Background: Sideroblastic anemia (SA) can present as congenital SA (CSA), acquired clonal SA, and acquired reversible SA. Patients (pts) with SA have anemia and ring sideroblasts (RS). Acquired clonal SA is often linked to myelodysplastic syndromes (MDS) or myelodysplastic/ myeloproliferative neoplasms (MDS/MPN). Clinico-pathologic overlap features, unmet morphologic and/or cytogenetic criteria complicate the diagnosis of SA leading to delayed therapies. Currently the diagnosis of SA is based on bone marrow (BM) examination and routine blood tests. There is a need to find easily testable biomarkers that can lead to faster diagnosis of clonal and non-clonal SA. Somatic mutation in splicing factor 3b, subunit 1 (SF3B1) are frequent in MDS-RS and MDS/MPN-RS and have been closely associated with RS. Objective: SF3B1 mutations can be a useful diagnostic biomarker for pts with acquired clonal SA who present with cytopenias and/or minimal morphologic changes suspicious of MDS and MDS/MPN but not sufficient to make a definitive diagnosis. Patients and Methods: Six pts with SA at presentation and seen at Cleveland Clinic were included in this study. The median age was 38 years (range, 6-75). Blood tests and BM biopsy showed persistent anemia [Hgb, 10.5 g/dL (range, 8.8-13)], RS [numerous (3 pts), 15% (1 pt), rare (1 pt) and >15% (2 pts)], 3/6 pts had minimal erythrodysplasia with 1 pt having a mild megakaryocytic dysplasia, 3 pts had hypercellular (60-90%), 2 pts had normocellular (50%, 80%) and 1 pt had hypocellular BM (30%) for age, and < 5% BM (2%=2 pts; 1%=1 pt; 3%=1 pt). Two pts had PLT count > 400 k/uL, 3 pts had > 100 k/uL, and 1pt <100 k/uL. Leukocytosis was observed in 2, leukopenia in 1, and neutropenia in 2 pts. Iron studies were unremarkable Molecular analysis was performed on SF3B1 (all exons), PRPF8 (exons 15, 25, 33), U2AF1 (exons 1, 6, 7), SRSF2 (exons 1, 2), DNMT3A (18-23), EZH2 (exons 18, 19) and IDH1/2 (exon 4). BCR/ABL p210 transcripts were assessed by RT-PCR. JAK2 (exon 14) was assessed by melting curve analysis. Results: All pts presented with sustained anemia, RS w/o or with minimal BM dysplasia, and normal karyotype. SF3B1 mutations (K666N and K700E) were detected in 2 pts (pts#2 and 6) before the clonal disease manifested. The first pt was a 75-year-old man with leukocytosis, transfusion-independent anemia, and fatigue. At the time of SF3B1 molecular testing the BM was hypercellular (60%) with <10%, megakaryocytic dysplasia, 2% blasts and 15% RS. JAK2 and BCR-ABL were wild type (WT). Later on, the patient developed hepatomegaly, his cytopenias worsened requiring RBC transfusions, and a repeat BM biopsy showed more prominent dysplasia of erythroid cells and megakaryocytes. The second pt was a 51-year-old man with fatigue, low WBC= 3.0 k/uL, ANC=0.7 k/uL, and PLTs=113 k/uL and macrocytic anemia (MCV=106 fL and Hgb=12.2 g/dL). His spleen was borderline enlarged (14 cm). BM biopsy showed 90% cellularity, erythroid hyperplasia, rare dyserythropoiesis and 60% RS. Suspicion for a possible MDS was raised although morphology was inconclusive. SF3B1 mutation (K700E) confirmed the clonality of the disease. A few months later, the pt developed worsening counts and overt MDS was confirmed by BM biopsy. All SF3B1WT pts were WT also for other genes known to be mutated in MDS and MDS/MPN like PRPF8, U2AF1, SRSF2, DNMT3A, IDH1/2, and EZH2. Only genetic tests showed an ALAS2 mutation in pt#1 and a paternally inherited duplication of 10q23.31 in pt#3. Both pts were diagnosed with CSA. Clinical history and work up assessed the same diagnosis for pt#4. Pt#5 presented with neutropenia and anemia. BM biopsy showed no dysplasia, 50% BM cellularity, trilineage maturation, and 25% RS. The presence of RS appeared to be induced by the use of an antibiotic prior the BM biopsy. Patient’s cytopenias and RS resolved after drug cessation. Conclusion: This case series although conducted in few pts provides important insights in the diagnostic use of molecular genetics in clinical practice. Often SA pts come to the clinic with inconclusive morphologic features of MDS or MDS/MPN. The presence of SF3B1 mutations may serve as an additional tool that can help differentiating between clonal and non-clonal cases of SA. In both pts, SF3B1 mutations anticipated the subsequent overt manifestation of clinical MDS or MDS/MPN phenotype. The collection of a larger cohort of pts is ongoing in order to further confirm this finding. Disclosures No relevant conflicts of interest to declare.
The identification of the JAK2V617F mutation in myeloproliferative neoplasms (MPN) paved the way for the pivotal studies that led to the FDA approval of a JAK1/2 inhibitor, ruxolitinib (rux) in patients (pts) with myelofibrosis (MF). Improvement in splenomegaly and debilitating disease-related symptoms were the primary clinical responses observed with rux. Although JAK2 mutational status did not impact response/survival in MF pts, cytogenetics had an impact on prognosis. In a related myeloid neoplasm specifically myelodysplastic syndromes, molecular mutations (TET2/DNMT3A) predict for better therapeutic response to DNA methyltransferase inhibitors. We hypothesized that somatic mutations and single nucleotide polymorphism array (SNP-A) lesions are frequent in MF pts treated with rux and may affect their clinical outcomes. To further investigate the predictive and prognostic impact of SNP-A lesions and somatic mutations in MF pts in the rux era, we studied 54 MF pts who received at least 12 weeks of rux therapy (tx) using a modified dose escalation approach (Tabarroki A et al. 55th ASH; Abstract 1586). Clinical (total symptom score [TSS], spleen size), cytogenetic (metaphase cytogenetics [MC], SNP-A), hematologic and survival data were collected before and 12-weeks post rux tx. Categorical data were analyzed using X2 test. A p-value of <.05 was considered statistically significant. Sanger sequencing for genes relevant to myeloid neoplasm pathophysiology like TET2, CBL, LNK, DNMT3A, TP53, SF3B1, U2AF1, SRSF2, ASXL1, EZH2, JAK2, CALR, and IDH1/2 was performed. The median age of the cohort was 66 yrs (41-89); male/female: 28/26. The median follow-up time after initiation of tx was 17 months. The median overall follow-up of the cohort from the time of diagnosis was 35 months. Using DIPSS-plus, pts were stratified as high (24, 44%), int-2 (22, 41%) and int-1 (8, 15%) risk groups. Baseline median WBC=9.4k/μL, Hgb=10.2g/dL, PLT=212k/μl, TSS=20, and median palpable spleen size=13cm. Post-tx median WBC=9.9k/μL, Hgb=10.1g/dL, PLT=150k/μL, TSS=4, and spleen size=6cm. MC identified cytogenetic abnormalities in 24/54 (44.4%) pts. The most frequent chromosomal defects included del(20), +8, and +9. Serial MC was available for 20 pts and no cytogenetic evolution was identified. SNP-A data were available for 29 pts, of which 28 pts had SNP-A lesions. The most commonly involved chromosomes were 9 (15.1%), 20 (14.1%), and 14 (8.5%). Compared to MC analysis, additional SNP-A lesions were found in 66% of pts. Of note 39% of the pts had normal karyotypes but with pathologic SNP-A lesions; another 27% had pathologic SNP-A lesions besides the abnormal MC. Serial SNP-A analysis was available in 10 pts who while on rux tx did not develop any additional/new SNP-A lesion. There was no difference in spleen response rates or TSS between those who carried SNP-A lesions versus those who did not. Molecular analysis was possible for 34 pts. The most frequent somatic mutations observed involved JAK2 (70.6%), ASXL1 (24%), CALR (24%), SRSF2 (15%), and U2AF1 (9%). Pts with int-2 and high DIPSS plus scores were more likely to carry at least 1 mutation in any gene compared to pts with int-1 scores (int-2 vs int-1, p=.05; high vs int-1, p=.06). After a median follow-up of 35 months from diagnosis, 95% of the pts were still alive. 3 pts died from disease progression: 1 had a sole SRSF2 mutation, 1 had an SRSF2 plus CALR mutation, and 1 had a TET2 plus TP53 mutation. SRSF2 mutant pts had more severe thrombocytopenia pre-rux tx (91 vs. 203k/μL; p=.04). ASXL1 mutant pts had increased spleen sizes pre-rux (21 vs. 15cm, p=.06), but had similar response post-rux (10 vs. 8cm, p=0.6) compared to the wild-type. SRSR2 mutant pts had higher DIPSS-plus score (4.4 vs. 3; p=.05). Our study showed that MF pts treated with a rux modified dose escalation approach resulted in meaningful clinical and splenic responses regardless of molecular mutation status. Frequently found cryptic SNP-A lesions in MF pts may explain their poorer outcomes compared to pts with other MPNs. The fact that pts did not acquire additional/new SNP-A lesions during rux tx may be one of the mechanisms of improved survival in these pts. ASXL1, CALR, SRSF2 and U2AF1 were the most frequent non-JAK molecular mutations in MF pts treated with rux and were more frequent in high risk pts. Further studies are necessary to elucidate the clinical/ biological effects of these mutations in MF pts treated with rux. Disclosures Tiu: Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees.
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