5-azacytidine (51% of GFI136S homozygous carriers compared with 52% of GFI136N carriers). However, the response to treatment was much shorter in GFI136N carriers than in GFI136S homozygous carriers (Fig. 2E). This observation is not surprising because treatment with hypomethylating agents, such as 5-azacitidine, would not revert the increased levels of H3K9 acetylation and H3K4 methylation seen in cells with a GFI136N allele .
The suitability of GFI136N as a prognostic marker should be verified in prospective studies and, if the findings can be confirmed, the status of GFI136N should be determined routinely in MDS patients. Considering the role of GFI1 in myeloid development, GFI136N status could also be of prognostic value for patients with myeloproliferative diseases and chronic myeloid leukemia. Indeed, the frequency of GFI136N is also elevated among patients with chronic myeloid leukemia. In summary, GFI136N could be a useful therapeutic and prognostic marker for myeloid malignancies.
CK was supported by a Max Eder Grant from the Deutsche Krebshilfe, Germany, and the IFORES fellowship from the University Clinic Essen, Germany. TM's lab was supported by a grant from the Leukemia and Lymphoma Society (USA). TM holds a Tier 1 Canada Research Chair and grants from the Canadian Institutes of Health Research (CIHR, Grants MOP-84238 and MOP-111011). JB and AP are supported by Bloodwise (UK).
We thank the patients for their consent to participate in the different studies and Saskia Grunwald and Renata Köster for excellent technical assistance.
LB, LCM, HM, JM, and CK contributed equally to this work.
Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.exphem.2016.04.001.
Cyrus Khandanpour received travel reimbursement from Amgen and Chugai for attending scientific conferences. Jaroslaw Maciejewski received speaker honoraria from Celgene and Alexion.
|Variable||Wald χ2||p value||Hazard ratio||95% CI for hazard ratio|
|IPSS intermediate 1||4.607||0.032||0.725||0.540||0.972|
|IPSS intermediate 2||1.586||0.208||1.229||0.892||1.694|
CI = confidence interval.
|Variable||Wald χ2||P value||Hazard ratio||95.0% CI for hazard ratio|
|Blast (% BM)||7.141||0.008||1.045||1.012||1.078|
|IPSS intermediate 1||2.561||0.110||0 .728||0.494||1.074|
|IPSS intermediate 2||0.205||0.651||0.909||0.603||1.371|
|Cytogenetic poor||1.278||0 .258||1.469||0.754||2.862|
CI = confidence interval.