ALBERT

Description: Introduction: Myelodysplastic syndrome with del5q (MDSdel5q) is the only cytogenetically defined MDS category recognized by WHO in 2001, 2008 and 2016 and is defined as a MDS with deletion on the long arm of chromosome 5 and less than 5% of blast cells in bone marrow. It is known that for patients with MDSdel5q and transfusion dependence (TD), Len (LEN) is the first choice of treatment. However, data regarding factors that may impact on the development of TD or disease evolution in patients diagnosed without TD are scanty. In our study a retrospective multicenter analysis on patients with low-int 1 MDSdel5q without TD at diagnosis has been performed in order to answer these questions. Patients and methods: We performed a multicenter collaborative research from the Spanish (RESMD) and German MDS registries. Data from 153 low risk MDSdel5q without TD at diagnosis were retrospectively analyzed. Statistical analysis: Data were summarized using median, range, and percentage. The event of TD was defined as the development of TD according to the IWG criteria (2006) and/or the beginning of a treatment which could modify disease course (LEN or ESA). Transfusion or treatment free survival (TFS), overall survival (OS) and leukemia free survival (LFS) were measured from diagnosis to TD or treatment, the first occurred (or to last follow up if none), last follow up or death from any cause and evolution to AML, respectively. TFS, OS and LFS were analyzed using the Kaplan Ð Meier method. The Log-rank test was used to compare variables and their impact on survival for univariate analysis.Multivariate analysis was performed using Cox's proportional hazards regression model. For comparison of Kaplan Meier curves the long rank test was used, with statistical significance with p350 109/L) (p=0.007), and neutropenia (

Description: Background and Aim Although new agents have been approved for the treatment of MDS, the only curative approach for these patients is allogeneic hematopoietic stem cell transplantation (HSCT). Nevertheless, in these patients this approach has only obtained 40-60% of overall survival. Somatic mutations in MDS have recently been analyzed in order to confirm clonally and also prognostic impact in MDS patients. In this regard, TP 53 mutated gene is present in MDS in less than 10% of patients and is associated with advanced disease and high-risk features. Recent studies confirms poor outcomes in patients with TP 53 mutated receiving allogeneic stem cell transplantation1,2. The present study try to analyze if the development of chronic graft versus host disease (cGVHD) could modify, due to graft versus leukemia effect, the adverse prognosis of these high-risk patients (TP53 mutated patients). Design and Methods Results of HSCT in 92 MDS patients from 5 centers in Spain were retrospectively studied. Samples were collected 1 month prior to transplant. 280ng of the genomic DNA from BM cells was screened for somatic mutations in TP53 gene. The study was done by NGS on a GS Junior Instrument (Roche) according to an amplicon sequencing design. For each sample, eight exons (4-11) were amplified with preconfigured primer plates provided within the IRON II study network. Data analysis, were carried out using the Sequence Pilot software version 3.5.2 (JSI Medical Systems) and GS Amplicon Variant Analyzer software, versions 2.7 and 2.9 (Roche Applied Science). Minimum coverage of sequenced exons was 100 reads and the sensitivity of variant detection was set to a lower limit of 〉2% for bidirectional reads. Only those variants that resulted in amino acid change in the protein sequence were considered. OS and RFS were calculated using the Kaplan-Meier method. The log-rank test was used for comparisons. All calculations were done using SPSS 18.0. Cumulative incidence of relapse was also calculated by xlstat version 2014 program. Results Median age was 54 years (17-69), 71.7% were "de novo" MDS and regarding IPSS, 53% were in the int-2/high-risk category. Other characteristics were in Table 1. In the pre-transplant evaluation, 15 patients out of 92 (16,3%) were TP 53 mutated. The mutations were located in exons 5, 6, 7, 8 and 10. These variations were present in a variable percentageof the cell population (3 to 84%). All mutations were specific nucleotide changes except for two cases. At the time of the last update, 16 patients had relapsed (17.4%) and 40 had died (43.5%). After a median follow up of 15.5 months, OS was 56.5%. Median OS for patients with mutated TP53 trend a toward to be shorter than survival for patients without mutated TP53 (median of 7 mo vs median not reached, respectively, p=0.156). Multivariate analysis for OS confirmed complex karyotype (HR 5,588, 95CI 1,794-17,407, p=0.003) and no developement of cGVHD (HR 3,531, 95IC 1,634-7,632, p=0.001) as predictors for poor outcome. Cumulative incidence of relapse was 20.3% (+/-4.3%) at 1 years. Mutational status of TP53 significantly influenced on relapse (53.3% +/-12.9% vs 13.7% +/-4% at 1 year for patients with vs without TP 53 mutation (Gray test=0.001, Figure 2). Regarding Relapse Free Survival (RFS), after a median of follow up of 17 months, RFS was 67.9% and as previously suggested, the presence of TP 53 mutation had an impact on RFS (41.7% for mutated (median RFS of 6 months) and 75% for non mutated patients (median RFS not reached), p=0.009). Multivariate analysis for RFS confirmed age (HR 1.054, 95CI 1.005-1.106, p=0.032) and TP 53 mutated (HR 3.054, 95IC 1.145-8.149, p=0.026) as predictors for lower RFS. Regarding 15 patients with mutated TP 53, 7 did relapsed and 9 had died. Developement of cGVHD showed a trend toward to improve outcome among TP 53 mutated patients, with a better OS and RFS for those developing cGVHD as compared to those who did not (OS of 55% vs 17% for patients with and without cGVHD, p=0.039, Figure 2 and RFS of 71% vs 50%, respectively, p=0.3). Conclusions Mutated TP53 pre-allo patients presents poor outcome as compared to not mutated, as previously described Bejar1 and Kim2. Nevertheless, the developement of cGVHD could overcome the adverse impact of this factor due to the developement of graft versus tumor efect, improving survival curves (OS and RFS) as compared to previous published results. Study supported by GRS-1033/A/14 P53. 1.-BŽjar, JCO 2014, 32(25). 2.-Kim, BBMT 2015, Epub ahead of print. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Sanz: JANSSEN CILAG: Honoraria, Research Funding, Speakers Bureau. Valcarcel:AMGEN: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CELGENE: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Díez-Campelo:CELGENE: Research Funding, Speakers Bureau; JANSSEN: Research Funding; NOVARTIS: Research Funding, Speakers Bureau.

Description: Cytogenetic abnormalities are found in around half of MDS patients (pts) and have both clinical impact and may be subtype-defining, e.g. in 5q-syndrome. Interstitial deletion of the long arm of chr.5 [del(5q)] is the most common aberration (almost 20% of cases with abnormal cytogenetics). Del(5q) is heterogeneous, occurring as a sole abnormality or in combination, with the deleted region often truncated within or extended and/or beyond the CDR boundaries. Isolated del(5q) is frequently shorter and confers a more favorable prognosis with regard to survival and lenalidomide (LEN) responsiveness, while del(5q) in the context of a complex karyotype (CK) imparts a poor prognosis. In addition to chromosomal lesions, somatic mutations can contribute to the pathogenesis of MDS, including del(5q). We theorized that recognition of molecular defects in MDS with del(5q) may clarify the pathogenic mechanisms behind this lesion and help explain the clinical heterogeneity. We analyzed 225 pts with myeloid neoplasia and del(5q) using WES (n= 107 samples) and targeted multiplexed PCR (top 60 most frequently mutated genes) (n =133 samples); serial analysis was performed in 15 pts studied at ≥2 time points, 11 during LEN therapy and 4 upon relapse/progression. A total of 116 samples had a CK with other lesions such as -7/del(7q) found in 31% cases, and 18% had -17/del(17p). WES (average depth 〉60x) was followed by a bioanalytic pipeline, detecting ≥1 mutated gene in 71% of cases. Candidate somatic alterations were found in 357 genes and selected for further analysis. When focused on hemizygous mutations within the retained 5q allele, CSNK1A1 mutations were the most common, found in 4 pts, while other genes were only sporadically affected. Among heterozygous mutations on the non-deleted portion of del(5q) and other chromosomes (Chr), we found several novel mutations, in addition to TP53 (n=26), DNMT3A (n=8), PRPF8 (n =8), RUNX1 (n=5), TET2 (n=5), and ASXL1 (n=4), among others. Furthermore, LOH/haploinsuffciency of genes on 7q (e.g., LUC7L2, CUX1, EZH2 and MLL3) appears to be a common defect seen in pts with non-isolated del(5q), suggesting synergistic functional defects. When functionally grouping gene mutations, DNA methylation family (8 cases) and transcription factor mutations (29 cases) were associated with advanced disease (AD) and a CK. Heterozygous mutations in TP53 (34%) or deletions involving the TP53 locus (23%) resulted in total of 42% of cases carrying either TP53 LOH or mutation. TP53 lesions were more common in pts with AD vs. low risk. (21 vs. 5 p =.0008). In contrast, TP53 mutations are found in 8-10% of cases of MDS. A total of 34 pts were treated with LEN and subgrouped into responders (n=17) vs. refractory (n=9) with an overall response rate of 65%. When mutational profiles were compared, the presence of TP53 mutations did not preclude responsiveness to LEN. CK was present in 12% of responders vs. 67% of refractory pts. The most frequent Chr abnormalities were -7/7q (0% vs. 67% in responders vs. refractory) and 17p-(6% vs. 67% in responders vs. refractory) suggestive of their role in LEN resistance. In addition to cross sectional analysis, our WES study using paired Germline/tumor samples followed by deep sequencing facilitated analyses of clonal architecture by examining clonal dynamics over time. Assessment of del(5q) clone size by allelic imbalance combined with clonal burden by VAF allowed us to reconstruct the clonal hierarchy: in 73% of cases, del(5q) appeared to be the initial defect followed by subsequent mutations (e.g., TP53, DNMT3A, IDH2). In contrast, in 24% of cases, TP53, RUNX1, JARID2, were the primary defect followed by a subclonal del(5q) events. Serial samples collected before and after therapy demonstrated that responses were associated with decreased clonal burden for del(5q) but persistence of certain mutations. In refractory cases, persistent subclonal lesions and the appearance of new lesions were associated with progression. For example, pts with TP53, LAMB4, EPHA6 progressed and acquired additional lesions such as CSMD2 or KCND2, and did not see the disappearance of TP53 alterations upon treatment. In conclusion, no unifying somatic defect was found in pts with del(5q) regardless if the deletion event was primary or subclonal. Most commonly associated lesions were not present on the retained 5q alleles but rather other chr yet modified clinical behavior, including responsiveness to LEN. Disclosures Bejar: Celgene: Consultancy, Honoraria; Alexion: Other: ad hoc advisory board; Genoptix Medical Laboratory: Consultancy, Honoraria, Patents & Royalties: MDS prognostic gene signature. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Description: CONTEXT: Microenvironment of Hodgkin and Reed Sternberg(H-RS) cells is a current focus of interest to define risk and predict evolution of Hodgkin′s Lymphoma. OBJECTIVES: To determine the role of infiltrating CD8+ cells, using flow cytometry (FCM), in the evolution of patients with HL. DESIGN: Cell suspensions obtained after mechanical disintegration of ganglion biopsies of patients with newly diagnosed HL were analyzed using FCM. Definitive diagnosis was made by conventional histology and immunohistochemistry. Clinical data were collected from medical records. Statistical analysis was performed using SPSS 20.0 software. SETTING: In the University Hospital of Salamanca, we consecutively analyzed by FCM all lymph nodes with suspected lymphoma at diagnosis. There was no selection bias when collecting patients, except for some cases with inadequate quality(insufficient cells). PATIENTS OR OTHER PARTICIPANTS: From 1996(earliest available FCM data) to 2014, 104 of that samples had a definitive diagnosis of HL. Treatment depended on stage: a)early diagnosis received ABVD(x3) & local radiotherapy(RT) (20-30 Gy), b)advanced: ABVD(x6 to 8), plus RT in selected cases. Median follow-up was 10 years. INTERVENTIONS :This was a retrospective observational study. MAIN OUTCOMES MEASURES: Primary end points were overall survival(OS) and freedom from treatment failure(FFTF), considered from diagnosis to progression or relapse. RESULTS: Most cells obtained were lymphocytes (Median17.1±86.9%) with a T/B/NK distribution of 72%/27%/1.6%(median) and predominance of CD4+ (median CD4:51%-CD8:12%). Median CD8+ cells(12%) was used to divide patients into 2 groups.FFTF was longer in patients with more than 12% of CD8+cells(93% vs. 71%, p=0.01). When analyzed separately patients with early/advanced disease, the clinical benefit remained in the group with advanced disease (p=0.006), whereas the statistical significance was lost in the group with early disease, possibly due to the excellent prognosis for those patients(FFTF 10 years 〉 95%). No differences were observed in the OS, because second line therapy was highly effective. In the multivariate analysis using Cox regression, advanced stage (HR=9.6 with 95% CI 1.2 to 73.9) and〉12% CD8+ tumor infiltrating T-cells were independent variables(HR=0.26, 95% CI 0.07 to 0.9). CONCLUSIONS: Increased number of CD8+ in the H-RSC microenvironment of HL is associated with better FFTF, particularly in the advanced-disease group. This should be considered as a new biomarker to identify high-risk patients. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Viviparity in teleosts involves, invariably, the ovary in a gestational role. This type of viviparity is due to the combination of unique aspects, different from those found in the rest of vertebrates. These aspects are: The ovary has a saccular structure; the germinal epithelium lines the ovarian lumen; the absence of oviducts; and the intraovarian insemination, fertilization, and gestation. The communication of the germinal zone of the ovary to the exterior is via the caudal zone of the ovary—the gonoduct. The germinal epithelium is composed of oogonia and oocytes scattered individually or in cell nests among somatic epithelial cells. In the ovarian stroma the follicles are included which are formed by the oocyte, which is surrounded by follicular cells and the vascularized theca. The oogenesis comprises three stages: chromatin-nucleolus, previtellogenesis, and vitellogenesis. There is no ovulation, as the oocyte is retained in the follicle. During the insemination, the spermatozoa enter into the ovarian lumen and the intrafollicular fertilization occurs, followed by intrafollicular gestation. The intraovarian gestation of poeciliids involves morphological characteristics associated with the intrafollicular embryogenesis and types of nutrition, such as lecithotrophy and matrotrophy. In lecithotrophy, the nutrients come from the yolk reserves stored during oogenesis, whereas in matrotrophy the nutrients are provided by supplies from maternal tissues to the embryo during gestation. The maternal–embryonic metabolic interchanges converge through the development of the association of maternal and embryonic blood vessels, establishing a follicular placenta.

Description: INTRODUCTION: In the rituximab era, the prognostic influence of bone marrow (BM) infiltration in patients with diffuse large B-cell lymphoma (DLBCL) has been hardly studied. In this retrospective observational study, we aim to investigate the prognostic influence of concordant and discordant BM infiltration in patients with histological diagnosis of DLBCL. In addition, we analyzed the possible clonal relationship between BM and lymph node tumor cells in the cases with discordant histology. PATIENTS AND METHODS: All patients diagnosed of DLBCL in our center from January 1, 1999 were included in the study. Histological BM infiltration pattern was classified as concordant (DLBCL infiltration) or discordant (small or low-grade B-cell lymphoma) based on the diagnostic reports from the Pathology department. All cases were reviewed for this purpose by an expert pathologist. To further characterize the discordant cases, flow cytometry (FCM) reports of BM infiltration were reviewed. In the discordant cases, the clonal IGH rearrangement was studied in both BM and lymphadenopathy, by amplification of the VDJ genes as recommended in the BIOMED-2 protocol. For survival analysis, only patients treated with R-CHOP or similar were included. RESULTS: 236 patients diagnosed of DLBCL were included in the study; of them, 31 (13%) had concordant histological BM infiltration and 18 (7.6%) discordant. Phenotypic characterization by FCM of the discordant cases was heterogeneous: chronic lymphocytic leukemia (N = 2), follicular lymphoma (N = 5), marginal zone lymphoma (N = 2), non-specific phenotype (N = 5) or non-infiltration (N = 2). Clonality studies were performed in the discordant cases. Good quality DNA was obtained in 17 out of 18 patients. We confirmed the same clone in both BM and lymphadenopathy in 12 patients (70%); different clones were observed in two, and a polyclonal pattern was obtained in the remaining three patients. Survival analyzes were conducted only in the 186 patients treated with R-CHOP or similar. With a median follow up of 58 (1-135) months, PFS was significantly worse in patients with concordant (35% at 5 years, p = 0.001) or discordant (35% at 5 years, p = 0.04) histology, compared to patients without infiltration (64% at 5 years) (Figure 1). OS was significantly lower in patients with concordant histology (53% at 5 years, p = 0.05), whereas there was no significant difference between patients with discordant infiltration (62% at 5 years, p = 0.8) and non-affected BM (75% at 5 years). In the multivariate analysis, concordant BM infiltration (HR = 2.25, 95% CI 1.2 to 4.3, p = 0.01) had a negative influence on PFS (but not on OS), independently of the age, ECOG and LDH, while discordant histology was close to statistical significance (RR = 2; 95% CI 0.95 to 3, p = 0.1) CONCLUSIONS: Our results indicate that BM infiltration, both concordant and discordant, is associated with a lower PFS in DLBCL patients treated with R-CHOP or similar. Cases with discordant BM infiltration have a very heterogeneous phenotype, but we found a clonal relationship between the two different histologies in a high proportion of cases, indicating a probable histologic transformation from a low-grade lymphoma. Further studies are needed to determine the sequence of biological events that might be involved in this transformation. Figure 1. Progression free survival (PFS) according to the concordant or discordant histology of the BM Figure 1. Progression free survival (PFS) according to the concordant or discordant histology of the BM Disclosures No relevant conflicts of interest to declare.

Description: INTRODUCTION MDS are a heterogeneous group, and it is necessary an adequate prognostic stratification in order to the best management. The new revised international prognostic scoring system (IPSS-R) has improved prognostic ability for survival and AML evolution comparing with the previous prognostic indexes. But, it is not clear the prognosis of patients included in the intermediate group, 20% of MDS, patients with a median OS of 3 years according to Greenberg et al, are they in the high or in the low risk category? The aims of the present study were to describe characteristics of patients included in this intermediate group of the IPSS-R in the Spanish MDS cohort and to identify which factors could have an impact on survival. A new score prognostic system (GESMDi score) in order to a better stratification should be proposed in this subset of patients that will be useful for determine the best therapeutic approach for them. METHODS: All patients were included in the GESMD, diagnosed of Primary MSD and Intermediate IPSS-R. The Statistical analyzes were performed using SPSS version 21, Cox models and Kaplan-Meier curves were used to demonstrate clinical outcomes. Regarding the new score proposed, GESMDi score, modeling of prognostic risk was based on multivariate analysis of survival time. Cox model for survival was built to derive the relative weights within the score. RESULTS: Data from 957 patients of 69 centers of GESMD were evaluated. Their median age was 73.9 years (p25/p75 66-80), 61.6% males (N=590), and median follow-up 21,4 months (p25-p75 de 11-41). Regarding WHO 2001 classification: 31% were RAEB-1, 21% CMML, 18% RCMD, 14% RAEB-2, 3% RCMD-RS, 3.1% RARS, 2.5% RA, 2% 5q-syndrome, 2% AML, 1% unclassified. Median hemoglobin at diagnosis was 9.8 g/dL (p25/p75:8.3-11.6), median bone marrow (BM) blasts 6% (p25/p75:3-8) and median platelet count 99x109/L (p25/p75:66-180). According to IPSS, 5% of patients were classified as low risk, 78% as intermediate-1, 16% as intermediate-2 and 1% as high risk. Cytogenetic were very good in 2% of patients, good in 76%, intermediate in 17%, poor in 5% and in 1% very poor. IPSS-R score classified patients in 3 different groups, with a punctuation of≤ 3.5 (35.6%), 〉3.5 and ≤ 4 (35.8%) and〉 4 and ≤ 4.5 (28.5%). Median OS was 30.1 months, the estimated 1-year and 2-y OS were 79.2% and 57.8%, respectively. In the univariate analysis for OS older age (〉74y, p3.5 and ≤ 4 and 〉4 and ≤ 4.5, p=0.023 and p=0.004, figure 1) had a deleterious impact on survival. In the multivariate analysis, only age, Hb level, PB blast, ferritine level and IPSS-R value retained statistical significant impact on OS (table 1a). In the multivariate analysis, Hazard ratio, a new score system (GESMDi score) was established for all patients. Patients with adverse features were added points in order to stratify the risk of death: age500 ng/ml and/or IPSS-R of 〉3.5 (1 point), table 1a. The GESMDi score was performed in 685 patients with all data available and 7 groups of patients were defined with different median OS (p30 months) and high risk patients (