The Role of JAK2 Mutations in RARS and Other MDS

Myelodysplastic syndromes (MDS) are characterized by cytopenia due to ineffective hemopoiesis and an increased risk for leukemic evolution. Erythroid failure resulting in anemia is the most common feature in MDS and may range from mild, with only slightly decreased hemoglobin levels and increased red cell volume, to severe, with a complete inability to produce red blood cells. Defective erythropoiesis in MDS could be divided into a hypoproliferative/ suppressed and a hyperproliferative/ineffective form.¹ Hypoproliferative erythropoiesis, characterized by a decreased relative number of erythroid progenitors in the bone marrow, is typically seen in advanced MDS, in hypoplastic MDS, in some cases with 5q- syndrome, and in MDS with severe marrow fibrosis. Ineffective hyperproliferative erythropoiesis is characterized by an increased percentage of marrow erythroblasts, of which many undergo intramedullary apoptosis before they mature into erythrocytes. This type of erythropoiesis is typically observed in refractory anemia with ringed sideroblasts (RARS), but is also common in a subset of refractory anemia (RA) and sometimes in RA with excess blasts (RAEB) with a moderate increase of marrow blasts.

The WHO classification from 2001² has recently been revised, and the updated classifications of MDS and mixed MDS/MPD are available as working documents. A new subgroup; refractory cytopenia with unilineage dysplasia was suggested, encompassing three entities; RA, refractory neutropenia and refractory thrombocytopenia. The 5q- syndrome subgroup has been renamed “MDS associated with isolated del(5q),” but still requires < 5% marrow blasts. The classification of “mixed myelodysplastic/myeloproliferative neoplasms (mixed MDS/MPN) was also updated. Within this group remains the provisional entity RA with ringed sideroblasts and marked thrombocytosis (RARS-T), defined by < 5% marrow blasts, ≥ 15% ringed sideroblasts and a persistent platelet count of > 600 × 10⁹/L. In the 2008 proposal, the cut-off value for thrombocytosis was decreased to 450 × 10⁹/L, which may be problematic as the majority of published articles on the subject used the higher cut-off level. The revised WHO classification of MDS and mixed MDS/MPN are outlined in Tables 1  and 2 .

The sideroblastic anemias constitute a heterogeneous group of inherited and acquired disorders characterized by anemia of varying severity and the presence of ringed sideroblasts in the bone marrow.³ Ringed sideroblasts are erythroblasts with iron-loaded mitochondria visualized by Prussian blue staining as a perinuclear ring of blue granules and with most of the iron deposited in the form of mitochondrial ferritin.⁴ The presence of ringed sideroblasts in the bone marrow (15% or more of erythroblasts) is a marker of the myelodysplastic syndromes subgroup RARS, refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), and RARS-T.² 

The division of the previous French-American-British (FAB) RARS subgroup into the WHO subgroups RARS and RCMD-RS has significant clinical relevance. RARS patients do not show pancytopenia and have an overall good prognosis, with a 5-year survival well above 50% and a very low risk for transformation to AML.⁵ By contrast, patients with RCMD-RS have a 5-year survival of 37%, a cumulative risk for AML transformation of 9%, and a substantial risk for developing more advanced MDS.⁶ The two subgroups also differ in their response to treatment with erythropoietin (EPO) plus granulocyte colony-stimulating factor (G-CSF), as described below.⁷ The different clinical profiles of RARS and RCMD-RS indicate that at least partly different biological mechanisms are active in these disorders.

Ringed sideroblast formation may be caused by exogenous factors, such as lead intoxication and treatment with isoniazid, both of which inhibit δ-aminolevulinic acid (ALA) dehydratase activity, block hemoglobin formation and cause ringed sideroblast formation.³ 

A number of hereditary conditions are associated with ringed sideroblast formation. The most common of these inherited forms is X-linked sideroblastic anemia (OMIM 301300), which is caused by mutations in the erythroid-specific ALA synthase gene (ALAS2).³ Defective ALAS2 enzyme activity in bone marrow erythroid cells leads to insufficient protoporphyrin IX synthesis, mitochondrial iron overload, and intramedullary death of red cell precursors. Most but not all XLSA patients are, to a variable extent, responsive to pyridoxine, which is metabolized to pyridoxal phosphate.

Another interesting inherited condition is the X-linked sideroblastic anemia associated with cerebellar ataxia (XLSA/A; OMIM 301310), characterized by neurological manifestations early in infancy with impaired gross motor and cognitive development. This X-linked sideroblastic anemia differs clinically from the classic XLSA (OMIM 301300), which does not have neurological manifestations, is associated with iron overload, and is generally at least partially responsive to pyridoxine. XLSA/A is caused by missense mutations in the human ABCB7 gene, which encodes a membrane-associated protein belonging to the superfamily of ATP-binding cassette (ABC) transporters.⁸ The ABCB7 protein functions to enable transport of iron from the mitochondria to the cytoplasm. It has been reported that ABCB7 is essential for hematopoiesis⁹ and that RNA silencing of the gene in HeLa cells causes an iron-deficient phenotype with mitochondrial iron overload.¹⁰ Moreover, in a recent study we showed that ABCB7 expression is also markedly reduced in acquired RARS and RCMD-RS, and that its expression levels are inversely related to the percentage of ringed sideroblasts in these conditions.¹¹ However, no gene mutations were observed in this cohort of patients, indicating that the ABCB7 gene may be suppressed via other factors and pathways.

A defective mitochondrial enzyme function of RARS bone marrow cells was suggested decades ago, and there is now accumulating evidence that mitochondria play a central role in the pathophysiology of ineffective erythropoiesis in MDS. Electron microscopy has demonstrated pronounced ultra-structural mitochondrial changes not only in RARS but also in other types of MDS.¹² In addition, the occurrence of mitochondrial DNA mutations has been reported in MDS; however, these are of uncertain significance.^13,14 Studies of an in vitro model of erythroid differentiation showed that early erythroid progenitor cells from low-risk MDS spontaneously release excessive amounts of cytochrome C from mitochondria, resulting in activation of caspase-9 and subsequent cell death.¹⁵ Inhibition of caspase-9 activity abrogated the enhanced sensitivity to Fas ligation; hence, the increased sensitivity of MDS progenitor cells to death receptor stimulation seems to be due to a constitutive activation of the mitochondrial axis of the apoptotic signaling pathway in these cells. Other investigators have shown involvement of the Fas-caspase-8 pathway and Fas-associated protein with death domain (FADD)-mediated erythroid apoptosis.¹⁶ Recently it was also shown that cultured erythroblasts in RARS accumulate mitochondrial ferritin during early differentiation, long before morphological signs of erythroid differentiation are visible.¹⁷ 

In spite of the isolated anemia and erythroid dysplasia observed in RARS, apoptosis seems to be initiated at the stem cell level.¹⁸ Moreover, stem cells have also been shown to be clonal, as assessed by HUMARA analysis, in pure sideroblastic anemia (WHO-RARS), further confirming that this disorder is a clonal stem cell disorder and that the initial pathogenetic event occurs in multipotent stem cells.¹⁹ Thus, the genetic defects present in RARS must both give rise to a proliferative advantage leading to expansion of the clone, but also to ineffective erythropoiesis and abnormal erythroid iron metabolism, leading to accumulation of the metal in the mitochondria of immature red cells. It is likely that this defect would encompass gene(s) involved in erythroid differentiation, mitochondrial function, or cellular iron metabolism.²⁰ 

Philadelphia chromosome–negative chronic myeloproliferative disorders (polycythemia vera, ET, primary myelofibrosis) are characterized by various combinations of erythrocytosis, leukocytosis and thrombocytosis, i.e., the opposite of the cytopenia found in MDS. These conditions are typical clonal disorders of hematopoietic stem cells. The occurrence of the unique V617F mutation of JAK2 exon 14,^21,–23 of several mutations of JAK2 exon 12^24,–25 and of MPL mutations^26,–27 in a multipotent stem cell generates a myeloid clone that expands to replace hematopoietic cells without the mutation.

Although MDS and MPD appear to have entirely different pathophysiological mechanisms, the existence of conditions with overlapping features is well established. In 2002, Schmitt-Graeff and coworkers²⁸ published an interesting study on 38 patients showing both thrombocytosis in peripheral blood and ringed sideroblasts in the bone marrow, a condition that was at that time defined as “essential thrombocythemia with ringed sideroblasts (ET-RS).” Findings of this study provided evidence that ET-RS includes a wide spectrum of conditions ranging from MDS in the strict sense to MPD (ET, prefibrotic primary myelofibrosis).

Szpurka and coworkers²⁹ studied 57 patients with MDS/ MPD and found that 11 of them carried JAK2 (V617F). In particular, this mutation was detected in 6 of 9 patients with RARS-T, and the authors suggested that RARS-T constitutes another JAK2 mutation–associated form of MPD. Several other studies on mutation analysis of JAK2 and MPL in MDS/MPD have been published in the last 2 years,^30,–38 and their findings are summarized in Table 3 . Schmitt-Graeff and coworkers³⁷ have recently reported findings from a study evaluating JAK2 (V617F) status in 23 patients with RARS-T by using allele-specific PCR. The mutation was detected in 11 of 23 patients, and in 6 patients with RARS-T the allelic ratio of JAK2 (V617F) was above 50%, indicating the presence of cells homozygous for the mutation. Interestingly, in 2 of these latter patients a transition from JAK2-V617F heterozygosity to homozygosity was documented, and this was accompanied by rising platelet counts in sequential samples. The MPL (W515L) mutation was detected in 1 JAK2 (V617F)-negative patient. This study clearly indicates that RARS-T has several features of MPD in addition to overproduction of platelets, including striking megakaryocytic proliferation, leukocytosis, abnormalities of chromosomes 8 and 20, vascular events, and marrow fibrosis. A recent preliminary report of the hitherto largest series of 47 patients showed the JAK2 mutation in 61% of patients with a platelet count higher than 600 × 10⁹/L, as compared with 12.5% in patients with a platelet count between 400 and 600 × 10⁹/L.³⁸ All patients who were JAK2 positive had a platelet count higher than 500 × 10⁹/L. Patients who were JAK2 positive had significantly higher hemoglobin levels and white blood cell counts, but no difference in survival or marrow fibrosis was observed (P = .38). Finally, an additional study investigated JAK2 status in 97 cases with MDS and del(5q).³⁹ A mutation was found in 6 patients, all with an isolated del(5q). These patients displayed higher platelet counts, but otherwise no major differences compared with patients without the mutation.

The mechanisms of anemia in a JAK2 (V617F)–positive RARS-T are unclear, considering that this mutation is normally associated with erythrocytosis. In order to properly answer this question, it should be first considered that the combination of ringed sideroblasts and JAK2 (V617F) mutation is very unlikely in terms of probability, considering the low prevalence of both RARS and ET in the general population. This means that this combination cannot be simply coincidental. Thus, there must be a pathophysiological mechanism that predisposes RARS patients to acquire JAK2 (V617F), or alternatively patients with JAK2 (V617F)–positive myeloproliferative disorder to develop mitochondrial iron loading and ineffective erythropoiesis. Further studies are required to elucidate which of the two mechanisms is more likely.

Published studies on the subject are in agreement that patients with MDS and a JAK2 mutation have significantly higher platelet counts than other MDS, and some, but not all, indicate that white blood cell counts and bone marrow cellularity also are elevated. Figure 1 (see Color Figures, page 491) shows a representative bone marrow from a patient with RARS-T. It should be remembered that slightly elevated platelet counts (350 to 500 × 10⁹/L) are common also in WHO-RARS. There is at present some confusion as to the best cut-off platelet count level for a diagnosis of RARS-T as compared to RARS. The 2001 WHO classification uses 600 × 10⁹/L, while the 2008 classification suggests 450 × 10⁹/L, to be in line with the criteria for ET (Tables 1  and 2 ). Most reports cite above have used a cutoff of 600 × 10⁹/L, and a few have used 500 × 10⁹/L, while a cut-off level of 450 × 10⁹/L has not been evaluated. One study³¹ failed to demonstrate any JAK2 mutations in RARS patients with a platelet count of 400 to 600 × 10⁹/L and the recent report by Raya et al strongly supports the present cut-off of 600 × 10⁹/L³⁸ (Table 3 ). We conclude that diagnostic criteria for RARS-T need to be better defined, as this disorder currently appears to represent a condition that is borderline to RARS, ET and primary myelofibrosis. As a practical recommendation, we suggest that a Perls staining on bone marrow aspirate should be performed in patients with myeloid neoplasm and thrombocytosis whether carrying mutations of JAK2 or MPL or not. In analogy, analysis of JAK2 mutational status should be performed in RARS patients with an elevated platelet count.

The first-line treatment for the anemia of low- and intermediate-1 risk MDS is erythropoietic stimulating agents: erythropoietin or darbepoetin, with or without the addition of G-CSF, leading to an erythroid response rate of 40% to 50%. The median response duration in patients achieving normalized hemoglobin levels sis around 2 years, with 30 months in patients with a low IPSS score.⁴⁰ The combined information of pretreatment transfusion need (≤ 2 units/month) and serum erythropoietin level (≤ 500 U/L) predicts for a response to treatment, and while patients with low transfusion need and S-EPO levels show a response rate of 74%, the corresponding figure for those with a high transfusion need and S-EPO level is 7%.⁴¹ Two retrospective studies comparing patients treated with EPO with or without the addition of G-CSF with untreated patients from the IPSS registry have demonstrated that treatment is safe and leads to no increased risk for leukemic transformation.^40,42 Moreover, the French study showed by univariate comparison that the survival of treated patients was better than that of the untreated cohort. We have recently performed an epidemiological study comparing treated and untreated but otherwise matched cohorts of patients with low- and intermediate-risk MDS enrolled from 1990 through 1999.⁴³ This study, which had information of and could control for all major risk factors, showed a very clear survival advantage for the EPO plus G-CSF–treated cohort (relative risk 0.61, 95% confidence interval 0.44–0.83), and also decreased risk of non-leukemic death (HR = 0.66, 95% CI 0.44–0.99, P = .042). There was no association with the risk of AML evolution (HR = 0.89, 95% CI 0.52–1.52, P = .66). A subgroup analysis revealed that EPO plus G-CSF treatment was associated with enhanced survival only in patients receiving more than 2 units per month at start of observation (HR_{<2 U/month} = 0.44, 95% CI 0.29–0.66, P < .001, HR_{=2 units/month} = 1.04, 95% CI 0.57–1.89, P = .91). This group of patients corresponds well with the good prognosis group, according to the predictive model.⁴¹ 

Immunosuppressive treatment and thalidomide, which may be efficacious in subsets of RA, have proven relatively inefficacious in RARS.⁴² Lenalidomide has been shown to abrogate transfusion need in around 25% of patients with EPO-refractory RARS, with a median duration of response of 43 weeks, and is presently evaluated in combination with EPO and other drugs in clinical trials.⁴³ Interestingly, while several reports have shown that the response rate to EPO in RARS is lower than the response in RA or early RAEB, the effect of the combination of EPO and G-CSF is better in RARS than in other subsets of MDS, with an overall and complete erythroid response rate of 50% and 38%, respectively.³⁹ RARS and RCMD-RS also showed significant differences in response to treatment with EPO+G-CSF.⁴³ The survival of patients with WHO RARS was 121+ months, with a response rate of 71% and a response duration of 28 months (8–116+ months). Interestingly, RCMD-RS patients showed a lower response rate, 30%, but a similar response duration (25 months, range 27–95) and a shorter survival, 31 months (14–43 months). No patients with WHO RARS developed AML as compared to 10% in the RCMD-RS cohort. Hence, treatment with EPO+G-CSF in patients with MDS and ringed sideroblasts is safe and leads to prolonged responses. It is, however, clear that patients with RARS show a higher response rate than those with RCMD-RS, 71% versus 30%, confirming previous results from a smaller study.⁷ These response rates indicate that while the biology of RARS is relatively homogeneous, that of RCMD-RS shows a larger variation.

The current literature does not provide good information on the outcome of this patient group, partly because of the recent identification of the syndrome and the still unclear diagnostic criteria. Compared to patients with ET, patients with RARS-T have a worse outcome, more similar to that of patients with RARS in general. A recent study reported that 11 of 23 patients with RARS-T had JAK2 mutations, and that no AML evolution and a better survival were observed in this group compared to those without JAK2 mutation.³⁷ By contrast, a recent relatively large report³⁷ failed to show any difference in survival in the JAK2 cohort, despite significantly more myeloproliferative features. Other reports have observed AML transformation and progressive disease also in JAK2-positive RARS-T. Some studies have indicated a lower degree of anemia, while others see no difference. In our EPO+G-CSF–treated cohort we identified 4 patients with pretreatment platelet counts > 450 × 10⁹/L (452–585 × 10⁹/L, unpublished observations). This small group did not differ from the RARS/RCMD-RS cohort regarding response rate and duration. There is no published evidence that the thrombocytosis in RARS-T is associated with an elevated risk for thrombotic complications, and there is no reason to believe that treatment of very high platelet counts in RARS-T should differ from that of ET. Thus, the available evidence is insufficient for recommending any specific treatment. Whether there is a role for the new JAK2 inhibitors remains to be studied, but available data do not support that the myeloproliferative component of RARS-T contributes significantly to the morbidity of these patients.

More than 50 years have elapsed since Sven Erik Bjorkman described 4 patients with RA and accumulation of “iron granules” in the cytoplasm. These observations eventually led to the inclusion of acquired sideroblastic anemia in the classification of MDS. Although there has been progress as regard to the biology and biochemical alterations of this disorder, the mechanisms underlying accumulation of aberrant ferritin in the mitochondria and associated erythroid apoptosis and anemia still remain to be discovered. The occurrence of JAK2 mutations and thrombocytosis in RARS is too frequent to be the result of chance only, and it is possible that this link may provide a key to an increased understanding of the genetic abnormalities causing ringed sideroblast formation.