ALBERT

Description: Abstract 612 Background: Chromosome 5q deletion (del5q) is the most common cytogenetic abnormality in myelodysplastic syndrome (MDS). Although haplodeficiency of several genes may contribute to the disease phenotype, allelic deletion of the ribosomal protein S14 (RPS14) gene is a key effector of the hypoplastic anemia. Disruption of ribosome assembly arising from RPS14 deletion leads to nucleolar stress that triggers p53 activation. In a murine model of the human 5q- syndrome, TP53 inactivation was alone sufficient to rescue the hematologic phenotype, indicating that the molecular pathogenesis of del(5q) MDS is p53-dependent. The tumor suppressor TP53 gene is a key regulator of stem cell homeostasis and senescence. A well described single nucleotide polymorphism (SNP) located at codon 72 in the proline-rich, pro-apoptotic domain of TP53 has been linked to cancer and mutagen susceptibility, and treatment outcome. Substitution of a cytosine (‘C’ allele) for the more common guanine (‘G’ allele) results in translation of a proline rather than arginine residue at position 72, with diminished apoptotic potential. Given the pathogenetic role of p53 in del(5q) MDS, we hypothesized that homozygosity for the ‘C’ allele may be associated with disease predisposition. Methods: Bone marrow and blood samples were investigated from 118 del(5q) MDS patients, 102 non-del(5q) MDS patients, and 98 healthy controls. Genomic DNA was extracted and codon 72 of the TP53 gene was amplified by PCR. Forward and reverse Sanger sequencing was performed to determine genotype. Relationship to disease specific features at diagnosis including cytogenetic risk category, IPSS score, blast percentage, and age were investigated as well as the relationship to response to lenalidomide and AML transformation using SAS software (Version 9.2, SAS Institute Inc., Cary, NC, USA). Results: Genotype distribution significantly differed between del(5q) MDS patients (18% CC, 47% CG, and 35% GG), non-del(5q) MDS patients (9% CC, 58% CG, and 33% GG),and healthy controls (7% CC, 43% CG, and 50% GG) (p=0.01). The frequency of the homozygous CC genotype was 〉2x greater in del(5q) MDS (18%) compared to both non-del(5q) MDS (9%) and healthy controls (7%) (p=0.05). There was no significant frequency difference between non-del(5q) and healthy controls. Del(5q) MDS patients were 〉6 times more likely to carry the CC genotype vs. GG when compared to healthy controls [odds ratio (OR)=6.71, 95% CI: 1.56 to 28.86], whereas non-del(5q) patients were 〉3 times more likely to carry the CC genotype vs. GG when compared to healthy controls (OR=3.87, 95% CI: 0.66 to 22.71). The corresponding ‘C’ allele frequency was significantly greater among del(5q) MDS patients (41.7%) compared to healthy controls (28.6%) (p=0.006), and approached significance in non-del(5q) patients (37.8%) versus controls (p=0.06). There was no association between TP53 R72P genotype and cytogenetic risk group in either del(5q) (p=0.67) or non-del(5q) MDS patients (p= 0.60), IPSS (del5q, p=0.29; non-del5q, p=0.89), or response to lenalidomide (del5q, p=0.57; non-del5q p=0.89). Mean age at diagnosis was significantly (p=0.04) lower in del(5q) MDS (67.4 years; SD=11.1 years) compared to non-del(5q) MDS patients (70.8 years; SD=9.1years), although significant differences in age according to TP53 R72P genotype were not apparent in either MDS cytogenetic group (del5q, p=0.99; non-del5q, p=0.89). Conclusion: Our findings indicate that the TP53 R72P homozygous CC genotype occurs with significantly greater frequency in del(5q) MDS compared to both non-del(5q) MDS patients and healthy controls, suggesting that this polymorphism may play a key role in the pathogenesis of and predisposition to del(5q) MDS. Disclosures: Kurtin: Celgene: Honoraria. Maciejewski:Celgene: Research Funding; Eisai: Research Funding; Alexion: Consultancy. Nevill:Celgene: Honoraria. Karsan:Celgene: Research Funding. List:Celgene: Research Funding.

Description: Abstract 4001 Background: Low serum albumin level is known to be an adverse prognostic factor in patients with malignancies such as multiple myeloma. We previously reported that severe hypoalbuminemia ( 4.0 g/dl). The Kaplan–Meier method was used to estimate median OS. The log rank test was used to compare Kaplan–Meier survival estimates between two groups. Cox proportional hazards regression was used for multivariable analysis. Results: Between January 2001 and December 2009, 844 patients were captured by the MCC MDS database. The median age was 69 years. MDS subtypes were coded as refractory anemia (RA) (n=98;12%), refractory anemia with ring sideroblasts (RARS) (n=76;9%), del(5q) (n=20;2.4%), refractory cytopenia with multi-lineage dysplasia (RCMD) (n=96;11%), refractory anemia with excess blasts (RAEB) (n=255;30%), therapy related MDS (n=22;2.6%), and MDS-NOS (n=275; 33%). The distribution of IPSS risk groups was: 18.7% Low risk, 42.9% Intermediate-1 (Int-1), 19.9% Int-2, 5.3% High risk, and 13.2% unknown. Baseline characteristics for the three patient groups defined by serum albumin level are summarized in (Table-1). There was no difference in red blood cell transfusion dependency (RBC-TD) rate between the 3 groups (p=0.21). The median OS for all patients was 36 months (95% confidence interval (CI) 31.5–40.5 mo). Age, IPSS risk group, RBC-TD, Serum ferritin were statistically significant prognostic factors in univariable analysis. The median OS was 19 mo (95%CI= 14.9–23.1 mo), 35 mo (95%CI= 28.7–41.3 mo), and 53 mo (95%CI= 44.7–61.3 mo) for patients with serum albumin levels ≤ 3.5 g/dl, 3.6–4.0 g/dl, 〉 4.0 g/dl, respectively. (Figure-1) (p= 4.0 g/dl, respectively (p=0.003). Among patients in the Int-2/High IPSS risk group, the median OS was 16 mo (95%CI 13.3–15.7 mo), 22 mo (95%CI 18.0–26.0 mo), and 21 mo (95%CI 8.8–33.2 mo) respectively for patients with serum albumin levels ≤ 3.5 g/dl, 3.6–4.0 g/dl and 〉 4.0 g/dl, respectively p=0.03). Conclusion: In this retrospective analysis of a large single institution MDS database, serum albumin is found to be an independent prognostic factor for OS and AML transformation in MDS patients. The prognostic power of low serum albumin was greatest among patients with Low/Int-1 IPSS risk group, but remained an independent variable across all risk groups. Serum albumin may also be a surrogate marker of general health, co- morbidities, and performance status. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Lenalidomide is approved by the FDA for treatment of transfusion-dependent, lower risk, deletion 5q (del(5q)) MDS patients and used widely in practice for non-del(5q) MDS patients with anemia. Recently, subsequent primary malignancies (SPM) have been reported to be associated with lenalidomide treatment of multiple myeloma, and it is unclear if this observation is disease-specific or more broadly related to a particular therapy. The SPM risk in lenalidomide-treated MDS patients has not been evaluated previously. To investigate whether lenalidomide is associated with an increased risk of SPM in MDS patients, we conducted a large, retrospective cohort study of 1,248 MDS patients treated with or without lenalidomide at the Moffitt Cancer Center (MCC). Methods: Patients treated for MDS at MCC in 2004-2012 were identified through MCC's enterprise wide data warehouse which combined clinical information from a variety of sources, including the Cancer Registry, electronic medical records and disease-specific databases. A total of 1,248 MDS patients, ages 18+ years, were identified, corresponding to International Classification of Diseases for Oncology Third Edition (ICD-O-3) codes 99801, 99803, 99833, 99843, 99853, 99863, 99873, 99891 and 99893. A total of 41 cases of SPM were verified by two hematologists for confirmation of both the baseline MDS diagnosis and the SPM diagnosis. SPM incidence rates were estimated based on the Poisson distribution. Cox proportional hazards ratios (HR) and 95% confidence intervals (CI) were calculated to estimate the age-adjusted association between lenalidomide treatment and SPM in the overall cohort, and stratified by lower versus higher risk IPSS. To obtain additional details on lenalidomide treatment and potential confounders, medical chart abstraction was conducted for all SPM cases in addition to a sample of MDS patients from the baseline cohort who had not developed SPM; these controls were matched to cases 1:1 on age at MDS diagnosis (

Description: Age and mutagen related stem cell depletion may contribute to emergence of neoplastic clones. While telomere shortening or dysfunction may initiate replicative senescence and apoptosis, oncogene-induced senescence arising from induction of inflammatory cytokines may precede telomeric changes in neoplastic cells (Cell2008; 133: 1019). Activation of cellular senescence is associated with up-regulation of b-galactosidase activity at pH6 known as ‘senescence-associated’ b-galactosidase (SABG). To investigate the role of senescence in myelodysplastic syndromes (MDS), we evaluated SABG activity in bone marrow cells of newly diagnosed MDS patients (n=18) and age-matched controls (n=9). METHODS: MDS cases that received no prior treatment other than growth factors were included. All MDS diagnoses were histologically confirmed by bone marrow examination, classified by WHO and categorized into low risk {RA, RCMD, RCMD-RS and del (5q)} (n=10) and high risk groups { RAEB-1, RAEB-2} (n=8). Controls bone marrows were obtained from staging marrows for lymphoid malignancy after confirmed absence of medullary tumor involvement. Cytospins were prepared from bone marrow aspirates and stained using a SABG staining Kit. SABG expression was characterized by average staining intensity and quantitated by the percentage of positive cells according to cell lineage (i.e., megakaryocyte, myeloid, erythroid and lymphoid cells). Staining differences were compared using Vander Waerden Two-Sample Test. Correlations between myeloid SABG expression and, blast percentage or, absolute neutrophil count (ANC); megakaryocyte positivity and platelet count; average positivity and IPSS score among the two WHO groups was analyzed using Spearman coefficient. RESULTS: SABG expression as measured by mean percentage of positive cells was significantly higher in low risk MDS cases (mean (SD) = 9% (0.090476)) when compared with controls (mean (SD) =1% (0.030923)) (p 0.02). Percentage of SABG-positive myeloid cells was significantly also higher in low risk MDS cases (mean (SD) = 8% (0.077201)) when compared to controls (mean (SD) =1%) (p 0.02). High risk MDS cases also had increased SABG expression as measured by mean percentage of positive cells (mean (SD) = 14% (0.164099)) and percentage of SABG-positive myeloid cells (mean (SD) = 24 % (0.290369)). Similarly, the percentage of SABG positive megakaryocytes was higher in low risk MDS cases (mean (SD) =20 %( 0.312681)) and high risk MDS cases (mean (SD) = 14% (0.2258)) compared to controls (mean (SD) =0% (0.007559)) (p 0.13 and 0.21 respectively). In high risk MDS we found a positive correlation between the percentage of SABG staining myeloid cells and marrow blast percentage (mean (SD) = 9.3750(4.5019)) (0.07702). Moreover, in low risk MDS there was a negative correlation between percentage myeloid cell positivity and absolute neutrophil count (mean (SD) = 2.2 (1.27)) (−0.40122). There was no significant difference in average percentage of SABG staining among IPSS risk categories. SABG positive cells lacked cytologic features of apoptosis or mitosis. CONCLUSION: MDS is associated with activation of cellular senescence programs in affected hematopoietic precursors. The linkage between myeloid SABG expression and neutropenia and blast percentage suggests that cellular senescence may contribute to maturation impairment and ineffective myelopoiesis.

Description: Abstract 1890 The myelodysplastic syndromes (MDS) became reportable malignancies to U.S. population-based cancer registries including the Surveillance, Epidemiology and End Results (SEER) Program in 2001. Registries capture information on MDS cases through reports sent by hospitals and physicians’ offices. Electronic pathology (E-path) reports generated by private pathology laboratories are another potential source for finding cases; however, the sheer volume of E-path reports generated and the limited resources of cancer registries preclude the review of all E-path reports. Therefore, some registries rely on probability scoring based on keyword hits to identify reports most likely consistent with a diagnosis of cancer. Given the diverse morphologic features of MDS pathology and our earlier observation that MDS is often diagnosed and managed in the outpatient setting (Rollison, et al. Blood 2008), we hypothesized that MDS is often not captured by state cancer registries. To estimate the proportion of uncaptured MDS cases in Florida, all E-Path reports sent to Florida Cancer Data System (FCDS), the state cancer registry, in 2006 were queried using a unique keyword search strategy based on an algorithm of identifying bone marrow biopsy reports that met the inclusion and exclusion diagnostic terminology for MDS. Of 7,111 E-path reports identified, only 18% corresponded to individuals registered in FCDS as having been diagnosed with MDS. To estimate the percentage of uncaptured MDS cases in the remaining 82% of E-Path reports, a stratified random sample of E-path reports were reviewed by a single hematologist/oncologist to determine whether the E-path reports were consistent with MDS and to assign an MDS subtype. The strata for random sampling included: 1) reports that linked individuals registered as having been diagnosed with cancers other than MDS in FCDS (48%) versus those that did not link to FCDS (34%) and 2) four categories based on number of keyword hits. Overall, E-path reports corresponding to 285 individuals were reviewed, of which 71 were determined to have MDS. The percentage of uncaptured cases seemed to be lower for those individuals that were registered in FCDS as having a previous cancer (17%) than that for those who did not link to FCDS (28%) and increased with number of keyword hits. Based on the percentages of uncaptured cases estimated within each of the eight strata, and the distribution of those stratified factors in the total sampling frame, we estimated that 641 MDS cases were likely uncaptured, representing approximately 45% of the captured and uncaptured cases combined. Thus, current case finding mechanisms by population-based cancer registries capture approximately half of the true MDS cases. Compared to MDS cases captured by FCDS, uncaptured MDS cases were younger (〈 65) (p=0.01), less likely to have Refractory Anemia (RA) and more likely to have Refractory Cytopenia with Multilineage Dysplasia (RCMD) (p=0.002). Gender and race seemed to be similar between the groups. Together, these data indicate that current population-based case finding methods are not capturing a large percentage of MDS cases. Application of a keyword search strategy to identify cases among E-Path reports is a feasible technique to improve MDS case ascertainment in population-based cancer registries until greater resources are committed. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2823 Background: Mutations in TP53, or less often its regulators, increases risk for malignant transformation. Murine double minute protein 2 (MDM2), an E3 ubiquitin ligase, targets p53 for proteasomal degradation and is the most well studied negative regulator of p53. Recent investigations have highlighted the emerging importance of p53 in MDS. Haploinsufficiency for ribosomal protein S14 in deletion 5q MDS liberates free ribosomal proteins that bind to and promote degradation of MDM2, thereby activating p53 in erythroid precursors. A single nucleotide polymorphism (SNP) in an MDM2 promoter (SNP309) is linked to younger age of onset of several solid tumors and an increased risk for acute myeloid leukemia (AML) [Knappskog and Lonning. 2011. Transcription 2:207, Xiang et al. 2009. Leuk Res. 33:1454]. The thymine (T) to guanine (G) substitution introduces an additional Sp1 transcription factor binding site causing upregulation of MDM2 transcription. A second SNP in this promoter (SNP285) has also been linked to cancer susceptibility, where a guanine (G) to cytosine (C) exchange is associated with decreased ovarian and breast cancer risk (Knappskog and Lonning. 2011. Oncotarget. 2:251). The C-allele of SNP285 has diminished Sp1 promoter binding compared to the G-allele decreasing MDM2 expression. In this study we investigated genotype distribution of MDM2 SNPs in del(5q) and non-del(5q) MDS patients and compared results to healthy controls. Methods and Results: Using Sanger sequencing, we compared allele and genotype frequencies for SNP285 and SNP309 in 155 healthy controls, 97 non-del(5q), and 119 del(5q) MDS patients. For SNP285, we found no significant difference in genotype or allele frequency among non-del(5q) or del(5q) cases compared to controls (p=0.25 and 0.26, respectively). Although there was no difference in age at diagnosis by genotype in del(5q) MDS (p=0.82), there was a significant difference among non-del(5q) MDS cases [p

Description: BACKGROUND: Incidence rates for myelodysplastic syndromes (MDS) and chronic myeloproliferative disorders (CMD) in the United States were unavailable prior to the addition of these stem cell malignancies to the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) Program and other central cancer registries in 2001. Description of national incidence rates for 2001–2003 will provide an important baseline for future studies of secular trends and allow for the examination of rates by selected demographic factors to define risk profiles of these malignancies in the American population. METHODS: Incidence rates of MDS and CMD were calculated for 18 SEER areas between 2001–2003. These rates were stratified by disease subtype using the FAB classification (including chronic myelomonocytic leukemia [CMML]) with the addition of the WHO deletion 5q category, sex, age at diagnosis and race. Based on the observed SEER incidence rates, counts were estimated for the entire U.S. population. RESULTS: In 2003, 2,538 cases of MDS and 1,421 cases of CMD were observed for all 18 SEER areas combined. Similar numbers of cases were observed in 2001 and 2002. Age-adjusted incidence rates for 2001–2003 were significantly higher among males than females for MDS (4.5 per 100,000 in males vs. 2.7 per 100,000 in females, p

Description: Abstract 2777 Poster Board II-753 Telomeres are repeated nucleotide sequences at the ends of chromosomes that serve to preserve genetic integrity in hematopoietic progenitors (HPs). Abnormal telomere maintenance resulting from mutations in telomerase or other telomere repair genes have an established pathogenetic role in a subset of acquired and congenital forms of bone marrow failure (BMF). Excess telomere shortening of myeloid elements was reported previously in MDS, however it is unclear whether such abnormalities in telomere maintenance play a pathogenetic role in disease susceptibility or represent a disease specific phenotype arising from accelerated cellular proliferation of the myeloid compartment. Alterations in telomere maintenance and telomerase activity in T cells may offer insight into genetic susceptibility since these cells are not derived from the malignant clone. We examined telomere length and replication history in neutrophils and T cells in newly diagnosed MDS cases (n=66) and compared results to unrelated, age-matched controls (n=63) lacking a prior history of cancer. Telomere length in peripheral blood mononuclear cells was assessed by quantitative real-time PCR (qRT-PCR) and found to be significantly shorter among MDS cases compared to controls. Peripheral blood concentration of the senescence protein stathmin was analyzed in a subset of 48 patients and 48 controls and found to be higher in MDS cases compared to controls after adjustment for age and sex (mean 4.24 pg/ml ± 7.25 in cases vs mean 2.45 pg/ml ± 2.84 in controls, p=0.06) with no change observed with age. Mechanisms of cellular senescence and telomere attrition in T-cells may be related to antigen-driven or homeostatic proliferation. Therefore, telomere length was compared to an independent genetic marker of proliferation, i.e., the concentration of T cell receptor excision circles (TRECs). TRECs are episomal DNA fragments excised during TCR gene rearrangement within the thymus that do not transfer to daughter cells. Therefore, TREC DNA copy number has been shown to be diluted during each round of division and tends to be reduced with age due to impaired generation of new thymic T-cell emigrants. In controls, TREC concentration declined with age consistent with cellular proliferation and loss of thymic function. For every year increase in age, log TREC values decreased by 0.05 DNA copies (p=0.0012). More aggressive proliferation was evident in T cells from cases compared to controls with a 2-fold more rapid decline in TREC copy number each year (0.099 unit decrease in TREC copies per year among cases, p

Description: Introduction: Lenalidomide is currently approved by the FDA for treatment of transfusion-dependent, lower risk, deletion 5q (del(5q)) MDS patients. In practice, lenalidomide is often used for treatment of non-del(5q) anemic, lower risk MDS patients as endorsed by national guidelines. The risk of AML transformation among non-del(5q) MDS patients treated with lenalidomide has not been well studied, nor among del(5q) MDS patients outside the context of original clinical trials. We conducted a retrospective cohort study of 1,248 MDS patients treated with or without lenalidomide at the Moffitt Cancer Center (MCC) to investigate the association between lenalidomide and AML transformation. Methods: Patients treated for MDS at MCC in 2004-2012 were identified through MCC's data warehouse, including data from the Cancer Registry, electronic medical records and disease-specific databases. A total of 1,248 MDS patients, ages 18+ years, were identified, corresponding to International Classification of Diseases for Oncology Third Edition (ICD-O-3) codes 99801, 99803, 99833, 99843, 99853, 99863, 99873, 99891 and 99893. A total of 150 cases of AML transformation were verified by two hematologists confirming the MDS diagnosis and AML transformation. Incidence rates and 95% confidence intervals (CI) for AML transformation were estimated based on the Poisson distribution. Cox proportional hazards ratios (HR) and 95% CIs were calculated to estimate age-adjusted associations between lenalidomide treatment and AML transformation in the overall cohort, and stratified by lower (low and intermediate-1) risk versus higher (intermediate-2 and high) risk IPSS. To obtain additional details on lenalidomide treatment and potential confounders, medical chart abstraction was conducted for all AML cases and a sample of MDS patients from the baseline cohort who had not developed AML; these controls were matched to cases 1:1 on age at MDS diagnosis (

Description: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis with potential for progression to acute myeloid leukemia (AML). We compared natural killer (NK) cytolytic function in 48 MDS patients with 37 healthy donors and found reduced activity in the patient population (K562 cytolysis, 19% ± 21% SD versus 40% ± 17%) (P 〈 .001). NK cytotoxicity in MDS patients was reduced against 3 disparate tumor targets with differential activating receptor requirement, suggesting global defects in NK function. Reduced NK function in MDS was significantly associated with higher International Prognostic Score (P = .01), abnormal karyotype (P = .05), the presence of excess blasts (P = .01), and age-adjusted bone marrow hypercellularity (P = .04). MDS patients had a display of the activating receptor NKp30, and NKG2D down-regulation closely correlated with impaired NK function (P = .001). NKG2D ligands (MICA and MICB) were expressed on CD34+ cells from bone marrow of 30% of MDS patients and a leukemic cell line derived from an MDS patient (MDS1). Collectively, these findings suggest that impairment of NK cytolytic function derives in part from reduced activating NK receptors such as NKG2D in association with disease progression. Evasion of NK immunosurveillance may have importance for MDS disease progression.