ALBERT

Description: It has long been proposed that Myelodysplastic syndromes (MDS) arise into a context of genomic instability leading to accumulation of multiple mutations. However, the underlying mechanism remains elusive. Two different genetic instability pathways, chromosome instability (CIN) and microsatellite instability (MSI) can be study using repetitive polymorphic markers (STR). Mutations of CIN genes increase the rate of gross chromosomal changes and loss of heterozygosity (LOH), which is an important mechanism of tumor suppressor gene inactivation. MSI is manifested by alterations of the length of STR due to germline mutations or methylation-induced silencing of key DNA mismatch repair genes (MMR). To verify whether both mechanisms might be involved in MDS, microsatellite instability and loss of heterozygosity (LOH) were analyzed with 10 specific STR markers. Bone marrow samples from 21 de novo MDS patients (12 females/9 males) with a mean age of 70.7 years (range 38–93), including 13 RA, 4 RAEB, 1 RAEB-T, 2 RARS and 1 CMML, were analyzed. All patients gave informed consent and the study was approved by our Ethics Committee. Mononuclear cells were separated by density gradient centrifugation through Ficoll Hypaque and depleted of adherent cells by 1 h incubation at 37°C. Myelodysplastic DNA was obtained from non-adherent mononuclear cells, while normal DNA was extracted from polymorphonuclear cells. Ten STR were amplified at different annealing temperatures (55–58°C), depending on the average size of each primer. Four mono- or dinucleotide markers (BAT 25, BAT 26, D2S123 and D18S58) were chosen from the Bethesda reference panel for MSI studies. Six additional loci at CRTL (5q13-14), IRF (5q31), D5S209 (5q31-33), CSF1RT (5q33-35), D7S525 (7q21-31) and TP53 (17p13.1), were selected because of their location on specific genomic points involved in MDS and acute leukemia. PCR products were analyzed by medium size non-denaturing polyacrylamide (15%) gel electrophoresis and silver staining (0.1%). Allele variations between normal and myelodysplastic DNA were scored according international criteria. Six out of 21 cases (28.6%) presented an unstable phenotype with band shifts or losses. No STR alterations were observed with markers BAT 25, BAT 26, IRF and D2S123. Two cases (9.6%) presented LOH at TP53, D5S209 and D7S525. Bethesda panel revealed MSI only in one patient (4.8%) at D18S58. STR at hematological loci allowed the identification of 5 cases (23.8%) with amplifications or deletions of the repeat sequences at CSF1RT, CRTL and TP53. MSI was detected only at 1/9 or 2/9 STR per patient and therefore these cases were classified to exhibit low grade of MSI (MSI-L). The mean frequency of mutated STR/individual showed a significant difference between hematological markers (0.08±0.03) respect to Bethesda STR (0.01±0.03) (Student test, p=0.007), suggesting that colon markers are not useful to evaluate MSI/LOH in MDS. MSI-L was more frequent than LOH in patients with unstable phenotype, suggesting that putative tumor suppressor genes at the loci studied are not involved in MDS genomic instability. Our data suggest that a subset of patients with MSI-L, are not related to MMR mutations but are probably associated to other genetic alterations, critical in MDS development. Moreover, a STR panel specific for MDS should be defined in order to deep evaluate genomic instability in this disease.

Description: Abstract 743 Background There has been considerable recent focus upon the molecular classification of myeloma. However, the prognostic impact of molecular changes has mostly been assessed from small and/or incomplete studies from single institutions or groups. There has been no large scale analysis of molecular features linked to ISS stage. Methods In order to clarify the overall impact of molecular changes we undertook a collective analysis of 9,897 patients through the International Myeloma Working Group (IMWG). Within this population 2,295 patients had presence of cytogenetic abnormalities (Any CA); 1,713 hypodiploidy; 1,673 hyperdiploidy; 2,309 cytogenetic deletion 13; 3,226 deletion 13 by FISH; 1,573 FISH t(4;14); 1,486 FISH del p17; 1, 683 FISH t(11:14); and 366 FISH t(4;16). Enrolled patients had complete clinical and treatment details available including baseline standard prognostic factors, ISS stage, as well as both progression free survival (PFS) and overall survival (OS) information. Data came from 14 sites: 3 from the US and the remainder from Europe, Asia, and Latin America as for the ISS staging system analyses. Univariate and multivariate analyses were performed. Results Each of the known adverse molecular features had a negative impact upon both PFS and OS (p=002 -

Description: There is increasing evidence that, in addition to genetic aberrations, epigenetic processes play a major role in carcinogenesis. Particularly, hypermethylation of CpG islands of the promoter regions of tumor suppressor genes (TSG) is now considered as an important epigenetic mechanism for gene inactivation. Multiple myeloma (MM) is characterized by neoplastic proliferation of monoclonal plasma cells. The natural course of disease may progress through monoclonal gammopathy of undetermined significance (MGUS) to MM. During this process, multiple genetic alterations are sequentially acquired and aberrant promoter hypermethylation might be one of the steps involved in this progression. In this study, we have evaluated methylation status of the following TSG: p15INK4b, p16INK4a, p14ARF, SOCS-1, p27KIP1, RASSF1A and p73 genes, in order to determine if they were involved in the evolution of MGUS to MM. Forty four MM (21 males; mean age 67.5 years; Durie-Salmon clinical stages: I: 20%, II:14% and III: 66%) and 21 MGUS patients (6 males; mean age 68 years) were study. All patients gave informed consent and the study was approved by the Ethics Committee of our Institution. Peripheral blood samples from 10 normal individuals and CpGenome Universal Methylated DNA (Chemicon International) were used as negative and positive controls, respectively. DNA was extracted from bone marrow cells of patients and peripheral blood lymphocytes of controls using phenol/chloroform method. Methylation status was performed using Methylation Specific PCR (MSP) technique. For statistical analysis, Student t and Fisher exact tests were used. The methylation index (MI; ratio between the number of genes methylated and the number of genes analyzed) was also calculated. SOCS-1 gene methylation was significantly more frequent in MM (52%) than in MGUS patients (14%) (p=0,006). Frequencies of methylation of p14ARF, p15INK4b, p16INK4a and RASSF1A were comparable in both entities: 29%, 32%, 7% and 2%, respectively, for MM; and 29%, 29%, 5% and 0%, respectively, for MGUS. TP73 gene showed a tendency of higher methylation in MM (45%) than in MGUS (33%). All patients lacked methylation at p27KIP1 gene. Whereas the percentage of MM with at least one gene methylated (84%) did not showed differences to that of MGUS (66%), the mean MI of MGUS was lower (0.16; range 0.14-0.43) than that of MM (0.24; range 0.14-0.71) (p

Description: Telomeres are essential structures that protect the ends of linear chromosomes. When telomeres become dysfunctional, critically short ends are sensed by the DNA repair machinery triggering senescence or apoptosis as well as creating interchromosomal fusions. Telomere structure and functions depend on the telomerase enzyme (hTERT, hTERC and DKC1) for elongation, on the sheterin complex that regulates telomere length (TL) and protects them against degradation and fusion, and on the non-shelterin complex. The latter is constituted by a set of multifunctional factors, including RPA1 and the MRE11-RAD50-NBS1 (MRN) complex, that facilitate telomerase-based telomere elongation. Particularly, RPA1 might be a crucial link between telomere homeostasis and the replication of chromosome ends as it regulate the telomerase action during the cell cycle. The role of MRN complex in telomere maintenance parallels its function in DNA repair. In telomerase-positive cells, a reduction of the MRN complex results in G-overhang shortening, suggesting MRN complex involvement in the recruitment or action of telomerase. Previous data from our group have provided the first evidence of modifications in the mRNA expression of shelterin members in plasma cell disorders (Panero et al, Mol Med 2010; 16: 471-8; Blood Cells Mol Dis 2014; 52: 134-9). The aim of the present study was to examine the expression profile of the non-shelterin complex: RPA1, RAD50, MRE11, NBS1 and DKC1 in patients with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Results were correlated with hTERT (telomerase catalytic subunit) mRNA levels, TL, and clinico-pathological characteristics of patients. Bone marrow samples from 70 cases: 35 MM (21 females; mean age: 67.9 years; range: 30-87 years; 35.3% stage III) and 35 MGUS (21 females; mean age: 68.7 years; range: 39-88 years) were studied. All patients gave informed consent and the study was approved by the Ethics Committee of our Institution. Gene expression was quantified by Real-time Quantitative PCR, using Taq-Man methodology, and TL measurements were performed by Terminal Restriction Fragments. A significant increase in the mean mRNA levels of all non-shelterin genes in patients with MM respect to cases with MGUS was observed (Table 1). In order to evaluate whether the expression of these factors was associated to telomerase activity, hTERT transcripts were also quantified. An upregulation of telomerase expression, with significant differences between entities was found (p=0.03). In MM, higher DKC1 mRNA levels (0.10±0.02) in patients who over-expressed telomerase compared to patients with low hTERT expression (0.04±0.004) (p=0.008) was observed. No association between gene expression and TL or clinical parameters was found. To the best of our knowledge, our findings show for the first time a global modification in the expression of non-shelterin genes in MM and MGUS, suggesting that their upregulation in MM may occur as a result of the increasing need for DNA repair and telomerase recruitment, contributing to the progression of the disease. In addition, DKC1 was related to hTERTlevels in MM patients, supporting the role that DKC1 plays in telomerase complex stabilization and telomere maintenance. Table 1. Expression profile of non-shelterin genes in patients with plasma cell disorders Genes MGUS (X±ES) MM (X±ES) p value DKC1 0.036±0.003 0.052±0.005 0.02 RAD50 0.060±0.007 0.237±0.064 0.0001 MRE11 0.277±0.040 0.545±0.114 0.003 NBS1 0.448±0.066 0.894±0.148 0.02 RPA1 0.391±0.044 0.591±0.082 0.02 Disclosures No relevant conflicts of interest to declare.

Description: Introduction Little is known about the incidence and clinical features of Multiple Myeloma (MM) in Latin America. A clinical registry of Latin American (LA) patients with MM represents an opportunity to gain insight into the prevalence of the disease in this region, the patterns of care and the current treatment status in different LA countries. Objective To characterize the demographic and clinical features of patients with multiple myeloma from five LA countries (Brazil, Argentina, Chile, Mexico and Peru) and to create a LA database on MM; in addition to investigating the patterns of care for MM patients in Latin America. Patients and Methods This is an observational, non-intervention study, with a prospective evaluation of data. Eligible patients were diagnosed with multiple myeloma, between January 1, 2005, and December 31, 2007, at any one of the participating centers, regardless of disease stage or treatment modality. The follow-up period extended to at least 5 years for each patient (December 31, 2012). Results Eight hundred and seventy six patients were included. The median age was 60 years old (25-97), 53.4% male and 46.6% female. The median follow-up was 31.4 months, and the median overall survival was 57 months. The median overall survival to patients who received high-dose chemotherapy was 77 months and for patients who received conventional chemotherapy was 48 months (p

Description: Introduction: Activated factor XIII (FXIII) stabilizes fibrin clots at the end of the coagulation cascade by bridging fibrin molecules. Disproportionate surgery related bleeding has been reported in association with FXIII deficiency, in patients with normal coagulopathies screening tests, and without a history of previous bleedings. Methods: Retrospective case series. We performed immunological factor XIIIA (FXIIIA) studies in the inpatient setting of the Hospital Italiano de Buenos Aires, between January 2014 and March 2016. FXIIIA below 50% were considered deficient. Patients with suspicion of congenital Factor XIII deficiency or 2 years old or less were excluded. Descriptive statistics were used. Populations were compared with chi-square, Fisher and T test, or Mann Whitney tests with Stata13 software. Results: FXIII was studied in 52 patients; 37 of these (26 females and 11 males) met inclusion and exclusion criteria. Median age was 43 years (range 9-81). Twenty two patients were studied because of disproportionate surgery related bleeding, 11 because of spontaneous bleeding; and 4 not specified. All patients presented normal coagulopathies screening tests, normal Von Willebrand factor and ristocetine cofactor activity and normal platelet function tests. Eleven patients (30%) had FXIIIA less than 50%. Statistically significant differences between groups were found for median drop in hematocrit points [3.5 (IQR 3-10) vs 1.5 (IQR 0-2, p=0.02)] and median number of red cell units transfused [4 (IQR 0-6) vs 0 (0-2), p=0.01]. Differences in rates of minor and major bleedings were not statistically significant. Only one patient of eleven presented FXIII deficiency associated spontaneous bleeding vs 8 out of 20 patients in the postsurgical setting. FXIII concentrate was used in two patients to treat persistent bleeding, resolving in less than 24 hours. Three patients died, none because of bleeding. Five of the 11 patients with FXIII deficiency returned to normal values when FXIII assay was repeated away from acute setting. Discussion: FXIII acquired deficiency is associated with disproportionate bleeding in postsurgical setting in patients without apparent coagulation defects. FXIII deficiency could be underdiagnosed. Patients with FXIII deficiency in our series had more hematocrit drop and more transfusion requirements. We found no association with spontaneous bleeding. Alltough deficiency could be transient, treatment with FXIII concentrate could be useful to manage serious, persistent or life threatening bleedings. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Compliance with Multiple Myeloma (MM) recommendations regarding diagnosis and treatment is highly variable worldwide, outside clinical trials. This survey among hematologists from Latin America (LA) aims to describe real access to diagnostic and prognostic analyses and first line treatment for newly diagnosed MM (NDMM). Objectives To describe the access to diagnostic and prognostic tests and first line treatment options for MM in LA. To compare public versus private access to tests and therapies. Methods This is a multicenter cross-sectional study. A 16-question survey focusing on demographic characteristics of physicians, centers, and standard of care practices for NDMM was emailed to 182 hematologists from 11 LA countries. (Fig 1) The survey was open from Dec/17-Feb/18. Results We received 103 completed questionnaires (56.6%) from 8 countries: Argentina (45), Uruguay (28), Chile (15), Paraguay (6), Peru (3), Costa Rica (2), Mexico (2), Venezuela (2). Most physicians (85/103) work in private and public institutions; the majority (64.7%) treat benign and malignant diseases, 30% mainly malignant diseases, and 4.9% mainly plasma cell disorders. Access to diagnostic tests is shown in Table 1. In 〉 20% of public hospitals there is no access to serum protein electrophoresis (SPEP), serum immunofixation (IFX) or serum immunoglobulins quantification (Igs); in 57.3% serum Free Light Chain (sFLC) assay is not done. Lack of access to Fluorescent in situ hybridization (FISH) is 67%, Computed Tomography (CT) scan 23.5%, Magnetic Resonance Imaging (MRI) 45% and Positron Emission Tomography (PET/CT) 66.5%. In private centers, lack of access to SPEP is

Description: Despite 90% stage I Hodgkin Lymphoma (HL) patients can respond to current systemic therapy, this drops to 60%, when diagnosed in advanced stages. Nevertheless and independently of the lymphoma stage, the real challenge when treating these patients, is the refractory and relapsed disease. There is no molecular biomarker to identify patients that would be non-responsive to conventional treatment or that would relapse. Furthermore, rescue chemotherapy schemes for refractory and relapsed patients, associate with acute and late toxicity high risk. This highlights the need to deeper understand the HL molecular biology and the screening for predictive biomarkers as well as potential therapeutic directed-targets. We have previously reported that HL relies on the alternative NFkB pathway, mediated by RelB and NIK, to survive. Depletion of either RelB or NIK by shRNAs or pharmacological NIK inhibitors induce HL cell death. ChIP-Seq analysis uncovered RelB target genes showing RelB bound to BCL2 promoter. A significant downregulation of BCL2 mRNA and protein levels, following RelB or NIK knockdown was observed, indicating that RelB regulates BCL2 expression in human HL cell lines. Our molecular studies suggested that NFkB alternative pathway constitutive signaling could at least partially explain the non-responding HL cases. We aimed to analyze whether mediators of this pathway could be useful as predictive biomarkers and would represent potential targetable factors in both refractory and relapsed patients. We analyzed NIK and BCL2 citoplasm expression in Hodgkin Reed-Sternberg cells (HRS) in lymphatic node biopsies of 96 patients by inmunohistochemistry [50 female Md age and (range) 59 (6-82), 46 male 42 (9-78)]. The univariate analysis showed no correlation between NIK or BCL2 expression and the prognosis clinical and pathological parameters, neither the molecular markers routinely assayed. A positive correlation was found between NIK and BCL2 expression (p=0.01). NIK and BCL2 correlated with lack of response to conventional therapy and both early and late disease progression. The analysis of survival, applying the Kaplan-Meier Curves, showed 〉 60% NIK positive HRS cells associated with shorter Disease Free Survival (DFS) [Log Rank Test (p=0.000)] and predicted overall survival (OS) as well [Log Rank Test (p=0.01)]. Furthermore, 〉 60% BCL2 positive HRS cells correlated with poor prognosis in terms of OS [Log Rank Test (p=0.002)]. The statistical significance was maintained in the multivariate analysis [Cox Regression and Logistic Regression (p=0.001)]. NIK and BCL2 performed successfully as useful predictive markers to identify refractory or risk of relapse HL patients at diagnosis. They represent attractive molecules to further analyse their potential as directed-therapy targets, since we have already reported that HL is sensitive to NIK inhibitors and BCL2 blockers have already been approved for clinical use in other hematological pathologies. Disclosures Zerga: Bristol Myers Squibb: Other: Conference fees; Janssen: Other: Conference fees; Roche: Other: Conference fees; Takeda: Other: Conference fees.

Description: Background Monoclonal gammopathy of renal significance (MGRS) is a recently defined entity. It is a group of renal diseases due to paraprotein deposition from a small B lymphocyte or plasma cell clon, not meeting the criteria for an overt gammopathy-associated neoplasm. Despite this feature, the secondary kidney damage may be severe and irreversible; therefore, its early recognition and treatment are crucial. There are few studies on MGRS in the international literature, and no reported data from Latin America (LA). Aims To describe epidemiological and clinical characteristics of patients diagnosed with MGRS in LA. To evaluate patients outcomes. Material and methods This is an international multicentric retrospective case series study. All members of GELAMM (Grupo de estudio latinoamericano de Mieloma Múltiple) were invited to participate. Patients with diagnosis of MGRS according to the IMWG definition were included. All cases had pathological diagnosis provided by a renal biopsy. Epidemiological and clinical data were collected from clinical records in a standardized report form. Renal response was arbitrarily defined as the partial or total recovery of renal failure or renal symtoms at the end of treatment. Statistical analysis was performed by descriptive statistics using STATA 12. Results We received data from 18 patients, from centers in Chile, Argentina and Uruguay. Median follow up was 22,5 months. The patients characteristics are shown in table 1. The median age was 58 years (36 to 78 years). Male to female ratio was 1:1,25. Twelve had history of hypertension and one patient of renal transplantation. Anemia was present in 78% of cases (mean 10,7g/dL +/-2,3), hypoalbuminemia in 72% (mean 2,8g/dL +/-0,7), renal failure in 83% (mean creatinine of 4,6mg/dL +/- 4,8) with 47% of these (7 patients) requiring renal replacement therapy (RRT). Proteinuria was measured in 16 patients. Its average was 4,4gr (range 0,12 - 11,5gr/24hrs). LDH and calcemia were normal in all cases. Half of the patients presented as a nephrotic syndrome. Regarding histological subtypes, the most frequently diagnosed was the proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID). The paraprotein most frequently found was Kappa, and in the renal biopsy was IgG Kappa deposition (table 1). Serum protein electrophoresis (sPEP) was performed in all cases. Only 8 out 18 patients underwent urine protein electrophoresis (uPEP) and 14 had urine and serum immunofixation (IFX) done. Serum free light chain (sFLC) were performed in 94% of the patients. The paraprotein identification according to each of this exams is shown in figure 1. Seventeen patients received treatment; 13 received an anti-plasma cell drug, 7 a thalidomide based regimen and 6 a bortezomib based regimen. The patient with IgM MGRS was treated with a rituximab based regimen. Regarding renal responses, there were no data in 5 patients. Nine out 13 of the patients achieved renal response: 3 achieved partial recovery and 9 complete recovery. Three patients become RRT independent. There was no mortality in our cohort. No patient relapsed, but 3 progressed: 1 to multiple myeloma (MM), 1 to systemic amyloidosis and another to systemic light chain deposition disease (LCDD). Discussion Only 18 cases from 3 South American countries were collected. The lack of hematologists in some countries, difficulties in achieving a renal biopsy, non-availability of immunofluorescence in the histological studies and few experienced pathologists could be some of the problems in our region. Our cohort is of rather young patients, which is probably related to the fact that these patients are mostly undergoing renal biopsy. We believe, however, that the incidence of MGRS (similar to what happens with MGUS), increases with age, which could mean a problem of underdiagnoses. As expected, nephropathies frequently associated with MM were found: AL amyloidosis and LCDD. However, the most common renal pathology was PGNMID, a rare entity. This data must be corroborated with a larger study. The high sensitivity of sFLC to identify the paraprotein was corroborated and highlighted the importance of this test in the follow up. Half of patients achieved a renal response, which reinforces the fact that they must be promptly treated. Conclusion According to our knowledge, this is the larger cases series study in LA, and we hope it will be a contribution to the knowledge of this pathology. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 5117 Background- Thalidomide (Thal) is a pro-apoptotic, immunoregulatory and antiangiogenic agent for MM. Although it is used since the 90's, the optimal combined treatment remains inconclusive. This is a preliminary analysis of the first Latin American prospective open-label study comparing three different combinations with Thal for MM patients not eligible for autologous SCT. Patients and Methods- Eligible patients were randomized to one of the three arms and received nine 28-days induction cycles. All patients received Thal(100–200mg/d) and one of the following: A- Cyclo (50mg/d)+ Dexa (40mg/D1-4, 15–18 in cycles 1 and 2, D1-4 in cycles 3 to 9), B- Dexa (40mg/D1-4, 9–12, 17–20 in cycles 1,3,5,7 and 9, D1-4 in even cycles) or C- Mel (4mg/m2/7d)+ Pred (40mg/m2/7d). Thereafter, they were randomized to maintenance treatment until progression disease or unacceptable toxicity, as follows: A1-Thal (100mg/d)+Pred (50mg/each other day) or B1-(Thal 100mg/d). All patients received aspirin as thromboprophylaxis and sulpha-trimetroprim as prophylaxis for infection. If indicated, they received biphosphonate monthly for 24 months, and then quarterly. Before study initiation, informed consent was obtained from patients and evaluation was performed at the end of each cycle. The primary endpoint were response rate and response duration. The secondary endpoints were safety, OS and PFS. IMWG index was utilized to analyze response criteria. SPSS 15.0 for windows® were used for statistical analysis. Results- Enrolment started on February 2007, and a total of 60 patients have been included. Median age at randomization was 71 (56–84) years-old, and 52% of patients were female. Durie-Salmon stages were 12% II, 82% III and ISS stages were 47% II and 28% III. Fifty-one per cent of patients presented IgG monoclonal component. There were no significant differences comparing response 〉 VGPR and treatment arms after three, six and nine cycles. However, the PFS is significant when compared CTD/MPT and TD arms (p=0.01). There was no difference in OS. Discussion- This analysis showed that Thal combinations are effective in MM patient treatment. We have not observed different responses between the tree arms. The progression free survival is significant in favor of alkylating combination than thal+dexa (p=0.01). Toxicity was manageable and balanced between the tree different arms. We would like to thank Dr. Jesus San Miguel and Dra. Maria-Victoria Mateos who have collaborated in this study. Disclosures: No relevant conflicts of interest to declare.