Good Karyotype Research Articles

PB2089 THE GENETIC PROFILE OF THE PATIENTS DIAGNOSED WITH MYELODYSPLASTIC SYNDROMES IN ALBANIA (2009-2013)

Background: Myelodysplastic Syndromes (MDS) are a heterogeneous group of malignant myeloid neoplasms affecting especially the eldest population and are characterized by cytopenia, dysplasia and a high risk of progression to acute myeloid leukemia. The accurate diagnosis and risk stratification are the hallmarks of the therapeutic strategy. Knowledge of the different cytogenetic abnormalities gives us the opportunity to improve the stratification, prognosis and providing tools for more individualized treatment of the MDS patients. Aims: Our study aims to study the genetic profile of all the patients diagnosed with MDS in our Service of Hematology, UHC “ Mother Tereza” during the interval of time from 2009 to 2013. Methods We analyzed cytogenetically 120 patients diagnosed with MDS during the period 2009-2013. All the patients were hospitalized in our Service of Hematology during this period of time and have performed bone marrow aspiration (biopsy only in cases where the diagnosis was uncertain), FBC and flow cytometry. Cytogenetic analyses were performed from peripheral blood samples from the patients at the moment of the diagnosis and were analyzed in our Department of Genetics. The data were analyzed with SPSS statistics 20.0. Results: From 120 patients diagnosed with MDS, 60% has shown a normal karyotype, 35% of patients have an abnormal karyotype and in 5% no growth has been shown. 83 patients (69,1%) has a good karyotype, patients (9,10%)a intermediate karyotype, 20 patients (16,6%) a worse karyotype. We have found the anomaly of chromosome 7 in 42,8%(18) of patients, the anomalies of chromosome 5 in 21,4% (9)of patients, del(20q) in 33,3% (14), del(11q) in 9,5% (4), anomalies on chromosome 2 in 2,3%(1), trisomy 8 in 2,3%(1),Y- in 7,1% (3) and del(17p) in 14,2%(6) of patients. The frequency of abnormal karyotype was more elevated in the patients diagnosed with RCMD and AREB-2 respectively 11 patients and 10 patients and the anomalies were more frequent in the patients more than 60 years old (28 patients) versus the patients less than 60 years old (11 cases). Summary/Conclusion: Karyotype is one of the most important constituents of the International Prognostic Scoring System (IPSS) and of the revised-IPSS. Knowing the genetic profile of the patients diagnosed with MDS allows us to perform a very accurate diagnosis and to treat them with the best-individualized treatment.

191 - Lenalidomide Treatment in Lower Risk MDS – The Experience of One Czech Center (Positive Effect of Erythropoietin ± Prednisone Addition)

In 2008, the WHO combined the former categories RCMD (refractory cytopenia with multilineage dysplasia) and RCMD-RS (ring sideroblasts ≥15%). We studied the clinical impact and genetic background of RARS, RCMD, and RCMD-RS in 1082 patients. Good karyotypes (IPSS) were similarly frequent in RARS, RCMD, and RCMD-RS. 2-year overall survival (OS) rates were similar in RARS, RCMD, and RCMD-RS (85.9%/89.0%/91.7%; n.s.). The 2-year OS rate was better in good than intermediate or poor karyotypes (p < 0.001). These results support to combine RCMD and RCMD-RS as performed by WHO and emphasize the prognostic power of cytogenetic criteria for these MDS subtypes.

Immunosuppressive Therapy: Exploring an Underutilized Treatment Option for Myelodysplastic Syndrome.

Immunosuppressive therapy (IST) in low risk myelodysplastic syndrome (MDS) is known to achieve hematologic improvement but remains an underutilized treatment option. We report our experience using antithymocyte globulin (ATG) and cyclosporine A (CSA) to explore clinical predictive response factors. Patients treated with IST identified in the Moffitt Cancer Center MDS database were analyzed using baseline data, IST details, and response rates. Sixty-six patients treated with IST were identified. The median age was 61 years; the majority were at low risk and had a good karyotype. The median time to start IST was 1 year. All patients received ATG, 60% rabbit (r-ATG), 32% equine ATG (e-ATG), and 60% received CSA. Overall hematologic improvement was 42% with a trend favoring e-ATG over r-ATG (52% vs. 39%; P= .09). Erythroid improvement was evaluated in 30 patients, and 60% responded; neutrophil improvement was evaluated in 15, and 39% responded; platelet improvement was evaluated in 18, and 57% responded. Six of 18 pancytopenic patients experienced trilineage response. Mean time from ATG to next therapy was 12 months. None of the patients with very high risk or high risk revised International Prognostic Scoring System (IPSS-R) responded. Poor karyotype had a lower response rate, 25%, compared to 41% for intermediate and 44% for good karyotype. No difference in predicting response was found based on the National Institutes of Health Response Model; 38% with low and 45% with high probability responded. A trend favored treatment within 2 years from diagnosis, with 46% responding compared to 33% treated after 2 years. First-line ATG or after lenalidomide responded better than after azacitidine or third-line therapy. CSA provided an advantage: the disease of 51% responded compared to 27% with ATG alone (P= .05). Ten patients experienced transformation to acute myeloid leukemia, 7% with disease that responded to therapy and 24% with disease that did not respond to therapy (P= .08). Overall survival was 67.2 months without difference between those with and without response. Adverse events were reported in 55 patients. Infusion reactions occurred in 85% but was similar between ATG types. Infection rate was 25% and higher with e-ATG. Serum sickness was reported in 18% and significantly higher with r-ATG. IST has a hematologic improvement response rate in the range of other therapies approved for lower risk MDS. High risk IPSS-R, poor karyotype, and treatment after 2 years from diagnosis have unfavorable response trend. ATG with CSA has higher response than ATG alone. First-line ATG or after lenalidomide had better response trend compared to third-line therapy or azacitidine therapy.

Abstract 5304: A serum protein test for improved prognostic stratification of patients with myelodysplastic syndrome (MDS)

Abstract While existing scoring systems provide physicians with methods of assessing prognosis of patients with MDS and help with the choice of appropriate treatment regimens, some patients defined as lower risk still have poor outcomes and tests which can refine current stratifications are of clinical interest. Serum samples, clinical and survival data were available from 106 MDS patients with refractory anemia (RA) (n = 46), RA with excess blasts (n = 29), RA with ring sideroblasts (n = 14), and chronic myelomonocytic leukemia (n = 17). The international prognostic scoring system (IPSS) score distribution 0/0.5/1/&amp;gt;1 was n = 36/23/24/12. Matrix assisted laser desorption/ionization (MALDI) mass spectra were acquired from the samples using the deep MALDI method1, allowing a deep probing of the proteome. The spectra were preprocessed and spectral features defined. The integrated intensities of these features were combined with the survival data using deep learning based machine learning techniques to create classifiers able to stratify patients into groups with better and worse survival. Patients in the good prognosis group had median survival of 52 months, compared with 23 months in the poor prognosis group (hazard ratio = 0.37, p &amp;lt; 0.001). Within the subgroup of patients with refractory anemia (n = 46) it was possible to identify a group of patients with no death events before 30 months, while median survival in the other patients was only 25 months. Multivariate analysis stratified by MDS subtype found that the mass spectral (MS) classification was a significant independent predictor of survival even when adjusted for international prognostic scoring system (IPSS) score and cytogenetic status. MS classification was able to separate low to intermediate risk (IPSS 0-1) and good karyotype subgroups into groups with significantly better and worse survival. If validated in an independent dataset, this serum test would provide complementary information for physicians to consider in addition to existing scoring systems to aid in planning treatment for MDS patients. 1. M Duncan et al, Proceedings of the 61st ASMS Conference on Mass Spectrometry and Allied Topics. Minneapolis, MN June 8-9, 2013 MP 181. Citation Format: Joanna Roder, Judith Löffler-Ragg, Reinhard Stauder, Ulrich Germing, Wolfgang R. Sperr, Peter Valent, Heinrich Roder, Arni Steingrimsson, Heinz Zwierzina. A serum protein test for improved prognostic stratification of patients with myelodysplastic syndrome (MDS). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5304. doi:10.1158/1538-7445.AM2015-5304

Persistent molecular remission of refractory acute myeloid leukemia with inv(16)(p13.1q22) in an elderly patient induced by cytarabine ocfosfate hydrate

The prognosis of relapsed acute myeloid leukemia (AML) in elderly patients is dismal, even if the AML exhibits a good prognostic karyotype, such as inv(16)(p13.1q22). We present a 72-year-old female with AML with inv(16)(p13.1q22) who suffered five episodes of relapse with temporary complete remission. Maintenance chemotherapy with oral cytarabine ocfosfate hydrate eventually produced persistent molecular complete remission of her AML that had not been induced by conventional regimens including intensive chemotherapy and low dose cytarabine therapy. The high level of tolerability to oral cytarabine ocfosfate hydrate may offer elderly patients with this type of AML a good chance for a cure.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-014-0100-6) contains supplementary material, which is available to authorized users.

Analysis of ND4 gene mutations in acute myelogenous leukemia

To investigate the relationship of the mutational status of the ND4 gene and the clinical features of acute myelogenous leukemia (AML) patients with ND4 mutations. Using PCR combined with directly sequencing, we identified somatic mutations of ND4 in 121 primary AML patients to couple with their clinical features. There were 58 male patients and 63 female patients (median age 49 years, 10-86 years). Eight of 121 patients (6.6%) with de novo AML were found harboring missense mutation of ND4 gene, including 3 patients with A131V (3/8, 37.5%), 2 patients with A404T (2/8, 25%), 1 patient with F149L (1/8, 12.5%), 1 patient with G242D (1/8, 12.5%) and 1 patient with Y409H (1/8, 12.5%), respectively. Patients with ND4 mutations were associated with good karyotype (P=0.049), regardless of gender, age, white blood cell, hemoglobin, platelet, blast cells of bone marrow or immunophenotype (P>0.05). There were no statistical significance in mutations of FLT3-ITD, NPM1, CEBPA, c-KIT and DNMT3A between patients with ND4 mutation and wild-type (wt) ND4 (P>0.05). The median overall survival of patients with ND4 mutations and wt ND4 were all not reached. The median relapse-free survival were not reached and 29(2-53) months, respectively (P>0.05). There was no significance in the ratio of CR and RR patients between wt ND4 and ND4 mutated groups (P>0.05). It was concluded that novel ND4 mutations could be found in de novo AML patients, especially in patients with good karyotype. Thus, ND4 mutations might play an important role in AML prognosis. However, whether the mitochondria dysfunction contribute to leukemogenesis needs to be further investigated.

Cytogenetic Response Based On Revised IPSS Cytogenetic Risk Stratification and Minimal Residual Disease Monitoringby FISH In MDS Patients Treated By Low-Dose Decitabine

ObjectiveTo observe and analyze the response characteristics based on revised IPSS (IPSS-R) cytogenetic risk stratification in patients with myelodysplastic syndromes (MDS) who received low-dose decitabine treatment. Patients and methodsEighty-seven MDS patients received decitabine treatment (15-20mg/M2/d×5/per course) with a median of 4 courses (range, 1-10). The clinical and cytogenetic response after treatment were observed to evaluate whether the IPSS-R cytogenetic risk stratification could predict treatment response. For those patients with abnormal karyotypes who achieved clinical complete response (CR), percentage of clonal cells in bone marrow (BM) was calculated by G-banding together with fluorescence in situ hybridization (FISH) to determine the minimal residual disease (MRD). In addition, the effect of cytogenetic response on overall survival (OS) was also analyzed. ResultsSixty of 87 patients (69.0%) achieved treatment response including 25 cases (28.7%) with CR. For the 44 patients with abnormal chromosome, 27 cases (61.3%) achieved cytogenetic response including 18 with complete cytogenetic response (cCR). The patients carrying poor or very poor karyotypes presented better clinical and cytogenetic response than those with intermediate or good karyotypes. Among the patients with poor or very poor karyotypes, the ones carrying chromosome 7 aberrance showed better response than the others. Among 18 patients with abnormal karyotypes who achieved clinical CR, 4 cases presented cytogenetic PR or NR through G-banding analysis. FISH analysis revealed an over 5 % of clonal cells in BM in two patients who presented G-banding defined cytogenetic CR. In addition, longer median OS (24 months) were observed for patients who achieved cytogenetic response than those who did not (12 months) (P=0.007) ConclusionIPSS-R cytogenetic risk stratification could be used to predict the clinical and cytogenetic response to decitabine treatment in MDS patients. The predicting effect may be related to the chromosome 7 involvement rather than the number of abnormal karyotypes. FISH together with G-banding analysis should be available in determining MRD in MDS patients after treatment. Disclosures:No relevant conflicts of interest to declare.

Cytogenetic response based on revised IPSS cytogenetic risk stratification and minimal residual disease monitoring by FISH in MDS patients treated with low-dose decitabine

The features of cytogenetic response have been not well described in myelodysplastic syndrome (MDS) patients receiving low-dose decitabine treatment. In this study, we observed and analyzed the response characteristics based on the revised IPSS (IPSS-R) cytogenetic risk stratification in eighty-seven MDS patients who received low-dose decitabine treatment (15-20 mg/M(2)/d×5/per course). Twenty-seven of 44 patients (61.3%) with abnormal karyotypes achieved a cytogenetic response, including 18 cases with complete cytogenetic response (cCR). The patients carrying poor or very poor karyotypes achieved better clinical and cytogenetic response than those with intermediate or good karyotypes. Among the patients with poor or very poor karyotypes, those carrying chromosome 7 aberrance showed a better treatment response than the other patients. Four patients (22.2%) of the patients who achieved clinical CR presented with a cytogenetic PR (partial response) or NR (no response). Over 5% of the clonal cells determined by FISH analysis were in the two patients who presented cytogenetic CR. Longer median OS (24 months) were observed in the patients who achieved a cytogenetic response than in those who did not (12 months) (P=0.007). The patients with poor or very poor karyotypes could achieve survival comparable to that of the patients with good or very good karyotypes after decitabine treatment (18 vs. 20 months, P=0.365). IPSS-R cytogenetic risk stratification could be used to predict the clinical and cytogenetic response to decitabine treatment in MDS patients, and the predicting effect may be related to chromosome 7 involvement. Analysis with FISH and G-banding should be available in determining the minimal residual disease in MDS patients after treatment.

Evaluation of revised IPSS cytogenetic risk stratification and prognostic impact of monosomal karyotype in 783 patients with primary myelodysplastic syndromes

Cytogenetic classification by the revised international prognostic scoring system (IPSS-R) and the prognostic value of monosomal karyotype (MK) were assessed in 783 patients with primary myelodysplastic syndromes (MDS). At 22 months median follow-up, 562 (72%) deaths were recorded. Percentages of patients with IPSS-R "very good," "good," "intermediate," "poor," and "very poor" cytogenetic categories was 5, 63, 18, 4, and 10%, respectively. The corresponding median survivals were 21, 40, 24, 18, and 6.5 months and the inter-group differences (good vs. very good/intermediate/poor vs. very poor; P < 0.01) or similarities (very good vs. intermediate vs. poor; P = 0.79) were not significantly modified in multivariable analysis. Results were similar when analysis was restricted to 602 patients managed by supportive care. MK adversely affected survival in both poor and very poor karyotype groups (P < 0.01). In conclusion, we were unable to confirm the prognostic superiority of IPSS-R-very good karyotype or prognostically distinguish between very good, intermediate and poor karyotypes. Furthermore, we show additional prognostic information from MK in poor/very poor karyotype.

P-135 Low RPS14 expression negatively conditions the prognosis of patients affected by not 5q- myelodysplastic syndromes during azacitidine treatment

We examined the efficacy of cyclosporin A (CsA) in 50 patients with myelodysplastic syndrome (MDS) consisting from 47 of RA, 1 of RARS, and 2 of RAEB. These patients showed various marrow cell types including hypo-, normo-, and hypercellularity. Patients belonged to the following International Prognostic Scoring System (IPSS) risk groups: 4 of low, 41 of intermediate-1, and 5 of intermediate-2. The median CsA dose was 4.58 mg/kg, and treatment responses were classified according to the International Working Group (IWG) criteria. Hematological improvement (HI) was observed in 30 (60%) patients, and all of them were belonged to RA. In the patients with RARS or RAEB, no efficacy was observed. Four (8%) of the responders achieved partial remission (PR) with granulocytes ≥1500 μl−1, Hb >11 g/dl and platelets ≥100,000 μl−1. Higher response rate (53%) was shown in erythroid lineage (HI-E) compared to platelet (HI-P, 36%) or neutrophil lineage (HI-N, 35%). When we analyzed the correlation between the response to CsA therapy and the karyotype or HLA type, there were significantly more responders with good karyotype or DRB1∗1501 than with intermediate/poor karyotypes or with other HLA types. These results indicate the usefulness of CsA therapy for MDS patients with any marrow cellularity, especially for erythroid lineage and patients with good karyotype or DRB1∗1501.

Evaluation of IPSS-Revised (IPSS-R) Cytogenetic Risk Stratification and Prognostic Impact of Monosomal Karyotype in 1,014 Patients with Myelodysplastic Syndromes (MDS)

AbstractAbstract 423 Background:Cytogenetic abnormalities at diagnosis are seen in 20–70% of patients with myelodysplastic syndromes (MDS). The International Prognostic Scoring System -revised (IPSS-R) classifies these cytogenetic abnormalities into five prognostic groups: very good (del(11q), ΠY with overall survival (OS) of 61 months); good (normal, del(20q), del(5q), del(12p) with OS 49 months); intermediate (+8,7q-, i(17q),+19,+21 with OS 26 months); poor (der(3)q21/q26, −7, complex with 3 abnormalities with OS 16 months); and very poor (complex with >3 abnormalities with OS of 6 months) (Schanz J, JCO 2012, Greenberg P, Blood 2012). Monosomal karyotype (MK) is defined as the presence of two or more autosomal monosomies or a single monosomy associated with a structural abnormality. We have previously shown that MK identifies a prognostically worse subgroup of MDS patients with a complex karyotype (Patnaik M, Leukemia 2010). Objectives:i.To validate the IPSS-R cytogenetic risk groups in an independent cohort of patients with MDS.ii.Evaluate the prognostic impact of MK to refine the current IPSS-R cytogenetic risk classification. Methods:The Mayo Clinic database for MDS was used to identify patients in whom bone marrow histologic and cytogenetic information was obtained at the time of diagnosis. WHO criteria were used for MDS diagnosis and leukemic transformation. Results:i) Patient characteristics: A total of 1,014 patients met the above-stipulated criteria. Median age of the cohort was 71 years (range: 18–98 years) with 684 (67%) males. 819 patients (81%) had primary MDS with 195 cases (19%) of therapy-related MDS. 759 patients (75%) are dead at a median follow-up of 18 months (range: 0–223 months) with leukemic transformation documented in 125 cases (12%). IPSS-R risk distribution was as follows: very low (n=149, 15%), good (n=261, 26%), intermediate (n=221, 22%), high (n=131, 13%), and very high (n=252, 25%).ii) IPSS-R karyotype (Entire cohort; n=1,014): The five cytogenetic groups by IPSS-R were as follows: very good (n=43, 4%), good (n=577, 57%), intermediate (n=187, 18%), poor (n=83, 8%), and very poor (n=124, 12%). We evaluated the prognostic impact of the above groups by Kaplan Meier and Cox proportional hazards analysis. OS for the five cytogenetic groups was as follows: good (36 months), very good (28 months), intermediate (20 months), poor (13 months), and very poor (6 months) (Figure 1). OS of patients with very good karyotype was significantly better than patients with very poor and poor karyotype (p<0.0001, RR 3.2 and p=0.04, RR 1.6 respectively) but similar to patients with intermediate and good karyotype (p=0.22 and 0.28 respectively).iii) IPSS-R karyotype (Primary MDS; n=819): Cytogenetic risk groups by IPSS-R were as follows: very good (n=38, 5%), good (n=512, 63%), intermediate (n=153, 19%), poor (n=46, 6%), and very poor (n=70, 9%). OS of the five groups was as follows: good (39 months), very good (23 months), intermediate (21.5 months), poor (14.5 months), and very poor (6.5 months). OS of patients with very good karyotype was significantly better than patients with very poor karyotype (p=0.0002, RR 2.5) but similar to patients with poor, intermediate and good karyotype (p=0.23, 0.26 and 0.18 respectively).iv) Monosomal karyotype: MK was identified in 153 (15%) cases of which 4 cases were intermediate, 42 poor, and 107 very poor by IPSS-R. OS of patients with MK was similar to those with very poor karyotype (6 months). In addition, OS of patients with very poor without MK was akin to patients with poor without MK (p=0.21). Furthermore, among primary MDS we identified 83 (8%) cases of MK of which 4 were intermediate, 21 were poor, and 58 very poor by IPSS-R. We confirmed that OS of patients with MK was similar to patients with very poor karyotype (6.5 months). Moreover, survival of patients with very poor without MK was similar to patients with poor without MK (p=0.51). Conclusions:In this independent analysis of 1,014 patients with MDS, IPSS-R cytogenetic risk characterization failed to discriminate between very good, good and intermediate prognostic groups. In addition, patients with very poor without MK have a similar prognosis to patients with poor without MK and should be included in the poor category whereas the presence of MK should warrant inclusion into the very poor risk category. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Significant Improvement in Overall Survival Among Patients Diagnosed with Low-Risk Myelodysplastic Syndrome (MDS) Treated with Selective-Serotonin-Reuptake-Inhibitors (SSRIs)

AbstractAbstract 3818 Background:Clinical outcomes in patients diagnosed with low risk MDS are heterogeneous and molecular characterization can distinguish subgroups with a more aggressive disease progression. Those patients determined to have a higher risk for transformation would seek therapy. Given a median age of 65, many of these elderly patients are not suitable for intensive interventions and options such as SSRI’s are reasonable. Gene expression profiling in low-risk MDS patients suggests deregulation of apoptosis and cytokine signaling pathways. SSRI treatment promotes normalization of deranged proinflammatory cytokines in patients diagnosed with depression such as TNF α and IL12. (Sutcigil et al. Clin and Dev Immunol 2007) In animal model of arthritis, sertraline abrogated manifestation of autoimmune arthritis by reducing TNF α (Baharav et al. Neuroimmunomodulation.2012) In addition, SSRI’s have been shown to induce anti-proliferative effect on colon and prostate cell lines.(Huang et al. Basic Clin Phar Tox.2011; Gil et al. Int J Onc. 2008) Here, we report our single institution retrospective epidemiological analysis of low grade MDS patients treated with SSRIs agents. Methods:After Institutional Review Boards approval, all patients over 18-years-old with an initial morphological diagnosis of low grade MDS per bone marrow examination from January 1, 2000 to December 31, 2010 were identified from the Michael E. DeBakey VA Medical Center cancer registry. 118 patients were included for analysis. Patients were considered to be in the selective serotonin reuptake inhibitor (SSRI positive) group if they had received at least 6 months of an SSRI (including fluoxetine, sertraline, paroxetine, or citalopram) during the 12 month period prior and after the diagnosis of MDS. Patients who did not meet the above criteria were assigned to the SSRI negative- group. We analyzed Overall Survival (OS) for patients receiving or not receiving SSRIs agents. Results:The median OS for patients with lower-risk disease was 1750 and 728 days in the SSRI+ and SSRI- group, respectively (P=0.0104, HR=0.493, 95% CI=0.287–0.847) (Fig. 1). To avoid any possible confounding effect of concurrent treatment, the same analysis was repeated after excluding patients who had received any disease-modifying agents (including azanucleosides, lenalidomide, thalidomide, anti-thymocyte globulin, or induction chemotherapy). Among patients with best supportive care (BSC) only (9 SSRI+ vs. 53 SSRI-), the improvement seen in the median OS remained significant at 1919 and 506 days for the SSRI+ and SSRI- group, respectively (P=0.0359, HR=0.477, 95% CI=0.239–0.952)(Fig. 2). Of interest, within the 9 SSRI+ patients, they were all males with a median age of 72 (range 55–82), and a predominance of RCMD category 6/9 (67%) and good karyotype 8/9 (89%). In order to further examine the impact of SSRI agents on clinical outcomes, we examined the OS per individual agent. There was a non-significant trend of improved OS for patients receiving sertraline (P=0.0519) (Fig.3). Conclusions:In this retrospective cohort analysis, we detected a significant improvement in OS for lower-risk MDS patients treated with SSRIs (such as sertraline), regardless concurrent administration of other MDS directed therapy. This data suggests a biological benefit from SSRIs in this entity and supports the possible combination of these agents with MDS targeted therapy. Our findings suggest a selective survival benefit from treatment with sertraline. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Adverse Effect of Very Poor Cytogenetics and Monosomal Karyotype On Outcomes Following T-Deplete Reduced Intensity Conditioned Stem Cell Transplant for MDS and AML.

AbstractAbstract 3141Cytogenetics are an important predictor of outcomes for patients diagnosed with MDS and AML. The new 5-group cytogenetic classification for MDS and identification of adverse prognosis associated with monosomal karyotype (MK) has enhanced risk stratification. Recent data have validated these factors to predict outcomes post-haematopoietic stem cell transplant (HSCT) in a multicentre study of predominantly T-replete transplantation. We sought to evaluate the 5-group cytogenetic classification and prognostic significance of MK at diagnosis and pre-HSCT in patients with MDS and AML secondary to MDS (MDS-AML) following T-cell deplete reduced intensity conditioned HSCT.We reviewed karyotypic profiles in 267 patients transplanted between 1999 and December 2011. Conditioning regimens consisted of FBC (fludarabine 150mg/m2 iv, busulphan 8mg/kg oral or 6.4 mg/kg iv, alemtuzumab 100mg iv) or FBATG (fludarabine 150mg/m2 iv, busulphan 6.4 mg/kg iv, ATG 6mg/kg iv) in 190 (71%) and 18 (7%) of patients respectively. A double dose of busulphan was used in 50 (19%) FB2C and 9 (3%) FB2ATG cases. Cyclosporine was used for GVHD prophylaxis.Diagnoses included RA/RCMD (n = 66, 25%), RAEB (n=65, 24%), MPN/MDS (n=22, 8%) and MDS-AML (n= 114, 43%). Using the IPSS, 176 (66%), 50 (19%) and 41 (15%) patients had good, intermediate and poor prognosis karyotypes respectively. Using the 5-group cytogenetic classification, patients were stratified: very good (n=3, 1%), good (n= 178, 67%), intermediate (n=46, 17%), poor (n=25, 9%) and very poor (n=15, 6%). MK was observed in 20 (8%) of patients.Seventeen (6%) patients had >5% blasts and 42 (16%) had cytogenetic abnormalities detectable pre-HSCT. Those without karyotypic abnormality pre-HSCT had normal cytogenetics (n=185, 69%), no analysable metaphases (n=20, 8%) or unavailable results (n=20, 8%). Cytogenetic abnormalities pre-HSCT in the 42 patients revealed fewer very good (n=3, 7%) and good karyotypes (n=3, 7%) and a higher preponderance of intermediate (n=22, 52%), poor (n=8, 19%) or very poor (n=6, 14%) karyotypes. MK was present in 7 (17%) cases. Their conditioning regimens comprised FBC (n=23, 55%) or FBATG (n=2, 5%); 17 received double-dose busulphan regimens: FB2C (n=15, 36%) or FB2ATG (n=2, 5%).Overall, 5 year OS, EFS (death from any cause, relapse and graft failure) and cumulative incidence of relapse (CIR) were 45%, 34% and 48% respectively. Graft failure occurred in 17 (7%) patients. Non-relapse mortality rates were 18% and 27% at 1y and 5y respectively.Grouping by traditional IPSS cytogenetics at diagnosis did not significantly influence survival or relapse. Classification of cytogenetics at diagnosis into 5 groups demonstrated patients with very poor karyotype to have significantly inferior OS, EFS and CIR compared with the other 4 groups. None of the 15 cases with very poor karyotype are alive at 5y; 5y OS was 48%, 49% and 43% for very good/good, intermediate or poor risk groups respectively (p<0.01). Patients with MK had 5y OS of 13% vs 48% for those without MK (p<0.01). CIR was 100% and 68% for the very poor karyotype and MK patients respectively.Persistent cytogenetic disease pre-HSCT was associated with significantly inferior 5y OS of 33%, vs 49% without (p=0.01); 5y EFS and CIR were 23% vs 37% (p=0.013) and 72% vs 43% (p<0.01) respectively. Patients with >5% BM blasts pre-HSCT had worse 5y OS at 32% vs 46% for those with <5% blasts (p=0.027) and higher 5y CIR (68% vs 47%, p<0.01).MK, BM blasts >5% at HSCT and residual cytogenetic abnormalities pre-HSCT all retained significance on multivariate analysis with respect to OS (HR 1.97 [95% confidence interval (CI) 1.13–3.45], 1.92 [95% CI 1.07–3.43] and 1.33 [95% CI 1.02–1.73] respectively); very poor karyotype did not retain significance (with HR 1.08 [95% CI 0.82–1.41]).These data demonstrate the adverse impact of very poor karyotype and MK at diagnosis on outcomes post-T-deplete HSCT for MDS and MDS-AML, emphasizing the effect of cytogenetics regardless of therapeutic intervention. In addition residual cytogenetic disease pre-transplant predicts inferior results despite higher intensity conditioning used in these patients. Future directions to improve outcomes for these poor prognostic groups should include prospective trials to determine the value of complete eradication of residual cytogenetic disease pre-HSCT and identification of novel strategies directed at preventing relapse. Disclosures:Marsh:Alexion Pharmaceuticals, Inc.: Honoraria. Mufti:Celgene: Consultancy, Research Funding.

Prognoses of MDS subtypes RARS, RCMD and RCMD-RS are comparable but cytogenetics separates a subgroup with inferior clinical course

In 2008, the WHO combined the former categories RCMD (refractory cytopenia with multilineage dysplasia) and RCMD-RS (ring sideroblasts ≥15%). We studied the clinical impact and genetic background of RARS, RCMD, and RCMD-RS in 1082 patients. Good karyotypes (IPSS) were similarly frequent in RARS, RCMD, and RCMD-RS. 2-year overall survival (OS) rates were similar in RARS, RCMD, and RCMD-RS (85.9%/89.0%/91.7%; n.s.). The 2-year OS rate was better in good than intermediate or poor karyotypes (p<0.001). These results support to combine RCMD and RCMD-RS as performed by WHO and emphasize the prognostic power of cytogenetic criteria for these MDS subtypes.

Tedavi Almamış 221 Miyelodisplastik Sendromlu Hastada Sitogenetik Değerlendirme

Objective: Myelodysplastic syndromes (MDS) are a heterogeneous group of malignant clonal hematopoietic stem cell disorders characterized by bone marrow failure, ineffective hematopoiesis, peripheral blood cytopenias, atypic cytological profile, increased apoptosis, and increased likelihood of evolution to acute myeloid leukemia (AML). Cytogenetic findings are major determinants in the diagnosis, classification, pathogenesis, prognosis, and treatment in patients with MDS. Cytogenetic analysis is a mandatory step in the full evaluation of a newly diagnosed patient. The aim of the present study was to retrospectively evaluate the cytogenetic findings of 221 MDS patients in İstanbul University Cerrahpaşa Medical Faculty, Medical Biology and Genetics Department. Material and Methods: Cytogenetic analyses of 221 patients (89 female, 132 male) were performed on bone marrow cells using a trypsin-Giemsa banding technique. Metaphase cells were obtained from short-term unstimulated cultures. When possible, at least 20 metaphases were analyzed and 10 of them were fully karyotyped. Results: Among the 221 patients, 122 had no karyotype anomalies (55.20%) and 99 (44.80%) had clonal cytogenetic abnormalities; with 46 (20.81%) having one, 19 (8.59%) having two and 34 (15.38%) having complex (?3) abnormalities. According to the International Prognostic Scoring System (IPSS) cytogenetic categories, 130 (58.82%) patients presented with a good karyotype, 54 (24.44%) patients with intermediate karyotype and 37 (16.74%) patients with poor karyotype. Conclusion: Although some cases appear to have a normal karyotype, the technical failures such as inability to obtain sufficient analyzable metaphases may reduce the actual proportion of abnormal cases. The examination of 20 or more metaphases could further increase the sensitivity of cytogenetic analyses with clinical impact in individual cases by identifying additional abnormal clones or subclones.

Prognosis of MDS Subtypes RARS, RCMD and RCMD-RS Does Not Differ by Cytomorphologic Criteria but Cytogenetics Allows to Delineate a Subgroup with Inferior Clinical Course,

Abstract 3796 Background:In 2008 the WHO classification combined the former categories RCMD (refractory cytopenia with multilineage dysplasia) and RCMD-RS (with ring sideroblasts ≥15%) thus not separating according to ring sideroblasts anymore in MDS with multilineage dysplasia, while the category refractory anemia with ring sideroblasts (RARS) was maintained separately. One aim of this study was to evaluate whether or not a separation with respect to ring sideroblasts is reasonable. Study Design: To investigate the clinical impact and genetic background of these MDS subtypes, we studied outcomes, cytogenetics, and molecular genetics in 1082 de novo MDS pts (153 RARS, 606 RCMD, 323 RCMD with ring sideroblasts ≥15% termed “RCMD-RS“): 703 m/379 f; median age, 73.1 yrs; 21.0–90.4 yrs. Cytogenetic risk groups were defined according to the International Prognostic Scoring System (IPSS; Greenberg et al., 1997). Results: Sex ratio (male preponderance in all subtypes; male/female ratio 1.9 in the whole cohort) did not differ significantly between the 3 MDS subgroups. Mean age (RARS: 71.8; RCMD: 70.1; RCMD-RS: 72.6 yrs) reached significant difference between RARS vs RCMD (p=0.020) and between RCMD vs RCMD-RS (p=0.004). Mean WBC count differed between all subgroups (RARS: 6.1; RCMD: 4.4; RCMD-RS: 5.3×10(9)/l; RARS vs RCMD p<0.001; RARS vs RCMD-RS p=0.039; RCMD vs RCMD-RS p<0.001) whereas mean platelet count (RARS: 232; RCMD: 144; RCMD-RS: 218 × 10(9)/l) and Hb level (RARS: 96; RCMD: 105; RCMD-RS: 98 g/l) differed between RARS vs RCMD and RCMD vs RCMD-RS (all p-values <0.001). IPSS categories were available in 854/1082 pts as follows: 514 pts (60.2%) low-risk, 312 (36.5%) intermediate-1, and only 28 (3.3%) intermediate-2 risk (no pt was assigned to high risk IPSS). Cytogenetics was available in all 1082 pts: The majority (918/1082; 84.4%) had good karyotypes (KTs) according to IPSS mainly due to high rates of normal KTs (821/1082, 75.9%); 112 pts had intermediate (10.4%); and only 52 (4.8%) had poor KTs. In detail, good KTs were equally distributed in RARS: 123/153; 80.4%; in RCMD: 530/606; 87.5%; and in RCMD-RS: 265/323; 82.0%. 3-yr overall survival (OS) rates did not differ significantly between the three MDS subtypes (RARS: 78.1%; RCMD: 86.7%; RCMD-RS: 80.6%). Also when entities with ≥15% ring sideroblasts (RARS + RCMD-RS) were compared to RCMD (<15% ring sideroblasts), 3-yr OS rate was similar (80.0% vs 86.7%; n.s.). Further, multilineage dysplasia per se did not impact on 3-yr OS rate (RCMD + RCMD-RS: 83.8% vs 78.1% in RARS; n.s.). In contrast, 3-year OS rate was better in good KTs compared to intermediate or poor KTs (91.4% vs 60.0% vs 29.3%; p<0.001) in the total cohort. Also in the different MDS subtypes, good karyotypes showed better 3-year OS rate than intermediate/poor karyotypes (p-values for comparison of cytogenetic risk groups: RARS: p<0.001; RCMD: p=0.032; RCMD-RS: p=0.007). In subcohorts genes were analysed and were found to be mutated only with low frequencies: RUNX1 mut: 14/213 (6.6%), NRAS mut: 1/283 (0.4%), MLL -PTD: 4/294 (1.4%), FLT3 -ITD: 0/285. This underlines the low risk profile of the cohort. In univariable analysis, good + intermediate vs poor KTs (p<0.001), aberrant vs normal KT (p<0.001), age (p=0.080), and Hb level (both continuous; p=0.007) had a significant impact on OS, while WBC count, platelets, and percentage of ring sideroblasts (all as continuous variables) were not significant. In multivariable analysis, IPSS cytogenetic risk groups (p=0.005) and Hb (p=0.008) only remained significant. Conclusions: As investigated here in 1082 pts, RARS, RCMD, and RCMD-RS all show high rates of good karyotypes as defined by IPSS and show similar clinical outcomes which clearly supports to skip the RCMD-RS category as done by the WHO in 2008. The karyotype and Hb level were the only independent prognostically relevant parameters in multivariable analysis. However, >80% of patients show a good risk cytogenetic profile making prognostication according to karyotype relevant only in a small subset of patients. To overcome this shortcoming only an increasing panel of new molecular markers in MDS can pave future investigations which presently becomes available by the advanced sequencing techniques. After diagnosed by morphology and cytogenetics especially molecular genetic information may therefore guide treatment in the near future for this low risk subgroup of MDS patients as investigated here. Disclosures:Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The Prognostic Role of Jagged-1 Protein Expression In Acute Myeloid Leukemia Patients

AbstractAbstract 2726 Objectives:Jagged-1 is a member of the Delta/Serrate/Lag-2 (DSL) family of proteins that are ligands for Notch receptors. Aberrant Jagged-1/Notch-1 signaling is posited to promote the development of AML by inducing excessive self-renewal with a concomitant block in cell differentiation. Moreover, Notch-1 signaling has been identified as a critical factor involved in the maintenance of a pool of self-renewing hematopoietic stem cells (HSC) as well as AML stem cells. So far there were no reports on the clinical role of Notch-1 and Jagged-1 expression in AML.In this study we evaluated the expression of Jagged-1 and Notch-1 proteins in AML blasts and CD34+ peripheral blood stem cells (PBSC) collected during mobilization procedures before autologous stem cell transplantation. In addition, in AML patients we correlated the expression of both proteins with known prognostic factors and response to treatment. Methods:The expression of Notch-1 and Jagged-1 proteins was examined in leukemic blasts isolated from bone marrow or peripheral blood of 53 de novo AML patients with median age 57 years (range 21–82). CD34+ collected from 13 lymphoma patients (11 multiple myeloma, 1 Hodgkin lymphoma, 1 non-Hodgkin lymphoma) with median age 57 (range 21–69 years) served as a control. All measurements were carried out using multi-colour flow cytometry. In parallel, the isotype controls were performed for all measurements. Protein expression was assessed as a percentage of Notch-1 and Jagged-1 positive cells. The cut-off 20% was used to subdivide patients into “low-expressers” and “high-expressers” group. Results:We found that the median expression of Jagged-1 was significantly higher in AML blasts (18,2%; range 0,9–62,4%) as compared to CD34+ PBSC (3,0%; range 0,9–21%); p<0.0001. In contrast, the expression of Notch-1 in AML patients (median 1,4%; range 0,1–24,8%) was lower than in the control CD34+ cells (median 3,85%; range 0,7–16%); p<0.004. There was no correlation between Jagged-1 and Notch-1 protein expression in both AML blasts and PBSC.Jagged-1 expression was significantly higher in AML patients with WBC ≤20G/L (median 21,2%) compared to group with WBC >20 G/L (median 9,85%); p<0.004. Consequently, we found the significant negative correlation between Jagged-1 expression and WBC count (p<0.02). Patients with good-risk karyotype according to SWOG classification showed significantly higher expression of Jagged-1 protein as compared to intermediate and poor risk group (medians 21,8% vs. 11,5% respectively; p< 0.02).Thirty two out of 53 AML patients received standard induction chemotherapy with daunorubicine and cytarabine (“3+7”), 21/53 received non-intensive therapy. Nineteen (61%) of intensively treated patients achieved complete remission (CR). We observed that the CR rate in “high-expressers” of Jagged-1 was significantly higher than in the “low-expressers” group (80% vs. 43% respectively; p=0.04). Additionally, a good karyotype and a high expression of Jagged-1 protein were the only factors associated with higher probability of CR (p=0.05, p<0.01, respectively) in univariate analysis.There was no statistical association between the Notch-1 expression and response to treatment, karyotype or tumour size associated risk factors as: WBC, percentage of leukemic blasts in bone marrow and LDH. Moreover, no correlations between Notch-1 and CD34 expression as well as Notch-1 and differentiation markers (CD13, CD14, CD15, CD33) expressions in AML blasts were found. Conclusions:Jagged-1 protein is highly expressed in AML blasts and correlates with better response to standard chemotherapy, favorable karyotype and lower WBC in AML patients. These data clearly demonstrate an important role of Jagged-1 in AML biology. A better understanding of autonomous Jadded-1 signaling in AML may create new options for therapeutic interventions in AML. Disclosures:Robak:Johnson & Johnson: Research Funding.

Invalidation of Stobnica chromosome race of the common shrewSorex araneus

In Central Poland, two similar chromosome races of the common shrewSorex araneus Linnaeus, 1758 were earlier described: Drnholec race (arm combinationsgm, hi, ko, nr) and Stobnica race (gm, hi, ko, np). Great similarity in size and G-banding patterns between thenr andnp metacentrics leave open to doubt the actual existence of both races in Poland. The present study, which is based on good quality karyotypes of common shrews from 18 sites, showed the presence of thenr arm combination. There is therefore strong evidence that thenp arm combination was wrongly described and thus the Stobnica race should not be considered valid.

Modification of WPSS for the Time of Diagnosis Retains Its Prognostic Impact and Confirms Transfusion Dependency as An Important Parameter Affecting Survival in Early MDS Patients with Isolated Erythroid Dysplasia.

We attempted to overcome a limitation of WPSS at the time of diagnosis by replacement of transfusion dependency with initial Hb level. Hb < 80 g/l was scored 1, Hb ≥ 80 g/l was scored 0. This adjustment was tested in a retrospective analysis of 107 non-transplanted patients with early MDS without of excess of blasts diagnosed between 1980 and 2004. Kaplan Meier estimates for 5 years survival were 58%, 57%, 36% and 12% for patients with very low, low, intermediate and high risk scored at the time of diagnosis according to modified WPSS. A significant difference in overall survival was present between patients with score < 2 and ≥ 2 and in leukemia free survival between patients with score < 1 and ≥1 (P=0.02). An analysis of individual WPSS parameters revealed a significant difference in median survival between patients receiving ≤ 2 units and those receiving > 2 units of RBC transfusions/month (46.1 vs. 26.9 months, respectively, P=0.03), between patients with RA+RARS+5q-syndrome and those with RCMD+RCMD-RS (45.4 vs. 27.8 months, respectively, P=0.02) and between patients with good karyotype and intermediate or poor karyotype according to IPSS (48.0 vs. 22.0 months, respectively, P=0.02). An impact of transfusion dependency on survival was validated by univariate and multivariate regression analysis of different clinical and laboratory variables. Leukemic progression was the most important parameter affecting survival in both univariate (χ2=47.7, P=0,001) and multivariate (P<0.0001) analysis and was the only independent variable affecting survival in patients with progressive disease. In patients without MDS progression, administration of > 2 units of RBC transfusions/month was the only independent variable with adverse impact on survival in patients with unilineage erythroid dysplasia (P=0.02). In patients with multilineage dysplasia only heavy transfusion dependence (> 3 TU RBC/month) in combination with serum ferritin > 2000 μg/l adversely affected survival (P=0.03). In conclusion, modification of WPSS by replacement of transfusion dependency with initial Hb level < 80 g/l retained its prognostic relevance and allowed to identify even at the time of diagnosis a potentially risk subset of early MDS patients with intermediate and high score and limited survival (< 40% at 5 years). Our results also confirmed a significant negative impact of transfusion dependency on survival in at least a subset of patients with early MDS. This subgroup is characterized by unilineage dysplasia limited to erythropoiesis in combination with dependency on > 2 TU of RBC per month. These patients have usually a prolonged survival enabling development of heavy transfusion iron overload and thus represent the main target group for intensive chelation therapy. The study was supported by scientific grant NR 9235-3 from the Czech Ministry of Health.

Metaphase Spreads and Spectral Karyotyping of Human Embryonic Stem Cells

INTRODUCTIONSpectral karyotyping is a powerful technique for enumeration of chromosomes in plant, animal, and human cells with high precision. It is also used as a tool for detecting and diagnosing abnormalities in individual chromosomes, such as deletions, duplications, and translocations. There are three basic steps to obtaining good spectral karyotypes: (1) making metaphase spreads, (2) hybridizing probes to the DNA, and (3) analyzing the spectral characteristics of the chromosomes. There are two techniques for spectral karyotyping that are widely used: spectral karyotype (SKY) and multicolor fluorescence in situ hybridization (M-FISH). Both techniques use multicolored fluorescent probes which emit at different wavelengths (200-700 nm) upon light excitation to hybridize to the target DNA sequences. The methods differ in the way in which light is captured and analyzed by the corresponding software. In this protocol, methods for making metaphase spreads from human embryonic stem cells (hESCs) for spectral karyotyping (SKY) using the spectral imaging system and software from Applied Spectral Imaging are described.