Cytogenetic and molecular predictors of response in patients with myeloid malignancies without del[5q] treated with lenalidomide

<p>Abstract</p> <p>Background</p> <p>While lenalidomide (LEN) shows high efficacy in myelodysplastic syndromes (MDS) with del[5q], responses can be also seen in patients presenting without del[5q]. We hypothesized that improved detection of chromosomal abnormalities with new karyotyping tools may better predict response to LEN.</p> <p>Design and methods</p> <p>We have studied clinical, molecular and cytogenetic features of 42 patients with MDS, myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes and secondary acute myeloid leukemia (sAML) without del[5q] by metaphase cytogenetics (MC) who underwent therapy with LEN.</p> <p>Results</p> <p>Fluorescence in situ hybridization (FISH) or single nucleotide polymorphism array (SNP-A)-based karyotyping marginally increased the diagnostic yield over MC, detecting 2/42 (4.8%) additional cases with del[5q], one of whom were responded to LEN. Responses were more often observed in patients with a normal karyotype by MC (60% vs abnormal MC; 17%, <it>p </it>= .08) and those with gain of chromosome 8 material by either of all 3 karyotyping methods (83% vs all other chromosomal abnormalities; 44% <it>p </it>= .11). However, 5 out of those 6 patients received combined LEN/AZA therapy and it may also suggest those with gain of chromosome 8 material respond well to AZA. The addition of FISH or SNP-A did not improve the predictive value of normal cytogenetics by MC. Mutational analysis of <it>TET2, UTX, CBL, EZH2, ASXL1, TP53, RAS, IDH1/2</it>, and <it>DNMT-3A </it>was performed on 21 of 41 patients, and revealed 13 mutations in 11 patients, but did not show any molecular markers of responsiveness to LEN.</p> <p>Conclusions</p> <p>Normal karyotype and gain of chromosome 8 material was predictive of response to LEN in non-del[5q] patients with myeloid malignancies.</p

SNP Array Karyotyping Allows for the Detection of Uniparental Disomy and Cryptic Chromosomal Abnormalities in MDS/MPD-U and MPD

We applied single nucleotide polymorphism arrays (SNP-A) to study karyotypic abnormalities in patients with atypical myeloproliferative syndromes (MPD), including myeloproliferative/myelodysplastic syndrome overlap both positive and negative for the JAK2 V617F mutation and secondary acute myeloid leukemia (AML). In typical MPD cases (N = 8), which served as a control group, those with a homozygous V617F mutation showed clear uniparental disomy (UPD) of 9p using SNP-A. Consistent with possible genomic instability, in 19/30 MDS/MPD-U patients, we found additional lesions not identified by metaphase cytogenetics. In addition to UPD9p, we also have detected UPD affecting other chromosomes, including 1 (2/30), 11 (4/30), 12 (1/30) and 22 (1/30). Transformation to AML was observed in 8/30 patients. In 5 V617F+ patients who progressed to AML, we show that SNP-A can allow for the detection of two modes of transformation: leukemic blasts evolving from either a wild-type jak2 precursor carrying other acquired chromosomal defects, or from a V617F+ mutant progenitor characterized by UPD9p. SNP-A-based detection of cryptic lesions in MDS/MPD-U may help explain the clinical heterogeneity of this disorder

Increased Frequency of Polymorphisms in XRCC3 and OGG1 Genes in Patients with MDS

Abstract The effects of genetic factors on susceptibility to MDS are not well understood. The predisposition may be a result of complex genetic traits, various theories can explain how inherited genic sequence alterations could result in a higher susceptibility to this disease. In theory, genes involved in the metabolism of genotoxic chemicals, DNA repair genes and immunogenetic factors could all play a role. Possibly, the predisposition can be multifactorial and overall risk for MDS modified by acute or cumulative effects of environmental exposures. Alterations/variants of genes involved in MDS may result from mutations, which due to LOH or “gene dose effect” could lead to functional consequences. In addition SNPs, may be present in a variety of genes and by modifying their function result in a disease predisposition. For example, genes coding for enzymes involved in the metabolism or detoxification of cancirogens may show polymorphisms associated with low functional capacity. Based on previous reports, we have selected 4 genes for which specific SNPs have been implicated in increased risk of malignancies. Genes involved in DNA repair constitute rational targets of analysis in MDS as their dysfunction could explain increased frequency of chromosomal aberrations characteristic for this disease. For example, OGG1, XRCC1 and XRCC3 have been implicated in sensitivity to DNA damage following radiation and their variants may increase radiation-induced risk of malignancies. The NQO1 variant (involved in the protection of DNA from oxidative damage) was found to be associated with secondary AML (sAML). We have studied the frequency of homo- and heterozygous SNPs of these genes in MDS to determine whether they constitute genetic factors predisposing to MDS. Experimental cohort included 62 patients with MDS (35 RA/RS, 19 RAEB/t and 8 CMML). An allele specific Taqman PCR assay was designed to distinguish between SNPs in OGG1 (S326C), XRCC1 (R399Q), XRCC3 (T241M) and NQO1 (P187S). When XRCC3 was analyzed, C/T and T/T genotype was found in 75% of MDS patients (vs. 47% in controls; N=175; p<.001). Interestingly, 3 out of 4 patients with sAML were homozygous for the T/T genotype. When patients with RCMD were separately analyzed, 8/10 patients showed at least one allele with XRCC1 G→A SNP (80% vs. 47% p<.001). Based on historically established large cohorts of controls, we did not find an increased frequency of homo- or heterozygous variants of NQO1, XRCC1 or OGG1 in the MDS group as a whole. However, 9/12 (80%) patients with RAEB-2 showed at least one allele with C→G SNP (vs. 29% in controls, N=31). Interestingly, we have found 4 MDS (6%) patients homozygous for OGG1 variant (G/G) that has not been described in healthy controls. Three of these 4 patients had MDS/MPL overlap and one showed evolution to AML. In general, we did not find any correlation between the presence of the gene variants tested and evolution of karyotypic abnormalities. Although we have analyzed only 4 selected DNA repair genes in MDS, our findings suggest that genetically-determined decreased function of these genes may constitute a predisposition factor for the development of this disease. Increased frequency of XRCC3 C/T SNP and presence of patients homozygous for OGG1 G/G may represent examples of such susceptibility. More comprehensive analysis may reveal further polymorphisms that could alone or in context of other defects explain occurrence of MDS

Defects of Base Excision Repair Involving OGG1 Can Be Detected in a Subset of Patients with MDS and May Convey Propensity to Chromosomal Breaks

Abstract Increased frequency of chromosomal lesions in MDS points towards defects in the DNA repair machinery as pathogenetic factors responsible for clonal evolution. Base excision repair (BER) is a possible mechanism of genomic instability. Upon genotoxic stress or due to defective function of the components of the BER, single-stranded breaks accumulate and are converted into double stranded breaks during mitosis. Oxidative damage may result in generation of 8-oxoguanine (OG), the most abundant DNA lesion. OG can be quantitated in hematopoietic cells using flow cytometry and we found significantly increased content of OG in patients with MDS (n=29, p=.0088). We stipulated that this observation may be a result of overwhelmed repair mechanism due to its insufficiency or inability to handle significantly increased oxidative stress. Here, we investigated this phenomenon in a subgroup of patients with increased genomic OG content (i.e. OG fluorescence >mean+2SD of controls). Remarkably, these patients were characterized by advanced stage of MDS (p=.0027), more profound cytopenias (neutropenia p=.001, thrombocytopenia p=.014) and propensity for AML transformation (p=.036). As OGG1 is the initial glycosylase that excises OG, we investigated the expression levels of this enzyme in highly purified CD34+ progenitor and stem cells in controls and patients with a high OG score; we found two expression patterns allowing for further sub-classification of affected patients. Low OGG1 (24/34) pointed towards a subset of patients with an inherent lesion that required further investigation. In contrast high OGG1 levels, found in 10/34 patients, were either consistent with an appropriate response to ongoing oxidative stress or the effect of a positive feedback due to low enzymatic activity. The SNP S326C of OGG1 has been associated with impaired function of the enzyme and compensatory upregulation of OGG1 transcription. This SNP is present in heterozygous form in controls at the frequency of 29%, but we found it in 8/10 patients with a high OGG1 expression, while 9/12 patients with wild-type had decreased expression (p=.01). In general, MDS patients (n=63) showed 6% homozygosity (vs. 0% in controls, p=.15) and 41% heterozygosity (vs 29% in controls, p=.24). Consequently, increased OG content could indicate a dysfunction of OGG1 resulting from the S326C SNP that cannot be adequately compensated. In fact, in 3/5 patients with OGG1 variant, an increased OG content and expression of OGG1 were found. Regardless of the activity of the OGG1, its upregulation could have various consequences to the downstream elements of the BER such as Polβ, which fills in the gaps left by the activity of endonucleases. Using Taqman RT-PCR performed on CD34+ cells; we tested Polβ expression in patients with upregulation of OGG1 and identified 2 groups of patients. The high Polβ group points toward overwhelming oxidative stress despite of adequate feedback response. In contrast, the low Polβ group could represent a subset of patients with defective upstream elements of the BER including endonuclease. This theory can be supported by elevated numbers of apurinic sites, detected by Elisa (0.54 +/− 0.70 in controls vs 1.5 +/− 2.1 in MDS AP sites/105bp). Taken together our results indicate that various lesions can contribute to the dysfunction of BER that occurs in MDS in the context of increased oxidative stress

Detection of Recurrent Uniparental Disomy and Cryptic Chromosomal Abnormalities in MDS/MPD-U and MDS/MPD-Derived Secondary AML

Abstract The WHO classification distinguishes MDS/MPD as a distinct entity. The JAK2 V617F mutation is present in a minority of these patients (pts). UPD9p characterizes pts homozygous for the V617F mutation. Other chromosomal abnormalities can also be detected in pts with typical MPD and MDS/MPD, and it is likely that cytogenetic methods with higher resolution could detect additional defects. We applied 250K SNP-arrays to examine genomic composition and identify previously cryptic defects and molecular abnormalities in pts with MDS/MPD-U and secondary AML (sAML) arising from MDS/MPD-U both in pts wild-type for V617F and those with the mutation. Any deletions, duplications, and/or UPD found by SNP-A in 76 controls or on available internet databases were considered copy number variants (CNV) and non-pathogenic. First, we used pts with typical MPD to assess the ability of SNP-A to detect UPD. All pts homozygous for V617F showed UPD9p by SNP-A. In pts with MDS/MPD, several additional cryptic lesions were detected, including segmental micro-deletions on chr 1, 5, 9, and 12. UPD was common, occurring on chromosomes other than 9 in 9/28 patients (32%, i.e. on chr 1,11,12). Shared/overlapping lesions (in >2 pts) included small segmental lesions on chr 7 (N=3), and a small cytoband (q14.1) of chr 11 (N=3). Overall, clonal lesions including segmental UPD were found in 23/28 (82%) pts by SNP-A in comparison to 17/28 (61%) by metaphase cytogenetics (MC). Pts with a history of MDS/MPD-U with (N=14) and without the JAK2 V617F mutation (N=14) were also analyzed. MC revealed chr aberrations in 10/14 (71%) of V617F+ pts, including common lesions such as +8 and del5q. With SNP-A, 1 additional pt with normal MC was found to have an abnormal karyotype (UPD 7 and 9), and 9/10 pts with abnormal MC had additional lesions previously undetected, including UPD on chrs other than 9 in 3/14 pts (21%). Examples of deleted regions include segmental losses within chrs 2, 7, 8 and 13, and UPD on chrs 1p, 7q, and 11. Likewise, additional lesions were identified in MDS/MPD-U pts negative for V617F. 8/14 pts (57%) showed abnormal MC; however SNP-A showed lesions in 12/14. In addition, 6/8 pts with abnormal MC had lesions in addition to those detected by MC. UPD was also common in V617F- pts, occurring in 6/14 (43%), predominantly on chr 11 (in 3/6 pts). No significant difference was found between the number or type of lesions found in pts with and without V617F mutation. SNP-A can also be used to identify lesions acquired during AML evolution. In 1 MPD pt at diagnosis, SNP-A showed UPD9p as a sole abnormality consistent with a homozygous V617F mutation. Upon transformation, repeated SNP-A showed a V617F- leukemic clone (normal chr 9) possessing microdeletions on chr 4 and 19. Similar evolution of a V617 negative leukemic clone was also observed in an MDS/MPD pt in whom a new UPD6 was detected. However, in 3 other MDS/MPD patients, SNP-A showed the presence of a V617+ leukemic clone (showing UPD9) in AML blasts. In summary, SNP-A-based karyotyping complements MC and allows for precise definition of chr aberrations in pts with MDS/MPD, including copy-neutral LOH. UPD is common in both JAK2 V617F+ and V617F- disorders, and is not restricted only to chr 9p, indicating other potential causative genes

TET2 Mutations Are Frequent in RARS-T

Abstract Abstract 3794 Poster Board III-730 Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative neoplasms (MDS/MPN). Since JAK2 V617F and MPL W515L mutations are present in a significant proportion of RARS-T patients, many investigators consider this entity to be more closely related to classical MPN. However, a significant minority of patients with RARS-T do not display either JAK2 V617F or MPL W515L mutations. We have studied a cohort of patients with RARS-T (N=20) characterized by the presence of ring sideroblasts, reticulin fibrosis and thrombocytosis (>450×109/L), that lack obvious causes of secondary thrombocytosis. While 8/20 patients harbored the JAK2 V617F, and 3/20 the MPL W515L mutations, the molecular pathogenesis for the remaining 9 patients was unexplained. Activation of JAK2 and MPL is associated with aberrant phospho-STAT5. Cases positive only for phospho-STAT5 may harbor other related, so far unidentified mutations. Many groups have recently observed a frequent area of somatic uniparental disomy (UPD) at 4q24, most commonly encountered in patients with chronic myelomonocytic leukemia (CMML), MDS/MPN, some typical MDS, and secondary acute myeloid leukemia (sAML). Overlapping microdeletions and UPD on 4q24 pointed towards possible mutations in the TET2 gene; such mutations were subsequently found in myeloid malignancies, most significantly MPN and MDS/MPN. Based on these findings, and the established correlation of RARS-T with JAK2 V617F and MPL W515L mutations, we evaluated the mutational status of TET2 in RARS-T patients. SNP-A allowed detection of copy neutral loss of heterozygosity (CN-LOH), such as UPD9p, which is associated with the JAK2 V617F mutation, and UPD1p, associated with MPL W515L. SNP-A facilitated detection of previously cryptic lesions; 11/20 patients showed an abnormal SNP-A-based karyotype (only 3 of these defects were also detected by MC). The new lesions seen by SNP-A included various UPD, such as, 1p, 2p, 3q, 6p, 8p, 9p and 10p. The presence of UPD9p/1p was consistent with homozygous JAK2 V617F/MPL W515L mutations. Likely, duplication of mutated alleles constituted a further permissive event during clinical evolution. However, none of the patients showed a somatic LOH at 4q24, suggesting that biallelic TET2 mutations were not involved in the pathogenesis of RARS-T. Simultaneously, lack of UPD11q suggested that CBL mutations were absent. Indeed, Cbl ring finger domain mutational screening revealed no mutations. An aberrant phospho-STAT5 staining pattern was present in all cases that were positive for either JAK2 V617F or MPL W515L mutations (N=10). However, 4 patients demonstrated abnormal megakaryocytic STAT5 phosphorylation, despite the absence of both JAK2 V617F and MPL W515L mutations. Within this group, a monoallelic TET2 mutation, delC 1480Sfs, was identified. In addition, we found a group of 5 patients without either JAK2 V617F or MPL W515L mutations, and also without association of the aberrant phospho-STAT5 staining. One of these patients had a monoallelic TET2 V1718L mutation; interestingly, another patient's specimen showed two novel non-synonymous SNPs: Y867H and P1723S. In total, 2/19 (11%) patients harbored TET2 mutations. These findings indicate involvement of TET2 mutations in RARS-T pathogenesis. RARS-T cases with MPN-associated mutations may not show obligatory phospho-STAT5 staining. The majority of patients were characterized by lack of splenomegaly, decreased white blood cell counts, increased thrombocytosis, and a normal karyotype. In summary, the majority of RARS-T patients harbor JAK2 V617F and MPL W515L mutations that strongly activate STAT5 phosphorylation. We described herein the third most common mutation in RARS-T, which can occur with or without abnormal STAT5 activation. Disclosures: No relevant conflicts of interest to declare

Hemochromatois-Associated Gene Mutations in Patients with Myelodysplastic Syndromes with Refractory Anemia and Ringed Sideroblasts

Abstract Complex interaction between a multitude of genetic variants may be responsible for differential susceptibility to specific diseases, and be responsible for phenotypic variability and heterogeneity of clinical presentations. Such a variability in clinical features confounded for many years investigations into the pathogenesis of myelodysplastic syndromes (MDS). We made a curious observation of increased ferritin levels in some newly diagnosed patients with MDS RARS (refractory anemia with ringed sideroblasts) in whom transfusional iron-overload was unlikely due to very low transfusion burden. Hence, we hypothesized that RARS patients may harbor hemochromatosis-related mutations, which could contribute to the pathophysiology of this particular subset of MDS. We studied a cohort of 109 MDS patients; 42 with RARS, and 67 with other forms of MDS (18 RA, 12 RAEB, 7 RAEB-T, 1 CMML, and 29 MDS/MPD overlap). All patients were genotyped using restriction fragment length polymorphism (RFLP) method, designed to detect presence of C282Y and H63D mutations of the HFE gene. We found significantly higher frequency of heterozygozity for the C282Y mutation in 21% of RARS patients (vs 9% in control population, n=2016, p= 0.017) while H63D genotype was not increased. The possible pathogenic role of this finding in RARS was supported by the normal distribution of mutant HFE alleles in patients with other forms of MDS (5% vs. 9%, p =0.35). Interestingly, 3/7 patients with RA not fulfilling the RARS criteria, but having increased numbers of ringed sideroblasts (<15%) also showed heterozygozity for either C282Y or H63D allele. To correlate the presence of C282Y allele with clinical features of RARS patients, we have performed a subset analysis. Within this group we have included patients with a rather nebulous and rare form of MDS, provisionally subclassified by WHO as RARS with thrombocytosis (RARSt); 7 of these patients (n=10) were found to have either C282Y or H63D allele resulting in a frequency of 30% and 40% of C282Y or H63D allele, respectively. The combined prevalence of either of these alleles in the control population is 33% (vs. 70% in RARSt, p=.01). Previously, we have demonstrated that RARSt patients are characterized by a high prevalence of the V617F JAK2 mutation (Szpurka et al, Blood 2006) suggestive of the pathophysiologic derivation of this syndrome from MPD rather than MDS. Consequently, we have tested the frequency of HFE gene variants associated with hemochromatosis in patients with MPD and Jak2 mutations. Of note is that patients with RARS harbored more C282Y alleles than those with other forms of MDS or MPD with Jak2 mutation (except for those with RARSt; (21% vs 5% and 3%, p =0.036 and .012, respectively). We conclude that hemochromatosis associated mutations may contribute to the pathogenesis of RARS. In patients with MPD and Jak2 mutation, concomitant presence of hemachromatosis-predisposing HFE variants may result in the unusual presentation associated with ringed sideroblasts

Presence of JAK2 Mutations in MDS/MPD-u WHO Classified Patients and Not Other Forms of MDS Suggests Their Derivation from Classical Myeloproliferative Syndrome

Abstract The WHO classification of myeloid neoplasms recognizes a category of myeloid disorders that overlaps traditional myelodysplastic syndromes (MDS) and myeloproliferative disorders (MPD) as the entity MDS/MPD disease. This disease category includes CMML, JMML, atypical CML and MDS/MPD unclassifiable (MDS/MPD-u). The provisional entity termed RARS associated with marked thrombocytosis (RARS-t) is currently best classified as MDS/MPD-u until further information is available regarding its pathogenesis. Recently a JAK2 mutation V617F (G→T) was identified as a pathogenetic lesion in typical myeloproliferative disorders (Kralovics et al, NEJM, 2005). Subsequent studies of the JAK2 mutation concentrated on more proliferative forms of MDS or MDS/MPD such as CMML and CNL (Steensma et al, Blood, 2005). These studies demonstrated that homo- and heterozygous JAK2 mutants are present in a rather small proportion of these patients. Based on similar clinical features, we theorized that JAK2 mutants might be also found in patients with MDS/MPD-u. We have collected a cohort of these patients (N=202) and analyzed them for the presence of JAK2 mutation using a molecular allele-specific PCR assay. Positive cases were confirmed by sequencing with a sensitivity of about 20% of mutated cells. A group of patients with PV, MF and ET served as positive controls (N=66). In agreement with previous reports, the detection rate of JAK2 mutants for PV, MF and ET was 92%, 55% and 55%, respectively. Our experimental group included 104 patients with MDS or MDS/MPD (53 RA/RS, 14 RAEB, 22 RAEB-t/sAML, and 15 CMML). Most significantly, the RA/RS group contained 13 patients with WHO-defined MDS/MPD overlap and 3 with RARS-t (among these patients, 3 were JAK2 mutants; all of them were heterozygous - 18.8%). Within 16 patients with CMML1 or 2 we found only 2 heterozygous for JAK2 mutation (12.5%). The remaining cohort of 73 patients in MDS categories revealed only 1 patient to be heterozygous for JAK2 mutation (RARS, 1.4%). As expected MDS/MPD patients with JAK2 mutation showed various degrees of BM fibrosis, splenomegaly and less pronounced cytopenias. Except for one patient, JAK2 mutants had normal cytogenetics. All had normal MCV and ANC. In 4/5 increased megakaryocytes with/or without atypia was seen. One CMML patient with abnormal cytogenetics showed an unusual translocation t(8;9)(q22;p24). JAK2 is located at 9p24 so it is possible that the JAK2 gene was involved in the translocation generating a novel fusion protein in addition to an activating JAK2 mutant. Our results showed that JAK2 mutations are rarely found in typical cases of MDS or CMML. However, further analysis of JAK2 mutational status in patients with MDS/MPD-u is warranted. The reported detection rate may suggest that the pathogenesis of these entities is more akin to myeloproliferative than myelodysplastic syndromes

Hemochromatosis-associated gene mutations in patients with myelodysplastic syndromes with refractory anemia with ringed sideroblasts

We observed increased ferritin levels in newly diagnosed MDS-RARS patients without transfusional iron-overload. Hence, we hypothesized RARS patients may harbor hemochromatosis-related mutations, which could contribute to the pathophysiology of this myelodysplastic syndromes (MDS) subset. We studied a cohort of 140 MDS patients: 42 with RARS, 10 with increased ringed sideroblasts, and 96 with other forms of MDS (43 RA, 27 RAEB, 17 RAEB-T, 8 MDS/MPD, 1 CMML). Patients were genotyped using restriction fragment length polymorphism, designed to detect C282Y and H63D mutations of the HFE gene. We found significantly higher frequency of heterozygosity for C282Y mutation in RARS patients compared with a large control population of matched race individuals (21 vs. 9.8% in controls, P = 0.03); H63D genotype was not significantly increased. Frequency of HFE variation in other MDS subtypes failed to differ significantly from controls. Within this group, we included patients with a rare form of MDS, provisionally subclassified by WHO as RARS with thrombocytosis (RARSt). 10/14 RARSt patients were carriers of either C282Y or H63D allele significantly increased compared with the combined prevalence in a healthy population (71 vs. 33%, P < 0.01). We found expected distribution of mutant HFE alleles in patients with other forms of MDS (9.1 vs. 9.8%, P = 0.82). Increased prevalence of HFE gene mutations is not a generalized feature of MDS, but some subgroups of MDS, especially those characterized by excessive accumulation of ringed sideroblasts, exhibit C282Y mutations at a higher frequency than in other forms of MDS and healthy controls

Acquired 1p Uniparental Disomy Is Associated with Biallelic MPL W515L Mutation in RARS-T

Abstract Refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative diseases (MDS/MPD). Since JAK2 V617F mutation is present in a significant proportion of RARS-T patients (Szpurka et al. Blood, 2006), many investigators consider this entity to be more closely related to classical MPD. However, a significant minority of patients with RARS-T do not display a JAK2 V617F mutation. We have studied a cohort of patients with RARS-T (N=18) characterized by the presence of ringed sideroblasts, reticulin fibrosis and thrombocytosis (>600×109/L), that lack obvious causes of secondary thrombocytosis. While 8/18 patients harbored a JAK2 V617F mutation, a molecular pathogenesis for the remaining patients was unexplained. The successful application of SNP-A to characterize the genomic lesions in MDS prompted us to use this technology to study RARS-T. SNP-A allows detection of copy neutral loss of heterozygosity such as UPD9p which is associated with JAK2 V617F mutation. SNP-A facilitated detection of previously cryptic lesions; 9/18 patients showed an abnormal SNP-A-based karyotype often involving multiple lesions (only 5 of these defects were detected by metaphase cytogenetics). The new lesions seen by SNP-A included gains of chromosome 11p, 20q and 21q; deletion of 2p and various areas of UPD including 1p, 9p, 6p, 2p and 8p. SNP-A allowed identification of seemingly invariant UPD1p in 4/18 patients. As this region includes the Mpl gene, we analyzed patients for the presence of MPL W515L/K mutations which have been described in MPD. We did not find any patients with MPL W515K, however MPL W515L mutation was present in 2/4 RARS-T patients with UPD1p; another patient showed monoallelic MPL W515L variant. In addition, 1 patient with UPD1p harbored both JAK2 V617F and MPL W515L mutations. To further delineate the molecular lesion we analyzed all patients for the presence of abnormal STAT5 activation. An aberrant phospho-STAT5 staining pattern was present in all cases that were positive for either JAK2 V617F or MPL W515L mutations (N=10); unexplained STAT5 activation was found in only 4 cases, pointing towards a molecular defect involving this pathway. In these 4 patients, and in 1 additional with UPD1p who did not harbor MPL W515L mutation, we searched for other genes which might explain the pathogenesis of this disease by potentially causing aberrant activation of STAT5. We sequenced Jak1T478S, Jak1V623A and Ntrk1S677N as well as the transmembrane, juxtamembrane and kinase domain of Tie1, Epha2 and Ephb2 genes, but no mutation was found. In addition, we found a group of phospho-STAT5-negative patients (N=4) that showed typical genetic features of myelodysplasia e.g. del(5), +8 and partial loss of chromosome X; these cases are probably best considered to be of MDS origin rather than MPD. To our knowledge, our work is the first description of biallelic MPL W515L mutation and UPD1p found in RARS-T patients. This data is important for understanding the clonal selection process and pathophysiology of activating mutations in MDS/MPD. Overall, our studies demonstrate that somatic UPD1p is associated with homozygous MPL W515L mutation in MDS/MPD cases. Localizing areas of somatic UPD by SNP-A may help identify candidate genes within the shared regions that are likely targets for mutations