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Clonal cytogenetic evolution (CE) (i.e., acquisition of new chromosomal aberrations over time) is relevant for the progression of myelodysplastic syndromes (MDS). We performed detailed analysis of CE in 729 patients with MDS and related disorders. Patients with CE showed shorter survival (median OS 18.0 versus 53.9 months; P P P P P = 0.020) and del (17p) (P = 0.002) were especially unfavorable. Extending the evolution patterns from Tricot et al. (1985) forming five subgroups, prognosis was best (median OS not reached) in patients with “transient clones/changing clone size”, whereas those with “CE at diagnosis” showed very poor outcomes (P

We genetically analyzed a group of high risk MDS/AML patients treated by a combination of azacitidine and lenalidomide. In our cohort, the extent of genetic rearrangements was associated with outcome and response to treatment. The size of total genomic aberrations as defined by molecular karyotyping (SNP-array analysis) was a predictive marker for overall survival. TP53 mutations were associated with therapy refractoriness only if accompanied by heavily rearranged chromosomes. This study suggests a potential value of molecular karyotyping as a method to objectivate comprehensively the extent of genetic alterations in high risk patients with complex karyotypes, especially if the clinical value of the size of total genomic aberrations and the fragmentation status of single chromosomes could be evaluated in larger therapy trials.

Loss of the Y-chromosome (LOY) is described as both a normal age-related event and a marker of a neoplastic clone in hematologic diseases. To assess the significance of LOY in myelodysplastic syndromes (MDS), we determined the percentage of LOY in clonal CD34+ peripheral blood cells in comparison to normal CD3+ T-cells of 27 MDS patients using fluorescence in situ hybridization (FISH) analysis. Results were compared with the percentage of LOY in CD34+ and CD3+ cells of 32 elderly men without hematologic diseases and in 25 young blood donors. While LOY could not be detected in CD3+ cells of young men, it was observed in CD3+ cells of elderly men without hematologic diseases (2.5% LOY) as well as in CD3+ cells of elderly MDS patients (5.8% LOY). The percentage of CD34+ cells affected by LOY was significantly higher in MDS patients compared to elderly men without hematologic diseases (43.3% vs. 13.2%, P = 0.005), indicating that LOY has an age-related basis but is also associated with MDS. Furthermore, we aimed to define a threshold between age- and disease-associated LOY in MDS. Statistical analysis revealed that a value of 21.5% LOY in CD34+ peripheral blood cells provided the best threshold to discriminate between these two conditions in MDS. We conclude that LOY is clonal in a substantial number of MDS based on an age-related predisposition.

In myelodysplastic syndromes (MDS), deletion of the short arm of chromosome 12 (del(12p)) is usually a small abnormality, rarely detected as a single aberration by chromosome banding analysis (CBA) of bone marrow metaphases. Del(12p) has been described in 0.6 to 5% of MDS patients at initial diagnosis and is associated with a good to intermediate prognosis as a sole anomaly according to current scoring systems. Here, we present the results of a systematic del(12p) testing in a German prospective diagnostic study (clinicaltrials.gov: NCT01355913) on 367 MDS patients in whom CD34+ peripheral blood cells were analysed for the presence of del(12p) by sequential fluorescence in situ hybridization (FISH) analyses. A cohort of 2,902 previously published MDS patients diagnosed by CBA served as control. We demonstrate that, using a sensitive FISH technique, 12p deletion occurs significantly more frequently in MDS than previously described (7.6% by CD34+ PB-FISH vs. 1.6% by CBA, P < 0.001) and is often associated with other aberrations (93% by CD34+ PB-FISH vs. 60% by CBA). Additionally, the detection rate can be increased by repeated analyses in a patient over time which is important for the patient´s prognosis to distinguish a sole anomaly from double or complex aberrations. To our knowledge, this is the first study to screen for 12p deletions with a suitable probe for ETV6/TEL in 12p13. Our data suggest that the supplement of a probe for the detection of a 12p deletion to common FISH probe panels helps to avoid missing a del(12p), especially as part of more complex aberrations.

Transfusion-dependent patients with low- or intermediate-1-risk myelodysplastic syndrome

International Prognostic Scoring Systems are used to determine the individual risk profile of myelodysplastic syndrome patients. For the assessment of International Prognostic Scoring Systems, an adequate chromosome banding analysis of the bone marrow is essential. Cytogenetic information is not available for a substantial number of patients (5%-20%) with dry marrow or an insufficient number of metaphase cells. For these patients, a valid risk classification is impossible. In the study presented here, the International Prognostic Scoring Systems were validated based on fluorescence in situ hybridization analyses using extended probe panels applied to cluster of differentiation 34 positive (CD34(+)) peripheral blood cells of 328 MDS patients of our prospective multicenter German diagnostic study and compared to chromosome banding results of 2902 previously published patients with myelodysplastic syndromes. For cytogenetic risk classification by fluorescence in situ hybridization analyses of CD34(+) peripheral blood cells, the groups differed significantly for overall and leukemia-free survival by uni- and multivariate analyses without discrepancies between treated and untreated patients. Including cytogenetic data of fluorescence in situ hybridization analyses of peripheral CD34(+) blood cells (instead of bone marrow banding analysis) into the complete International Prognostic Scoring System assessment, the prognostic risk groups separated significantly for overall and leukemia-free survival. Our data show that a reliable stratification to the risk groups of the International Prognostic Scoring Systems is possible from peripheral blood in patients with missing chromosome banding analysis by using a comprehensive probe panel (clinicaltrials.gov identifier:01355913). peerReviewed

Introduction: Complex aberrant karyotypes (CK, ≥3 cytogenetic aberrations, CA) are associated with an unfavorable prognosis and an increased AML transformation rate in MDS. However, even MDS with CK (CK-MDS) are heterogeneous in terms of genetic profile and prognosis. Recently, we demonstrated that a high number of CA as well as mutations in TP53 (TP53mut) are associated with increased risk in CK-MDS (Haase et al, 2019). However, as there is a strong association between CK-MDS and TP53mut, it is still a matter of debate whether the karyotype and TP53mut are prognostically independent genetic markers. Furthermore, loss of heterozygosity (LOH) of 17p13 (TP53LOH), due to loss of genetic material or to copy number neutral LOH (CN-LOH), is also associated with a poor prognosis. We here aimed to characterize TP53mut andTP53LOH in CK-MDS and to elucidate the impact of cytogenetics, TP53mut and TP53LOH on the outcome of CK-MDS. Methods: We included 178 pts with MDS (N=138), CMML (N=5) and secondary AML after MDS (AML with myelodysplasia-related changes, N=35), all with CK. The median precentage of bone marrow (bm) blasts was 11% (range: 0-90%). The median age was 72 yrs (range: 30-95 yrs). The male:female ratio was 1.23:1. The number of CA was determined by banding analysis in all cases. The karyotype was confirmed by multicolor FISH in 134 cases. TP53LOH was verified by FISH analysis of the TP53 locus in 17p13 (146 analyses) and/or molecular karyotyping (MK, 41 analyses). In 144 cases further FISH probes in addition to TP53 were used. TP53mut was identified by NGS (54 cases) or Sanger sequencing (124 cases). Follow-up data for survival analyses were available for 127 pts with MDS and oligoblastic AML with less than 30% bm blasts. Results: The median number of CA was 7 (range: 3-46), 98/178 pts (55%) showed a TP53mut (median VAF: 34%, range: 8-93%) and 64/178 (36%) a TP53LOH (median FISH clone size: 65%, range: 6-99%), including 9 pts with a CN-LOH in 17p13. The CN-LOH was either identified by MK (5/41 pts (12%) where MK was available showed a CN-LOH, 4/5 with TP53mut) or by NGS (4/54 pts (7%) where NGS was available showed a VAF >70% and normal TP53-FISH). In total, a TP53mut and/or a TP53LOH was identified in 116/178 pts (65%). Overall survival (OS) did not significantly differ between CK-MDS with TP53mut only, TP53LOH only, and TP53mut+TP53LOH (Fig.1). Therefore, we merged TP53mut and TP53LOH to TP53altered in all further analyses. Regarding the cytogenetic characterization of pts with TP53altered, the number of CA was significantly higher in pts with TP53altered than in pts with normal TP53 (median 9 CA (range: 3-46) vs 5 CA (range: 3-24), P The number of CA as well as the TP53 status contributed significantly to OS (Fig.2). The presence of anemia (Hb Conclusions: The presence of ≥5 CA is associated with reduced OS in CK-MDS. A TP53mut as well as a TP53LOH both further segregate outcome. The impact of the clone size of TP53mut and TP53LOH on survival is currently being evaluated. Our data imply that the TP53 status (TP53mut and/or TP53LOH) and the complexity of the karyotype are independent prognostic markers. Based on the presence of anemia, the TP53 status (TP53mut and/or TP53LOH), and the number of CA, the individual risk of CK-MDS can be estimated more accurately. This will allow to better tailor treatment decisions for individual pts with CA. Funding (FS): 2017 SGR 288-GRC Disclosures Germing: Jazz Pharmaceuticals: Honoraria; Novartis: Honoraria, Research Funding; Amgen: Honoraria; Celgene: Honoraria, Research Funding. Kaivers:Jazz Pharmaceuticals: Other: Travel Support. Kröger:Celgene: Honoraria, Research Funding; DKMS: Research Funding; JAZZ: Honoraria; Medac: Honoraria; Neovii: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Riemser: Research Funding; Sanofi-Aventis: Research Funding. Hertenstein:RS Media: Research Funding. Döhner:Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria; CTI Biopharma: Consultancy, Honoraria. Bug:Hexal: Membership on an entity's Board of Directors or advisory committees; Celgene Neovii: Other: travel grant; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Sanofi: Other: travel grants; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; Jazz Pharmaceuticals: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees. Nickelsen:Roche Pharma AG: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grant; Janssen: Membership on an entity's Board of Directors or advisory committees. Platzbecker:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Results of a multicenter prospective phase II trial investigating the safety and efficacy of lenalidomide in patients with myelodysplastic syndromes with isolated del(5q) (LE-MON 5)

The gold standard of cytogenetic analysis in myelodysplastic syndromes (MDS) is conventional chromosome banding (CCB) analysis of bone marrow (BM) metaphases. Most aberrations can also be detected by fluorescence-in situ-hybridization (FISH). For this prospective multicenter German diagnostic study (www.clinicaltrials.gov: #NCT01355913) 360 patients, as yet, were followed up to 3 years by sequential FISH analyses of immunomagnetically enriched CD34+ peripheral blood (PB) cells using comprehensive FISH probe panels, resulting in a total number of 19,516 FISH analyses. We demonstrate that CD34+ PB FISH correlates significantly with CCB analysis and represents a feasible method for a reliable non-invasive cytogenetic monitoring from PB.

Sequential combination of azacitidine and lenalidomide in del(5q) higher-risk myelodysplastic syndromes or acute myeloid leukemia: a phase I study

Background Few data are available on therapy-related myelodysplastic syndromes and acute myeloid leukemia developing after radioiodine treatment. Design and methods We retrospectively analyzed 39 patients with myeloid neoplasms following radioiodine treatment, whose data were reported to the Duesseldorf Myelodysplastic Syndromes Register (8 of 3814 patients) and five other German Myelodysplastic Syndromes centers (n=31) between 1982 and 2011. These data were compared with those from 165 patients from our Myelodysplastic Syndromes Register with therapy-related myeloid neoplasms following chemotherapy (n=90), radiation (n=30), or radiochemotherapy (n=45). Results With a median latency of 79 months, 18 patients developed therapy-related acute myeloid leukemia and 21 presented with therapy-related myelodysplastic syndromes (8 refractory anemia with excess blasts I/II, 6 refractory anemia with multilineage dysplasia, 3 myelodysplastic syndromes with del(5q), 1 refractory anemia, 1 refractory anemia with ring sideroblasts, 1 chronic myelomonocytic leukemia II, 1 myelodysplastic/myeloproliferative neoplasm unclassifiable). Risk assessment according to the International Prognostic Scoring System was low-risk in 23%, intermediate-1 in 29%, intermediate-2 in 35%, and high-risk in 13%. Karyotype was abnormal in 68%, with chromosomes 7 (30%), 5 (26%), 8 (26%) and 3 (17%) being most frequently affected. No differences in the distribution of gender, World Health Organization subtype, acute myeloid leukemia progression, International Prognostic Scoring System score, and cytogenetic risk were observed between patients with therapy-related myeloid neoplasms following radioiodine or other treatment modalities. Of 17 patients who received induction chemotherapy, 71% were refractory to this treatment or died from treatment-related toxicity. The median overall survival in the entire group was 21.7 months (95%-CI 10.5-33 months) and did not differ significantly in comparison to the survival of patients with therapy-related myeloid neoplasms following other cytotoxic treatments. Patients with therapy-related acute myeloid leukemia had significantly inferior overall survival (12.4 versus 28.7 months, P=0.002). Conclusions Patients developing a therapy-related myeloid neoplasm after radioiodine treatment usually present with biological characteristics similar to those seen in patients with therapy-related myeloid neoplasms following other cytotoxic treatment modalities, associated with a low response rate to induction chemotherapy and poor prognosis.

Introduction: Loss of the Y-chromosome (LOY) is frequent in myelodysplastic syndromes (MDS) and observed as a single aberration in 3-4% of male MDS patients (pts). It is often clonal and not only age associated and confers a very good prognosis and a very low risk for leukemic transformation (Greenberg et al, Blood 2012; Schanz et al, JCO 2012). But LOY does not prove a hematologic disease per se (Arber et al, Blood 2016). To facilitate a better discrimination between age-related and clonal LOY, the aim of this study was to identify molecular mutations and cytomorphological features which might be characteristic for MDS with isolated LOY. Methods: We included 291 pts in our analysis. The cohort comprised 199 pts with normal karyotype (NK) and morphologically proven MDS (excess blasts (EB) in 77/199 (38%) pts) and 92 pts with LOY. NK was defined by 20 normal metaphases or at least 10 normal metaphases and normal fluorescence in situ hybridization (FISH, Tab.1). Results from mutational analysis were available for all pts with NK and for 61 pts with LOY as single cytogenetic aberration in ≥3 metaphases. Seventeen core genes (Tab.2) were sequenced in all 260 pts by Sanger and/or next generation sequencing (NGS). In 134 pts further 28 genes (Tab.2) were analyzed using one of two NGS panels. In addition to these myeloid genes, the second NGS panel covered single nucleotide polymorphisms on the Y-chromosome which enabled determination of the LOY clone size. Detailed cytomorphology for the evaluation of dysplasia was performed by two experts (UG, UB) as previously described (Germing et al, Leuk Res 2012) in 41 pts, including pts with small LOY clones and with cytogenetic sub-clones. Results: Sequencing of 40 pts with LOY and morphologically proven dysplasia showed higher frequencies of mutations in TET2 (epigenetic regulator), ZRSR2 (splicing factor, located at Xp22.2), and CBL (kinase signaling) compared to MDS with NK (Fig.1). Amongst others, mutations in IDH1/2 (epigenetic regulators) and RUNX1 (transcription factor) were rare in MDS with LOY (Fig.1). The total number of mutated core genes did not significantly differ between MDS with LOY and MDS with NK and no EB (p=0.54), but it was significantly higher in MDS with NK and EB (p=0.014, Fig.2). To distinguish between LOY as ancestral or secondary mutation we sequenced 12 pts with MDS and cases we included as clonal cytopenia of undetermined significance (CCUS, pts with cytopenia(s) and molecular mutation and/or LOY≥75% of metaphases) (Wiktor et al, GCC 2000) using the second NGS panel that allowed determination of LOY clone size and detection of molecular mutations. Thereby, we identified four pts where LOY was most likely the founder aberration, two pts with LOY as secondary aberration in addition to ancestral molecular mutations, and six pts with co-dominance of LOY and a molecular mutation (Fig.3). Finally, we aimed to evaluate if the cut off of LOY≥75% (Wiktor et al, GCC 2000) can distinguish between age-related and clonal LOY in our cohort. In 41 pts analyzed in more detail, peripheral blood counts (hemoglobin: mean 10.4 vs. 9.7 g/dL; white blood count: 4.9 vs. 6.0x10(9)/L, platelets: 163 vs. 198x10(9)/L) and dysplasia of the individual cell lines (erythro-, granulo-, megakaryopoesis) did not differ significantly between LOY≥75% and Conclusions: Cytopenia with isolated LOY seems to comprise a heterogeneous mixture of cytomorphologic and molecular subtypes. We identified pts where LOY definitely is part of the MDS clone that also harbors molecular mutations, indicating that LOY is not only age-associated in these cases. The low total number of mutated genes corresponds to the previously described favorable prognosis of isolated LOY (Schanz et al, JCO 2012; Greenberg et al, Blood 2012). Our data support the current guidelines that LOY does not prove a hematologic disease in the absence of diagnostic morphologic features. In these cases LOY might fulfill the requirements of a clonal mutation as described for clonal hematopoiesis of indeterminate potential (CHIP) or CCUS. In summary, our data suggest that MDS with LOY shows a characteristic molecular mutation profile. Disclosures Germing: Janssen: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

In myelodysplastic syndromes (MDS), deletion of the short arm of chromosome 12 (del(12p)) is usually a small abnormality, rarely detected as a single aberration by chromosome banding analysis (CBA) of bone marrow metaphases. Del(12p) has been described in 0.6 to 5% of MDS patients at initial diagnosis and is associated with a good to intermediate prognosis as a sole anomaly according to current scoring systems. Here, we present the results of a systematic del(12p) testing in a German prospective diagnostic study (clinicaltrials.gov: NCT01355913) on 367 MDS patients in whom CD34+ peripheral blood cells were analysed for the presence of del(12p) by sequential fluorescence in situ hybridization (FISH) analyses. A cohort of 2,902 previously published MDS patients diagnosed by CBA served as control. We demonstrate that, using a sensitive FISH technique, 12p deletion occurs significantly more frequently in MDS than previously described (7.6% by CD34+ PB-FISH vs. 1.6% by CBA, P0.001) and is often associated with other aberrations (93% by CD34+ PB-FISH vs. 60% by CBA). Additionally, the detection rate can be increased by repeated analyses in a patient over time which is important for the patient´s prognosis to distinguish a sole anomaly from double or complex aberrations. To our knowledge, this is the first study to screen for 12p deletions with a suitable probe for ETV6/TEL in 12p13. Our data suggest that the supplement of a probe for the detection of a 12p deletion to common FISH probe panels helps to avoid missing a del(12p), especially as part of more complex aberrations.

Loss of the Y-chromosome (LOY) is described as both a normal age-related event and a marker of a neoplastic clone in hematologic diseases. To assess the significance of LOY in myelodysplastic syndromes (MDS), we determined the percentage of LOY in clonal CD34+ peripheral blood cells in comparison to normal CD3+ T-cells of 27 MDS patients using fluorescence in situ hybridization (FISH) analysis. Results were compared with the percentage of LOY in CD34+ and CD3+ cells of 32 elderly men without hematologic diseases and in 25 young blood donors. While LOY could not be detected in CD3+ cells of young men, it was observed in CD3+ cells of elderly men without hematologic diseases (2.5% LOY) as well as in CD3+ cells of elderly MDS patients (5.8% LOY). The percentage of CD34+ cells affected by LOY was significantly higher in MDS patients compared to elderly men without hematologic diseases (43.3% vs. 13.2%, P = 0.005), indicating that LOY has an age-related basis but is also associated with MDS. Furthermore, we aimed to define a threshold between age- and disease-associated LOY in MDS. Statistical analysis revealed that a value of 21.5% LOY in CD34+ peripheral blood cells provided the best threshold to discriminate between these two conditions in MDS. We conclude that LOY is clonal in a substantial number of MDS based on an age-related predisposition.

The karyotype represents one of the main cornerstones for the International Prognostic Scoring System (IPSS) and the revised IPSS-R (IPSS-R) that are most widely used for prognostication in patients with myelodysplastic syndromes (MDS). The most frequent cytogenetic abnormalities in MDS, i.e. del(5q), -7/del(7q), +8, complex karyotypes, or –Y have been extensively explored for their prognostic impact. The IPSS-R considers also some less frequent abnormalities such as del(11q), isochromosome 17, +19, or 3q abnormalities. However, more than 600 different cytogenetic categories had been identified in a previous MDS study. This review aims to focus interest on selected rare cytogenetic abnormalities in patients with MDS. Examples are numerical gains of the chromosomes 11 (indicating rapid progression), of chromosome 14 or 14q (prognostically intermediate to favorable), -X (in females, with an intermediate prognosis), or numerical abnormalities of chromosome 21. Structural abnormalities are also considered, e.g. del(13q) that is associated with bone marrow failure syndromes and favorable response to immunosuppressive therapy. These and other rare cytogenetic abnormalities should be integrated into existing prognostication systems such as the IPSS-R. However, due to the very low number of cases, this is clearly dependent on international collaboration. Hopefully, this article will help to inaugurate this process.

Cytogenetic aberrations are major markers in pathogenesis, diagnosis, and prognosis of patients with myelodysplastic syndromes (MDS). Their relevance for an individualized therapy in selected patients with MDS is increasing. Using conventional chromosomal banding analysis, aberrations are detectable in about 50% of patients. The application of additional methods like fluorescence in situ hybridization (FISH)-techniques improves the detection rate and diagnostic accuracy. In spite of the cytogenetic heterogeneity of MDS patients, the prognostic impact of the most frequent chromosomal changes is known and the understanding of rare abnormalities, independent clones, and double and complex aberrations is increasing. Future research will also focus on karyotype evolution, as it could give notice of disease progression and thus affect therapy decisions. International cooperations facilitate to unravel the remaining challenges for cytogenetics in MDS.

In myelodysplastic syndromes (MDS) chromosomal anomalies can be identified in 50-80% of patients. They have a diagnostic and prognostic impact and are increasingly considered for therapeutic decisions. Cytomorphology and cytogenetic analyses of bone marrow (bm) cells define the goldstandard to diagnose MDS patients and to document treatment response. We present a novel method using peripheral blood (pb) for frequent cytogenetic monitoring: after immunomagnetic cell separation circulating CD34+ cells were analysed by fluorescence in situ hybridization (FISH). We compared FISH analyses of enriched and non-enriched pb and bm cells with conventional chromosome banding analyses of bm metaphases: analysing circulating CD34+ cells by FISH is a sensitive, reliable method to measure the abnormal cell clones in pb. This method is practicable, non-invasive, representative for the clonal situation in the bm, and has a predictive value. Its feasibility was proven in a cohort of 27 MDS patients.

Introduction: Total (-7) or partial (7q-) monosomy 7 is the second frequent abnormality in MDS, occurring in around 12% of MDS/AML and up to 40% of therapy-associated MDS/AML. The present study was designed to analyze clinical features, prognosis and response to different therapeutic strategies in patients with -7 or del(7q) in a multicentric, retrospective cohort study. Patients and Methods: 471 patients with MDS/AML following MDS and monosomy 7 were registered and retrospectively analyzed. The median observation time was 3.6 years. Inclusion criteria were defined as follows: Morphologic diagnosis of MDS/AML following MDS, bone marrow blast count Results: 147 patients (31.2%) showed 7q-, 313 (66.5%) -7 and 11 (2.3) patients showed both abnormalities at the first cytogenetic examination. The abnormality was detected by CBA±FISH in 440 (93.4%) and by FISH only in 31 (6.6%). In the latter cases, the CBA was either unsuccessful or showed a normal karyotype. In 182 (38.6%) patients, -7/del7q was detected as a single abnormality, 77 (16.3%) showed two abnormalities and 184 (39.1%) showed a complex karyotype involving -7/7q-. As previously described (Schanz et al., 2012), untreated patients with an isolated 7q- as compared to an isolated -7 show a better prognosis regarding OS (median: 4.0 vs. 0.7 years; p Conclusions: To our knowledge, the study describes the largest patient cohort with MDS/AML and monosomy 7 published to date. Further data regarding the clinical characteristics of this subgroup of patients and the treatment regimes applied will be presented in detail. The study was supported by research funding from Celgene Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Schanz: Celgene: Honoraria, Research Funding, Travel grants: Celgene, Novartis, Lilly Other. Götze:Celgene Corp, Novartis Pharma: Honoraria. Nolte:Celgene, Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel grants Other.

Abstract 3816 Background Conventional chromosome banding (CCB) analyses of bone marrow (bm) metaphases represent the gold standard of cytogenetic diagnostics in myelodysplastic syndromes (MDS), but they are not suitable for frequent follow-up analyses. Most aberrations can also be detected by fluorescence in situ hybridisation (FISH), and they are provable in CD34+ cells from peripheral blood (pb). In our prospective multicenter German diagnostic study “Screening and genetic monitoring of patients with MDS under different treatment modalities by cytogenetic analyses of circulating CD34+cells” (ClinicalTrails.gov NCT01355913) we followed MDS pts by sequential FISH analyses. Methods CD34+ pb cells were enriched by immunomagnetic cell sorting (MACS®) and analysed by FISH using a “Superpanel” (D7/CEP7, EGR1, CEP8, CEP XY, D20, TP53, IGH/BCL2, TEL/AML1, RB1, MLL, 1p36/1q25, CSF1R, all Abbott® Products) at initial screening, every 12 months during follow-up and in case of suspected disease progression and a “Standardpanel” (EGR1, D7/CEP7, CEP8, TP53, D20, TEL/AML1, CEP XY, plus -if necessary- another informative probe) every 2 months in the 1st and every 3 months in the 2nd and 3rd year. If bm aspirate was available, additional CCB and FISH analysis of CD34+ and native bm cells were performed. Cut-off values for each FISH probe were evaluated in our lab. Cytogenetics, bm morphology, clinical course and therapies were documented in a database. All pts gave their written informed consent. The study was approved by all local ethic committees. Results After 3 years of study time, 361 patients (25 AZALE (University of Dresden), 110 LEMON5 (University of Duesseldorf), 226 CD34+FISH) have been included in the study, resulting in a total number of 19,516 FISH analyses: Median age, gender distribution and MDS subtypes were typical for the disease, median follow-up at the time of analysis was 8.2 (1–36) months. Chromosomal aberrations could be detected by FISH of CD34+ pb cells in 71.5% of pts (55% of CD34+FISH-cohort, 99% of LEMON5-trial pts, 100% of AZALE-trial pts). FISH and CCB were highly correlated: p Discussion Our interim results demonstrate that FISH analysis of circulating CD34+ pb cells provides relevant cytogenetic informations. It is a reliable novel method for screening and cytogenetic monitoring of MDS pts during the course of disease and under different therapies, and helps in cases where a bm biopsy is not possible or not successful. Disclosures: Braulke: Celgene: This study was supported by Celgene. Other. Götze:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bug:Celgene: Honoraria, travel support, advisory board Other; Novartis: Honoraria, travel support, advisory board, travel support, advisory board Other; Boehringer Ingelheim: Honoraria, travel support, advisory board, travel support, advisory board Other. Schafhausen:Novartis: Honoraria, travel support Other; BMS: Honoraria, travel support, travel support Other; Roche: Honoraria, travel support, travel support Other; Celgene: Honoraria, travel support, travel support Other; Alexion: Honoraria, travel support Other. Haase:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 921 This 3+3 dose escalation phase I trial by the German MDS study group aimed to determine the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of sequential azacitidine (AZA) 75 mg/m2 for 5 days followed by 14 days of up to 25 mg lenalidomide (LEN) in patients with higher-risk myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) and del(5q) cytogenetic abnormalities. Twenty patients (median age 69 years) were enrolled, including 16 (80%) with complex cytogenetic abnormalities and 65% of patients harboring at least one TP53 mutation. Nine patients (45%) had no prior treatment while relapse or progression to single agent azacitidine or lenalidomide treatment (30%) and allogeneic HSCT (15%) were the most common regimens among previously treated patients. Three of 6 patients treated with LEN at the 25 mg dose level experienced DLTs, with 20 mg subsequently identified as MTD. All eligible patients experienced treatment-related Grade 3/4 thrombocytopenia and neutropenia. Hematologic responses occurred in 4 of 9 (44%) previously untreated patients with complex karyotypes, including 3 with a TP53 mutation and were preceded by a significant decrease of del(5q) CD34+ progenitor cells in the blood during cycle 1. In fact, compared with baseline, the percentage of peripheral blood CD34+ cells with del(5q) clone was decreased in the hematologic responders after 1 cycle of azacitidine followed by lenalidomide therapy (P = 0.001) (Figure 1A). In contrast, the percentage of peripheral blood CD34+ cells with del(5q) remained unchanged in hematologic non-responders (Figure 1B). By applying TP53 mutation directed deep-sequencing technology we observed a concomitant early reduction and disappearance of minimal residual disease in MDS patients achieving a complete cytogenetic response. In one patient, a reemergence of the TP53 clone was observed later despite continuation of the treatment but by consolidation (lower dose and every 8 weeks compared to 4 weeks with induction) and it preceded the hematologic and cytogenetic relapse by several months. In conclusion, in a population of higher-risk MDS/AML patients with del(5q) that included a high proportion of patients with complex karyotypes and mutations of TP53, the sequential combination of azacitidine and lenalidomide was shown to be a feasible and potentially effective treatment strategy, even in those patients with TP53 -mutated clones. We additionally observed a correlation between the percentage of peripheral CD34+ cells with del(5q) and hematologic response, suggesting that monitoring of this cell population may be a surrogate marker of response in this setting. Our results encourage an application of sequential azacitidine and lenalidomide as first line therapy for higher-risk MDS patients in future trials. (A) HEMATOLOGIC RESPONDERS (B) HEMATOLOGIC NON-RESPONDERS (A) HEMATOLOGIC RESPONDERS (B) HEMATOLOGIC NON-RESPONDERS Disclosures: Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kuendgen:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Götze:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Germing:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Haase:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 4926 Introduction: Chromosomal banding analysis (CBA) of bone marrow metaphases is the gold standard to identify chromosomal abnormalities in myelodysplastic syndromes (MDS). We aim to comprehensively detect and follow chromosomal abnormalities during the course of the disease without the need of repeated bone marrow biopsies. In ongoing studies we attempt to achieve this goal by performing serial fluorescence in situ hybridization (FISH) analysis on CD34+ peripheral blood cells (PBC). The aim of this pilot study was to establish SNP-array-analysis (SNP-A) on CD34+ PBC to complement genetic analysis on peripheral blood by identifying chromosomal abnormalities not detectable by FISH and/or CBA. Methods: We immunomagnetically enriched CD34+ PBC of 20 patients (pts) with MDS (16 pts), suspected MDS (1 pts) and secondary acute myeloid leukemia (sAML, 3 pts). SNP-A was performed with arrays from Affymetrix (3x SNP 6. 0, 4x Cyto 2. 7, 13x CytoScanHD). Fresh or frozen CD34+ PBC of 10 pts and in methanol/acetic acid fixed CD34+ PBC of 9 patients were successfully processed. One whole genome amplified sample was included. CBA and FISH-A was done for all patients. Results: By CBA, 3 pts had no chromosomal abnormalities, 8 pts had one abnormality, 6 pts had 2–4 abnormalities and 3 pts had more than 6 abnormalities. By SNP-A on CD34+ PBC, additional abnormalities could be revealed in 13/20 pts. In two pts they were also confirmed by FISH-A. Most of them were micro-deletions not detectable by CBA. In addition, SNP-A revealed uniparental disomies (UPD) in 5/20 pts. Of the 3 pts with no detectable abnormalities in CBA, one had a micro-deletion in 4q24 (TET2). The other two had an insufficient number of metaphases. One of them showed a highly complex karyotype by FISH-A and SNP-A on CD34+ PBC. The other one had suspected MDS and did not show any abnormalities by SNP-A. The 17 pts with ≤ 6 abnormalities in CBA showed 55 abnormalities by CBA, FISH-A and SNP-A altogether. 34/55 (62%) abnormalities could be detected by SNP-A and/or FISH-A, but not by CBA. 24/55 (44%) abnormalities could only be detected by SNP-A. 4/55 (7%) of abnormalities were structural abnormalities or small clones and were only detected by CBA. Serial analysis indicated clonal evolution: A patient with 16 abnormalities detectable by CBA and additional three by FISH and SNP-A developed two further micro-deletions (del(2)(q31q32), del(4)(q24q26)) within four months. When a MDS patient with a known 20q-deletion (isolated by CBA and FISH) progressed to AML 25 months after first diagnosis we detected 3 micro-deletions by SNP-A of peripheral blood (0. 98 Mb on 4q, 1. 31 Mb on 12q, 2. 55 Mb on 12q) thus resulting in 4 cytogenetic alterations fulfilling the criteria of complex and prognostically unfavorable abnormalities. Conclusions: Recently it was shown that abnormalities detectable by SNP-A, but not by CBA, could worsen prognosis of MDS patients. We succeeded in detecting these additional abnormalities without the need of bone marrow biopsies out of peripheral blood. Nevertheless, by parallel FISH and SNP-A of CD34+ PBC, most abnormalities detectable by CBA of bone marrow metaphases could be detected. Comprehensive genetic analysis at close intervals thus is possible without the need of bone marrow biopsies to study clonal evolution. The information gained could be used for therapy decisions, to improve prognostication and to unravel genetic evolutionary steps towards acute leukemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 4872 Introduction: Chromosomal banding analysis (CBA) of bone marrow metaphases is the gold standard to identify chromosomal abnormalities in myelodysplastic syndromes (MDS). To detect and follow chromosomal abnormalities during the course of the disease without the need of repeated bone marrow biopsies, we are currently performing serial fluorescence in situ hybridization (FISH) analyses of CD34+ peripheral blood cells (PBC) in ongoing studies. To complement genetic analysis on peripheral blood we started a pilot study to establish SNP array analysis (SNP-A) on CD34+ PBC to identify chromosomal abnormalities not detectable by FISH. Methods: We analyzed eleven MDS and two AML-patients with known karyotypes and compared CBA with FISH and SNP-A of CD34+ peripheral blood and/or bone marrow cells. The FISH panel comprised up to 13 probes. The Affymetrix Genome-Wide Human SNP Array 6.0 and/or the Affymetrix Cytogenetics Whole-Genome 2.7M Array were used. We also included serial samples from a RAEB-II patient where bone marrow and/or peripheral blood were available from six time points collected over a period of 6 months. Results: In 13 patients analyzed using CBA, FISH, and SNP-A in parallel, 10/60 (17%) chromosomal abnormalities were exclusively detected by SNP-A. Using CBA and FISH we detected 28 chromosomal abnormalities in 12 patients. Additional SNP-A revealed 6 further aberrations (upd(7)(q11qter), upd(10)(q23.33q25.1), upd(17)(pterp11.2), del(9)(q22.33q31.1), del(13)(q12.3q22.2), del(15)(q15.1)). In one patient SNP-A increased the number of detectable chromosomal abnormalities from 22 to 26 (amplifications on 6p, del(15)(q11.2q21.1), del(18)(pterp11), upd(20)(q11.22q12)). Additional abnormalities were also detected in the serial sample: The major clone detectable by CBA at three different time points was 44,XX,del(5)(q13q33),-7,del(12)(p13p11.2),-17,-20,+der(20)t(17;20)(q10;p10). We could confirm all these abnormalities in CD34+ peripheral blood and bone marrow cells using a FISH panel that includes del(5q31)/EGR1, −7/CEP7, del(12p13)/TEL, del(17p13)/TP53, and del(20q12)/D20S108. FISH on CD34+ PBC confirmed a stable number of aberrant cells as 5q- was detectable in 94–98% of CD34+ PBC at all six available time points. We could also confirm all abnormalities of the major clone by SNP-A in CD34+ PBC in month 2 and in CD34+ bone marrow cells in month 6. Additional abnormalities occurring in sub-clones changed over time. A sub-clone exclusively detectable by CBA was identified in the first available sample in 2/25 (8%) metaphases: 44,idem,+der(3)t(3;6)(p10;q10),-6. Supplementary FISH and SNP-A revealed a 9.6 Mb del(13q14) in 44% of CD34+ PBC that was not detectable by CBA and had subsequently disappeared in the last available sample. SNP-A on CD34+ bone marrow cells of the last sample revealed two additional abnormalities in the absence of clinical signs of progression (del(2)(q31q32), del(4)(q24q26)). The del(4q)/TET2 could be confirmed by FISH. Conclusion: Detection and follow-up of chromosomal abnormalities during the course of the disease is possible without the need of bone marrow biopsies by parallel FISH and SNP-A of CD34+ peripheral blood cells. Detailed knowledge about the acquirement of chromosomal aberrations could be used to improve prognostication, to support therapy decisions and to unravel genetic evolutionary steps towards acute leukemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 3800 Introduction: In an ongoing diagnostic study we are currently following chromosomal anomalies in immunomagnetically enriched CD34+ peripheral blood cells in patients with suspected or proven myelodysplastic syndromes (MDS) at short intervals using fluorescence in situ hybridization (FISH) analysis every two to three months over three years. A loss of the Y chromosome was detected in 4% of these patients, as a single anomaly in 2%. Since it is controversially discussed whether loss of the Y chromosome is an age-related or a clonal event in patients with MDS, we aimed to examine whether a Y-loss is clonal or an age-related event in our patients. Methods: For patients with known Y-loss, we used peripheral blood not only to immunomagnetically enrich clonal CD34+ cells, but also CD3+ T-cells not belonging to the MDS clone. Subsequently, we performed FISH analysis to compare the clone sizes of cells with Y-loss in CD34+ and CD3+ cells. As our laboratory threshold for the FISH probe in CD34+ peripheral blood cells is 5%, we included 18 patients with clone sizes exceeding this threshold in CD34+ cells. The median age of the patients was 76 years (range 62–89). To establish a laboratory threshold for the FISH probe in CD3+ peripheral blood cells, we analyzed T-cells of 25 healthy men with a median age of 27 years (range 19–35). Furthermore, we just initialized an investigation of the laboratory threshold for the FISH probe in CD3+ peripheral blood cells of elder men by measuring the frequency of loss of the Y chromosome in T-cells of this control cohort not suffering from hematopoietic diseases. Until now we could recruit 15 men with a median age of 75 years (range 66–84) for this purpose, further will follow soon. Results: In patients with suspected or proven MDS, the number of cells with -Y was significantly increased in CD34+ cells compared to CD3+ cells (p Conclusion: Regarding the absence of Y-loss in CD3+ peripheral blood cells of young healthy men compared to up to 14% -Y in CD3+ peripheral blood cells of elder men, the low proportion of -Y in CD3+ cells of our patients suggests an age related Y-loss in normal T-cells. As the number of CD34+ peripheral blood cells with -Y exceeds the number of CD3+ peripheral blood cells with -Y in all patients, we assume that Y-loss is clonal to some extent in all of them. We established a reliable method to test if loss of the Y chromosome is disease- or age-related in individual MDS patients. It can be used to determine a clonal disease in patients with suspected MDS and Y-loss as sole abnormality. Disclosures: No relevant conflicts of interest to declare.

Abstract 4000 Lenalidomide has shown single agent activity in patients with MDS (Myelodysplastic Syndromes) and a del(5q) cytogenetic abnormality. Further, studies with the DNA methyltransferase inhibitor 5-azacytidine (5-aza) have been conducted in high-risk MDS (IPSS INT-2 or HIGH) and patients with acute myeloid leukemia (AML) resulting in considerable responses with a low rate of extramedullary toxicity compared to conventional induction chemotherapy (IC). Given the poor outcome of high-risk MDS and AML patients with chromosome 5 abnormalities, there is a significant clinical need to perform studies with new regimens in this patient population. We report first results of an ongoing phase I clinical trial evaluating the maximum tolerated dose (MTD) of lenalidomide in combination with 5-aza in patients with either high-risk MDS, refractory/relapsed AML or de novo AML not eligible for conventional IC with chromosome 5 abnormalities including monosomy 5 or del(5q). Given the mechanism of action of both drugs and also in contrast to a recent study in non-del(5q) MDS patients, a sequential approach was chosen. In fact, induction therapy consisted of 5-aza (75mg/m2 days 1–5) followed by increasing doses of lenalidomide (starting with 10mg p.o., days 6–19). In patients achieving a complete remission this was followed by a combined maintenance therapy every 8 weeks until disease progression. To determine the MTD, a standard “3+3” design was used. The dose limiting toxicity (DLT) is determined during the first cycle only and is defined as either inability to deliver the full dosing schedule of lenalidomide due to any ≥ Grade 3 non-hematologic toxicity or absence of hematological recovery after completing the 1st cycle despite complete marrow blast clearance or treatment delay of ≥ 4 weeks as a result of unresolved grade 4 non-hematological toxicity. Of 8 patients currently enrolled, median age was 67 years (range, 45 to 74 years), interval from primary MDS or AML diagnosis was 9 months (range, 1 to 100 months). IPSS categories were INT-2 (n = 1) and HIGH (n = 3) whereas 4 patients were included with advanced AML. It is of note, that all but two patients had a complex karyotype including a del(5q) abnormality. Prior treatment included IC (n=1), IC plus allogeneic HSCT (n=3) and/or single agent 5-aza (n=3) while 4 patients had received supportive care only prior to study entry. A median of 2 induction cycles were administered. During the first cycle of cohort I (10mg lenalidomide) and cohort II (15mg lenalidomide) grades 3 to 4 non-hematologic toxicities included febrile neutropenia (n = 3), enterocolitis (n = 1) and pneumonia (n=3) whereas therapy-induced grade 3–4 neutropenia or thrombocytopenia occurred in four and five patients, respectively. The MTD has not been reached yet. One patient (12.5%) with AML showed rapid progression while receiving the 1st cycle. Out of the remaining seven patients, one (12.5%) achieved a marrow CR together with a partial cytogenetic remission, and six patients (75%) had stable disease. Interestingly, two out of these achieved a partial cytogenetic remission. These preliminary data of an ongoing phase I trial demonstrate the safety and the potential of a combination of 5-aza and lenalidomide in patients with advanced MDS or AML and a del(5q). Disclosures: Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Haase:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Götze:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kuendgen:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giagounidis:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hofmann:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract 2937 Introduction: Chromosomal aberrations in myelodysplastic syndromes (MDS) play a major role in diagnostics, pathogenesis, prognosis, and, more recently, in treatment allocations. The acquisition of clonal abnormalities in pts with initially normal karyotype, the expansion of an aberrant cell clone with a given anomaly or the occurrence of new abnormalities are called karyotype evolution (KE). A model of stepwise cytogenetic changes is proposed, but only a few systematic studies had focused on this phenomenon as yet. Chromosomal anomalies can be detected by conventional chromosome banding analyses of bone marrow (bm) metaphases and most of them are provable by Fluorescence in Situ Hybridization (FISH) of circulating CD34+ cells from peripheral blood (pb). Aims: To closely follow chromosomal aberrations from pb, to assess karyotype evolution and rare anomalies and to correlate the molecular-cytogenetic results with pb blood counts, bm morphology and treatment modalities. For this purpose, we initiated a German multicenter prospective, non-interventional diagnostic study in October 2008. Methods: Sequential FISH analyses were performed on immunomagnetically enriched circulating CD34+ cells from pb as follows: a “super-panel” (D7/CEP7, EGR1, CEP8, CEP XY, D20, p53, IGH/BCL2, TEL/AML1, RB1, MLL, 1p36/1q25, CSF1R) was used for initial screening, every 12 months during follow-up and in every case of suspected progression. A “standard-panel” (EGR1, D7/CEP7, CEP8, p53, D20, CEP X/Y, TEL/AML1) was performed for analyses at short intervals every 2 months in the 1st year and every 3 months during the 2nd year. Results were also correlated with those of conventional banding and FISH analyses performed on bm samples, which typically were collected every 6 months. Results: As yet, 205 pts have been included in the study. Concerning median age, gender distribution, and MDS subtypes (according to WHO and IPSS) the study cohort was representative for the disease. Up to date, chromosomal aberrations were detectable in 126 pts. (62%) by FISH from pb. Most of them had 1 or 2 anomalies, but there were even pts with 4 or more aberrations. The most common aberrations- as known from other studies based on bm analyses- were del(5q) (64%), aberrations of chromosome 7 (del(7q)/-7) (25%), allelic loss of p53 (12%), +8 (11%), del(20q) (10%) and aberrations of chromosome 21 (del(21q)/-21/+21) (7%). Interestingly, a 12p-deletion was detectable as often as a trisomy 8 (11%), and in most cases as part of complex aberrations. A loss of p53 was significantly associated with aberrations of chromosome 7: 67% of pts with aberrations of chromosome 7 showed an allelic loss of p53 at the same time. In 4 cases, a coexistence of del(7q) and -7 in the same clone was observed. Within a median observation time of 7 months (2-21), a KE was detectable from pb by FISH in 17 pts (13%) out of 135 with at least 2 sequential analyses. In most cases del(7q)/-7 developed as a new anomaly, and was followed in frequency by aberrations of chromosome 21. Conclusions: FISH analyses of enriched circulating CD34+ cells are a feasible, less-invasive method to detect chromosomal aberrations in MDS pts and to follow the clone size from peripheral blood. By that method we are able to observe karyotype evolution as a step wise process in detail and to follow even rare abnormalities to learn more about their clinical and prognostic impact. Disclosures: No relevant conflicts of interest to declare.

Introduction: In myelodysplastic syndromes (MDS) the knowledge about chromosomal aberrations is important for diagnosis, pathogenesis, prognosis and treatment of cytogenetic subgroups of MDS patients. Usually, chromosomal anomalies in MDS patients are detected in bone marrow (bm) cells by chromosome banding analyses of metaphases. Alternatively or additionally they can be diagnosed by Fluorescence-in-Situ-Hybridization (FISH). We here present a novel method for cytogenetic monitoring of MDS patients from peripheral blood (pb) which is representative for the clone size in bm cells. Patients and methods: After initial diagnosis by chromosome banding analyses of bm cells, CD34+ cells from pb were enriched by immunomagnetic activated cell sorting (MACS®) and then analysed by FISH. Sixteen MDS patients (7 AML, 4 RAEB-2, 1 RAEB- 1, 3 RCMD, 1 RA) treated with 5-Azacytidine (5-Aza) and 3 patients (all 5q-syndromes) treated with Lenalidomide (Len) were included in this study. FISH analyses of circulating CD34+ cells were performed once a month in every patient during follow-up. Results: Our data show that, irrespective of FAB-/WHO subtypes or peripheral blast counts, the successful collection of enough CD34+ cells for FISH analyses from pb is possible: in average 80 000 to 400 000 CD34+ cells per sample (20ml pb) were enriched. Morphologic features of CD34+ cells by microscopic analyses were the same in all patients and did not depend on WHO subtypes or on the percentage of peripheral blasts: homogeneous, pure populations of cells with an immature appearance. Additionally, we compared the results of FISH analyses of non-enriched pb, enriched circulating CD34+ cells, non-enriched bm cells and enriched CD34+ bm cells with the results of classical cytogenetic analyses of bm using conventional banding techniques: Constantly, the smallest clone sizes were measured in non-enriched pb, followed by non-enriched bm cells, enriched CD34+ bm cells and enriched circulating CD34+ cells. In all cases the clone sizes of enriched circulating CD34+ cells measured by FISH were close to the results of classical cytogenetic analyses of bm metaphases representing the gold standard of cytogenetic examinations. In our patient cohort 10/16 patients treated with 5-Aza and 3/3 patients treated with Len developed a cytogenetic remission (cyR) detected by FISH analyses of circulating CD34+ cells. CyR was defined according to modified IWG criteria. Because there are no standardized response criteria for FISH analyses available, especially not for FISH from pb, as yet, we definded cyR as a reduction of more than 50% of circulating CD34+cells showing the respective aberration. Remarkably, in all cases with cyR under 5-Aza the hematologic improvement (HI) followed 1–3 cycles later. Under therapy with Len, cyR and HI were measured simultaneously. During follow up HI was still onlasting while there was a cytogenetic relapse detectable by a step-wise increase of aberrant circulating CD34+ cells. Conclusions: Analysing circulating CD34+ cells by FISH is a reliable method to survey the size of an aberrant clone in pb in high-risk as well as in low-risk MDS patients. It is representative for the clonal situation in the bm and allows a very frequent monitoring during therapy with obviously predictive value. It may reduce the need for repeated bm biopsies. In all cases where a bm biopsy is not possible or unsuccessful or metaphases for conventional banding analyses are not available, our method still allows the measurement of the clone size from pb.

In patients with myelodysplastic syndromes (MDS) iron overload caused by long-term red blood cell transfusions is an important factor for comorbidity especially in low-risk MDS. In this report we present the results of a comparative study based on colony formation assays of hematopoietic cells in MDS patients with and without iron overload. We demonstrate that iron overload suppresses the proliferation of erythroid progenitors cells (BFU-E), while the myeloid compartment (CFU-GM) was not found to be affected. Even patients with slightly elevated ferritin values show an impaired proliferation capacity in comparison to patients with normal ferritin levels. Furthermore, we show that this negative impact is reversible by sufficient iron chelation therapy. peerReviewed