ALBERT

Beschreibung: Multiple myeloma (MM) pathogenesis has been explained for many years by the cancer biology dogma introduced by Peter Nowell: first, a single plasma cell would be immortalized by an error in the immunoglobulin genes rearrangement process; then, a progressive stepwise acquisition of somatic cell mutations would induce a sequential selection and domination by the fittest clone. In line with this idea of “myeloma stability”, SNP arrays studies in diagnostic-relapse paired samples have revealed the presence of common clonal characteristics. Biologically, the M-protein remains usually constant across MM evolution and further, the variable domain of the rearranged immunoglobulin heavy chain genes (or CDR3 region) has been used as a patient-specific myeloma fingerprint in minimal residual disease (MRD) studies. However, massive genome studies with Next Generation Sequencing (NGS) have challenged this concept, showing a significant intraclonal heterogeneity at diagnosis with the possible presence of several clonal progenitors or tumor-initiating cells. In this study, we have characterized and compared the CDR3 region in 52-paired samples from 26 MM patients aiming: 1) to assess mono-clonality in MM evolution through the analysis of the CDR3 sequence and, 2) to validate ASO RQ-PCR approaches for MRD in MM, based on the constancy and specificity of the CDR3 region. Samples were obtained at diagnosis and progression (19 pairs) or at 2 different timepoints of progressive disease (7 pairs). Median time between sampling was 2 years. M-protein subtype remained stable in all pairs but 1, associated with a light-chain escape phenomenon. All samples proceeded from bone marrow (BM) except for 2 pairs, composed by BM and extramedullary disease (spleen and testes). Two major cytogenetic changes were identified: increased 13q14 deletion (from 7 to 54%) in 1 pair and increased 17p (p53) deletion (from 5 to 87%) in a further one. Treatments administered between sampling included most of the current approaches used in MM (data not shown). Genomic DNA isolation, PCR amplification and sequencing were performed following conventional methods. Germline VH, DH and JH segments were identified by comparison with public databases. CDR3 region was first identified in all samples and then compared between the two samples in the 26 pairs: the sequence of nucleotides was constantly identical in each pair, including those associated with major cytogenetic changes, a light-chain escape, extramedullar vs. BM infiltration and relapsed (and therefore, treatment selected) vs. refractory disease. Therefore, we can first conclude that the main tumor clone in MM retains a specific signature across all stages of disease evolution that allows the identification of samples as evolutionary related. This major clone signature is not modified by clinical or biological changes in the disease nor under different treatment pressures and would thus identify disease relapse and progression. Our results have also a clear impact on the validity of molecular MRD techniques. The high rate of complete responses (up to 50-60%) currently achieved in MM has prompted the use of new techniques for disease assessment. Today, ASO RQ-PCR, based on the use of specific primers and probes complementary of the VDJH rearrangement, continues to be the most sensitive approach. One pitfall of this technique would be the potential instability of PCR targets over time, which would induce false negative results. In B-cell precursor ALL, this is estimated to happen in 30-40% of cases but has not been deeply evaluated in MM yet. With the present study, we can also conclude that the junction region of the VDJH rearrangement in MM constantly identifies the myeloma cells responsible for relapse and therefore can be used as a reliable target for MRD assessment by ASO RQ-PCR and more recently, by NGS methods. If the CDR3 region remains stable, the novel concept of clonal tiding in MM should not be interpreted as a poly- or oligoclonal but subclonal. In MM, tides can be subclonal, but the ocean remains monoclonal. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Introduction VDJH usage description in B-cell hematological malignancies has brought new insights into the clonal differentiation and clinical implications for certain pathologies, improving patients' care, although no correlation has been made in MM. We present the largest-to-date VDJH gene repertoire analysis in MM, consisting in biological and clinical data from 413 patients. This database was used to comprehensively investigate the characteristics of VDJH rearrangements: gene segment usage, somatic hypermutation (SHM), CDR3 characterization, and immunoglobulin stereotyped clusters assessment. We also investigated the potential relationship of these molecular features with the outcome. Methods Newly diagnosed MM patients were included in the study. All were managed according to the recommendations of the GEM-PETHEMA Spanish MM group, including the inclusion of most patients in the GEM2000 and GEM2005 schemes. Monoclonal assessment was carried out with FR1 VH-JH amplification, following the BIOMED-2 methods. Moreover, 113 cases were also analyzed by NGS using the LymphoTrack IGH FR1 assay (Invivoscribe Tech, San Diego, CA). VDJH usage and mutational status were analyzed with IMGT-V-Quest. ClustalX2.0 was used to perform clustering analysis with CDR3 regions from our cohort and 1117 additional sequences obtained from IMGT/LIGM-DB corresponding to unique rearrangements from both normal and tumor human B cells. Molecular and clinical data were used to perform survival studies. Results The overall VDJH detection rate was 92.5% (382/413), including 376 patients with only one detectable rearrangement, five with a biallelic rearrangement, and one with a biclonal rearrangement, also detectable by flow cytometry. Thus, 388 rearrangements where detected: 362 were productive, and 26 unproductive. Gene segments were identified in productive rearrangements. VH repertoire in MM reflected its normal counterpart, with IGHV3 being the predominant selected family, followed by IGHV4, IGHV1, IGHV2 and IGHV5. IGHD and IGHJ segment usage showed a skewed distribution, with IGHD2 and IGHD3 families accounting for 55.5% of cases, and IGHJ4 and IGHJ6 accounting for 70.8%. SHM level could be identified in 349/362 productive sequences (mean: 9.16%, SD 3.95) with statistically significant differences between certain IGHV families, namely IGHV2 was less hypermutated than IGHV1 or IGHV4. We could also collect other rearrangement features: nucleotide addition by TdT appeared in 92.5% for N1 and 88.6% for N2, with evidence of exonuclease activity in all cases. CDR3 mean length was 16±4 aminoacids (median 15, range 6-29), with preference for Gly, Ala, Asp, Tyr (~10% each one). The R/S mutation ratio was 2.39 for CDRs versus 1.74 for FWRs, showing the high influence of antigen selection over the former. No stereotyped clusters were found in our cohort. Relationship between these finding and clinical evolution was done through univariate and multivariate analyses. A shorter Progression-Free Survival (PFS) related to well-known clinical factors: presence of plasmacytomas, poor performance status or ISS stages, response to therapy, and poor-risk cytogenetics (p

Beschreibung: MicroRNAs (miRNAs) are small non-coding RNA that regulate gene expression by inducing RNA degradation or translational inhibition of their mRNA targets. Recently, deregulation of miRNAs has been associated with cancer development. MiRNA expression profiling has been analysed in different hematological malignancies, but the information about miRNA expression in MM is limited. We investigated the expression levels of 368 miRNAs using a quantitative PCR-method (“TaqMan low density arrays-microRNA assay”, Applied Biosystems) in 49 patients with newly diagnosed MM. Moreover, 4 MM cell lines (MM1S, OPM2, U266 and RPMI) and 4 samples of normal human plasma cells (PC) were also included in the study. In all the bone marrow samples a CD138 positive PC isolation was performed. Relative quantification of miRNA expression was calculated with the 2−ΔΔCt method. The data were normalized respect to RNU48 and relative to a calibrator sample (average of normal human PC samples). Hierarchical clustering based on the average-linkage method with the centered correlation metric was used for unsupervised analysis. Differentially expressed miRNAs were identified using “Significant Analysis of Microarrays” (SAM) algorithm, the t test and the nonparametric Wilcoxon rank sum test. Primary myeloma cells displayed a distinctive miRNA expression pattern compared to normal PC, characterized by the downregulation of the miRNAs differentially expressed. Unsupervised analysis of the data revealed that the MM samples segregated mainly into two clusters: one contained 12 out the 14 MM cases with t(4;14), 20 out of the 30 MM cases with RB deletion, and the 3 samples with t(14;16) which were tightly clustered. The other cluster contained 5 out of the 7 MM samples without genetic abnormalities (IGH translocation, RB and P53 deletions and gain on 1q were ruled out by FISH analysis). MM samples with t(11;14) and those with gains on 1q were dispersed along the dendrogram. The comparative analysis of the miRNA expression profile between MM with t(4;14) and the remaining MM samples showed a set of 5 up-regulated miRNA (let-7c, miR-125a, miR-125b, miR-135a, miR-99a) in the t(4;14) group. Although MM samples with t(11;14) were not clustered together, the supervised analysis identified two upregulated miRNAs (miRNA-345 and miRNA-193a) compared to those MM cases without this translocation. None of the differentially expressed miRNA was located in the chromosomal regions involved in the specific translocations. Upon comparing the miRNA expression in cases with or withouth RB deletion it was found that the three most differentially expressed miRNA were miR-145, miRNA-378 and let-7b (downregulated in MM with RB deletion). There was no correlation between the expression level of miRNAs located on chromosome 13 and the RB status by FISH. In the same way, the miRNAs differentially expressed in MM with 1q gains were not located in 1q. In summary, these results indicate that miRNA expression is deregulated in myeloma cells, and what is more important, the miRNA pattern of expression in MM seems to be associated with specific genetic abnormalities, which indicate that they may play a relevant role in MM pathogenesis.

Beschreibung: Acute promyelocytic leukaemia (APL) is characterised by the t(15;17) leading to the fusion of the PML gene with the retinoic acid receptor a (RARa) gene. APL is associated with favourable prognosis, however, approximately 15–20% of patients ultimately relapse. With regards to pre-treatment characteristics, is now clear that long-established prognostic factors such as age and leukocyte count have a major impact on outcome. However, none of these parameters is robust enough, in order to adjust treatments strategies according to the risk of relapse. The quantification of PML-RARa fusion gene transcripts (leukemia-specific chimeric mRNA) can add important prognostic information useful for risk group stratification. However, few clinical studies have been carried out and there are discrepancies concerning the prognostic significance of the quantification of fusion gene transcripts of newly diagnosed patients. In this study we tested the PML- RARa fusion gene transcripts level in 126 newly diagnosed patients with t (15;17) in order to determine their correlation with disease characteristics at presentation and to identify a subset of patients at high risk of relapse. The presence of the PML- RARa fusion gene transcripts was analysed from 1mg of RNA by real-time quantitative RT-PCR (RQ-PCR) based on TaqMan probes and the 7700 Sequence Detector, performed according to the “Europe Against Cancer Program” (Leukemia2003, 17:2323; 2318–2357). Results: In 121 out of 126 patients, evaluable data were obtained: 73 (60,3%) cases had bcr-1, 4 (3,3%) cases had bcr-2 and 44 (36,4%) cases had bcr-3 fusion types. The expression of the fusion gene was reported as the PML- RARa normalized quantities (NQ, number of PML- RARa copies per 1x104 ABL copies as gene control). The median of NQ was 3030 and the expression was highly variable (range of 826 to 9605). The NQ was not significantly associated with disease characteristic at diagnosis, such as age, percentage of blasts cells, fusion types and platelet counts. Nevertheless, patients with NQ above 3000 had lower white cell counts (7,4±12,1 vs 24,2±39,3x109/L, p=0,03). Therefore, the fusion transcript copy number per leukemic cell was higher in patients with lower leukocyte count. Moreover, in survival analysis, the NQ did not correlate with disease free survival or overall survival. Conclusions: Our data confirms the wide range of differences in expression activity between individual patients. The NQ did not correlate with the clinical and biological disease characteristics except for white cell counts. Neither did it correlate with principal prognostic parameters (response and survival).

Beschreibung: Abstract 2554 Introduction: The Acute Myeloid Leukemias of Normal Karyotype (AML-NK) are a subgroup of hematologic malignancies with high heterogeneity, having some of them genetic markers with prognostic value. There is little information about the potential influence of different genetic markers in AML-NK on the immunophenotype of blast cells, particularly of immature populations. Objective: The aim of the present study is to analyze the potential influence of the presence or absence of different genetic markers (FLT3-ITD, mutNPM1) and gene expression levels of BAALC, ERG, EVI1, MN1, PRAME, FOXO3A and WT1) on the immunophenotype of AML-NK, focusing on the phenotypic characteristics of immature blast cells (CD34+ cell compartment). Material and methods: We included in the study 57 cases of de novo AML-NK confirmed by conventional cytogenetics and absence of known molecular alterations by FISH (chromosomes 5,7,8, and 11q23 probes), in which extensive molecular studies as well as a large panel of four colour multiparametric flow cytometry immunophenotype (CD34, CD45, CD19, cyCD79a, nTdT, cyMPO, cyCD3, CD7, CD2, CD56, CD65, 7.1, CD11b, CD13, HLADR, CD117, CD61, CD33, CD123, CD36, CD64, CD14, CD71, Glycophorin A, CD15, CD16) were performed at diagnosis. The CD34 and CD45 antigens were included in all four colour combinations. In 15/57 patients the FLT3 gene and 28/57 cases the NPM1 were mutated. The fold change in expression of the target genes relative to an internal control gene (ABL) was expressed as: Fold Change (RQ): = 2(–ΔCt sample) / 2(–ΔCt calibrator), where the DCt (Ct target gene – Ct ABL) of each patient sample is compared with a calibrator sample (mean expression of five bone marrow from healthy donors), using the DDCt method. The median gene expression was 1.228, 0.392, 3.253 and 0.758 for BAALC, FOXO3A, MN1 and ERG and the 75th percentile was 0.361, 33.87 and 259.77 for EVI1, PRAME and WT1 respectively. The threshold to define positivity for an antigen was set at 20% of blasts population analyzed. In addition, the complete immunophenotype was analyzed on the CD34+ blast cells when we identified a CD34+ blast cell subpopulation greater than 3% of the total blast cells, in order to include the minor subpopulations. The software SPSS v19 was used for the statistical analysis. Results: Upon comparing FLT3-ITD+ vs FLT3-ITD- patients, no differences were observed in the overall incidence of CD34+ and/or CD117+ cases. Nevertheless, if we considered cases with small CD34+ subsets (3%–20% of CD34+ blast cells) the frequency was higher among FLT3-ITD+ patients (40% vs 4%). Upon analyzing the phenotypic profile of the CD34+ cells according to FLT3-ITD status, in patients positive for this marker the CD34+ cells more frequently expressed CD7 (p=0,02) and CD36 (p=0,04). The same comparison was done according to NPM1 status (mutated vs non mutated). The incidence of CD34+ cases was significantly lower while the number of CD117+/CD34- blasts was significantly higher in the mutated subgroup. No differences were observed in the phenotypic profile of the CD34+ cells. For BAALC and MN1 expression, patients were divided according to the median value into two categories: high and low expression. In both markers, the high expression was associated with increased frequency of CD34+ cases (75 vs 17%, p=0,00) and (71 vs 21%, p= 0,01), respectively. The phenotypic profile of CD34+ cells did not differed between high and low groups. Patients with high ERG expression showed a tendency to be associated with increased frequency of CD34+ cells, but differences did not reached statistical significance (p=0,08). Of note, CD34+ cells expressed cMPO less frequently among the ERG high patients. We have no detected differences in the expression of blasts according to EVI1 or FOXO3A expression. Interestingly, in cases with low level of expression of these two markers, the CD34+ blast cell compartment more frequently expressed CD7 (p= 0,01 and p= 0,02, respectively). Finally, PRAME and WT1 expression were not related with the CD34 expression neither with the phenotypic profile of the CD34+ blasts. However, high WT1 was associated with a significantly higher number of CD117+/CD34- cells (p

Beschreibung: MM patients are living longer with increasingly effective therapies, but long-term complications including second primary malignancies (SPMs) are becoming new challenges in designing optimal patient care. It has been demonstrated in large studies that amongst others, risk is particularly high for SPMs such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Importantly, such increased risk of MDS/AML has also been observed in MGUS patients, suggesting that increased risk for MDS/AML may not only be treatment related but inheritably high in MGUS/MM. Thus, there is need to investigate for biomarkers that uncover cellular alterations predisposing for higher risk of MDS/AML in MM. Here, we started by investigating in 312 newly diagnosed MM patients the presence of MDS-like phenotypic abnormalities (MDS-PA) in bone marrow (BM) neutrophil, monocytic, and erythroid lineages, using multidimensional flow cytometry 8 color combinations (CD138, CD27, CD38, CD56, CD45, CD19, CD117, CD81; and HLADR, CD45, CD36, CD13, CD34, CD117, CD11b, CD71). Up to 33/312 (11%) patients showed MDS-PA at diagnosis, which were more frequently observed in the neutrophil lineage (6%), followed by monocytic (4%) and erythroid (4%) lineages. Four cases had multilineage MDS-PA. Afterwards, we investigated if the presence of MDS-PA was associated with underlying somatic mutations by performing targeted sequencing of 54 MDS/AML related genes (depth ≥500x) in 44 patients from the previous series (10 with MDS-PA and 34 without). Next generation sequencing was performed, at diagnosis and after HDT/ASCT in FACS sorted CD34+ hematopoietic stem cells (HSCs) and dysplastic cell lineages from patients with MDS-PA, as well as in HSC from cases without MDS-PA. CD138+ BM plasma cells (PCs) from both cohorts were also sequenced using the same panel. Six out of the 10 cases with MDS-PA showed somatic mutations. Namely, HSCs from one patient had two mutations in TET2 [allele fraction (AF): 18%, ≥ 26017x] one in CALR (AF: 14%, 1158x) and another in ASXL1 (AF: 7%, 1339x). None of these mutations were present in myeloid/erythroid cells. A second patient had NPM1 mutated in HSCs (AF: 7%, 12825x), which was absent in neutrophils. A third case had TET2 mutated in HSCs (AF: 16%, 1233x) as well as in dysplastic monocytes (AF: 27%, 16647x) and neutrophils (AF: 23%, 21719x). In the fourth case, a mutation in BCORL1 was noted in dysplastic erythroid cells (AF: 10%, 796x). The fifth patient had TET2 mutated in both HSCs and dysplastic monocytes (AF: 45%-63%; ≥21799x). The sixth case had PHF6 mutated in HSCs (AF: 8%; 800x). In none of the patients were the mutations found in HSCs and/or dysplastic lineages, present in PCs. Within the control cohort of the 34 patients without MDS-PA, only two of them displayed somatic mutations in HSCs; one case had DNMT3A mutated (AF: 26%, 1900x) and the other TET2 (AF: 13%, 3400x). After demonstrating a correlation between MDS-PA and MDS/AML-related somatic mutations, we sought to analyze the prognostic significance of such alterations in MM. Since the follow-up of the present series of 312 cases is relatively short, we focused on a large series of 965 patients with longer follow up (median of 6.5 years) enrolled in GEM clinical trials, and for which the presence of CD56+ aberrant monocytes could be readily investigated. Noteworthy, this particular MDS-PA was again observed in a similar frequency as noted above (n=63; 6.5%) and as compared to the overall MM population, patients with MDS-PA showed significantly higher age, lower hemoglobin values and higher BMPC infiltration at diagnosis. Furthermore, they experienced more frequently hematological toxicity including anemia and neutropenia during treatment. Most interestingly, as compared to the overall MM population, patients with MDS-PA had significantly inferior progression-free (medians of 24 vs 37 months; P=.006) and overall survival (medians of 47 vs 73 months; P=.01). In conclusion, we showed for the first time that a fraction of newly diagnosed MM patients harbors MDS/AML-related somatic mutations in HSCs and myeloid/erythroid lineages, and that such patients could be predicted through flow-based screening for MDS-PA. The presence of MDS-PA identifies a subset of patients that experience more frequently hematological toxicity and display inferior survival; accordingly, screening for MDS-PA could become an important biomarker to tailor treatment in MM. Disclosures Paiva: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding. Oriol:Amgen: Honoraria, Other: Expert board committee; Janssen: Honoraria, Other: Expert board committee. Mateos:Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria.

Beschreibung: Introduction The Wilms' tumor 1 (WT1) gene, located on chromosome 11p13, encodes a transcription factor with both oncogene and tumor suppressor functions. WT1 is reportedly overexpressed in 90% of patients with acute myeloid leukemia (AML) and thus can be used for minimal residual disease (MRD) monitoring by quantitative RT-PCR. The aim of the present study was to analyze the usefulness of WT1 as a marker for MRD in AML after chemotherapy and as a predictor of relapse and survival. Patients and Methods This retrospective and multicentric study included 114 patients with WT1-overexpressed AML (Table 1). Quantitative assessment of WT1 transcript levels was performed by quantitative RT-PCR in 283 bone marrow (BM) samples at diagnosis, post-induction and post-consolidation. WT1 gene expression was calculated by relative quantification using the normalized ratio of the target gene (WT1) related to a reference gene (GUS) and using cell line K562 as calibrator. Inter-laboratories methodological standardization was accomplished through a pilot study with 10 BM donor samples, 20 BM patient samples and commercial WT1 plasmids (ProfileQuant Kit, Ipsogen-Qiagen). Results No significant differences in WT1 gene expression (cycle threshold, Ct) were observed between different laboratories in the pilot study. The cut-off value of WT1 over-expression in BM was established in 0.55% (median+2SD values from healthy donors). Median WT1 expression in patient samples at diagnosis was 29.5% (range, 2-1220). Differential expression at diagnosis was not correlated with age, sex, leukocytes, karyotype (risk), however a higher expression in patients with AML-M1 and AML-M2 subtypes as well as patients with mutant NPM1 and/or ITD-FLT3 was observed. Most patients (88.6%; 101/114) received intensive chemotherapy as induction treatment. After induction, 80.2% (81/101) of patients had available WT1 data, of which 23.45% (19/81) were positive. In addition, 79.8% (91/114) received intensive treatment during consolidation. WT1 results were available for 75.5% (66/91), of which 22.7% (15/66) were positive. Post-induction WT1 positivity was correlated with a higher cumulative incidence of relapse (CIR; 2 years 76% vs. 28.2% p=0.002) and a lower overall survival (OS; 2 years, 44.9% vs. 78.2% p=0.022; Figure 1a,b). Similar results were obtained when patients intensified with allogeneic stem cell transplantation (allo-SCT) were excluded from the analysis: higher CIR (2 years 88.9% vs. 32.5%; p=0.005) and lower OS (40% vs. 76.2% p=0.17). Post-consolidation WT1 positivity was correlated with a trend to a higher CIR (2 years 60.3% vs. 41.4% p=0.21) and a lower OS (2 years 44.9% vs. 66% p=0.09; Figure 1c,d). Statistically significant results were obtained after consolidation when patients treated with allo-SCT were excluded from the analysis: higher CIR (2 years 100% vs. 40.1% p=0.005) and lower OS (2 years 20% vs. 66% p=0.003). Conclusions WT1 is a useful marker for MRD in AML patients undergoing chemotherapy (induction and consolidation) which allows anticipation of relapse and survival. Post-induction results were a strong risk factor of relapse and survival in all patients, including those intensified with allo-SCT. By contrast post-consolidation results are especially relevant in the group of patients not treated with allo-SCT. Intensification with allo-SCT overcomes the poor prognosis derived from positive post-consolidation WT1 results. Paper presented on behalf of the Hematological Molecular Biology Group (GBMH) of the Spanish Society of Hematology (SEHH). Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 1620 Poster Board I-646 Background So far, none of the high-density microarray studies in acute myeloid leukemia (AML) have provided a useful approach with relevant diagnostic and prognostic value for the routine clinical practice. We aimed to assess the clinical utility of a small combination of genes and fusion genes included in a microfluidic card by means of real-time quantitative PCR (RQ-PCR) in AML patients. Patients and methods Ninety-six clinically relevant markers were analyzed simultaneously using TaqMan® Low Density Arrays (TLDAs) (Applied Biosystems, Foster City, CA). This plataform includes: 3 control genes (ABL1, GADPH and GUS), 36 AML specific fusion transcripts described, NPM1 mutations and 55 genes which RNA expression levels have been associated with prognosis or pathogenic process in AML. To assess the feasibility of the designed TLDA we evaluated the gene expression profile of 191 leukemia patients at diagnosis: 167 AML (inv(16): n=11, t(8;21): n=9, t(15;17): n=7, with 11q23 abnormalities: n=27, with other cytogenetic abnormalities: n=20 and normal karyotype n=93), 16 pre-B-ALL, 5 T-ALL and 3 CML with rare BCR-ABL fusions (b2a3, b3a3 and e19a2). Results We observed a high correlation between TLDA results for fusion transcripts and those obtained by gold standard techniques (RQ-PCR based on Europe against Cancer protocols). Indeed, we identified 89 patients with different fusion transcripts, including 18 cases previously reported as negative by cytogenetics or FISH approach. Patients with a favorable karyotype showed a distinctive gene expression profile. Thus, inv(16) AML overexpressed: MYH11, CLIP3, CTNNB1, SPARC, SNAI1 and MN1; t(8;21) AML expressed high levels of ETO, POU4F1, CAV1, CD34, FOXO3A, PRAME and BAALC, and t(15;17) AML showed upregulation of HGF, FGF13, WT1 and PRAME. Further, the 51 NPM1 mutated patients (38 A, 5 B, 6 D and 2 DD1, confirmed by sequencing) also showed a specific gene profile, showing high NPM1, CD34, ABCB1, ABCG2, BAALC, PROM1 or BCL2 RNA levels and low HOXA7, HOXA9 and MEIS1 RNA levels. In contrast, AML with 11q23 abnormalities or with FLT3-ITD mutations did not exhibit a characteristic pattern. Several genes (EVI1, BAALC, ERG, PRAME or PIM1) showed prognostic significance in AML with normal karyotype and AML with NPM1 mutations, thus confirming TLDA as a useful approach for risk assessment in AML. Interestingly, PIM1 overexpression was an independent predictor for shorter relapse-free (RFS) and overall survival (OS) in patients with normal karyotype (p=0.012 and p

Beschreibung: Introduction: Detection of disparity in microsatellite DNA regions (STR - Short Tandem Repeats) between recipient and donor allows for sensitive and specific monitorization of the degree of haematopoietic chimerism. It is well known that disparities between donor and recipient in various polymorphic systems (mainly MHC) are associated with an increased incidence of graft-versus-host disease (GVHD). However, it is still unknown whether or not STR disparities could have a similar biological effect. Aim: To study the relationship between STR disparities and frequency of GVHD, overall survival and event free survival in patients who have received allogeneic transplantation. Patients: 161 consecutive patients transplanted with peripheral blood stem cells from identical MHC sibling donor at a single center were included in the study. Their characteristics were: median age 44 (17–69); Male/Female: 94/67; Sex disparity: 46%; Diagnosis: 39 AML, 26 ALL, 24 MDS, 19 MM, 17 CML, 14 NHL, 10 CLL, 10 HD, 1 CMPD,U, and 1 Hypereosinophilic Syndrome. The conditioning regimen was reduced intensity in 81 patients and myeloablative in the remainly 80 pts. All 161 patients engrafted and were evaluable for acute GVHD (aGVHD), while 128 were included in the analysis of chronic GVHD (cGVHD), according to the available follow-up. Methods: After genomic DNA extraction, PowerPlex®16 System kit (Promega Corporation, Madison, WI) was used to amplify 16 STR regions (15 plus gender marker, Amilogenin). The amplified products were analysed using GeneScan 2.1 (Applied Biosystems, Foster City, CA) after electrophoresis in the ABIPrism 377 (Applied Biosystems). The chi-square and y t-Student tests were used for statistical analysis. Log-rank analysis was applied for comparing differences in survival. Multivariate analysis was carried out according to the cox-regression method. Results: The number of STR disparities between recipient and donor ranged from 4 to 15 (median: 9). Discordances in D13S317, D18S51 and TPOX were associated with higher grades of aGVHD severity (p=0.024, p=0.027 and p=0.034, respectively). Disparities in D16S539 was associated with cGVHD (p=0.043). The number of loci discrepancies was not related to any clinical parameter included in the analysis (aGVHD, cGVHD, EFS y OS). However, when patients were grouped according to STR mismatches (11, n=127 and 17, respectively), shorter OS was associated in patients with 〉11 disparities (p=0.021). Conclusions: The presence of STR disparities could be associated with the development of complications during sibling allogeneic transplantation, including presentation of aGVHD. The data available only shows a marginal association and must be considered as preliminary.

Beschreibung: Introduction: The Wilms’ tumor 1 (WT1) gene, located on chromosome 11p13, encodes a transcription factor. WT1 is overexpressed in 90% of patients with acute myeloid leukemia (AML) and thus can be used for minimal residual disease (MRD) monitoring by quantitative RT-PCR. The aim of the present study was to analyze the usefulness of WT1 as a marker for MRD quantification in AML after chemotherapy. Patients and Methods: This retrospective and multicentric study included 148 patients with WT1-overexpressed AML. Quantitative assessment of WT1 transcript levels was performed by quantitative RT-PCR in 370 bone marrow (BM) samples at diagnosis (148), post-induction (125) and post-consolidation (97). WT1 gene expression was calculated by relative quantification using the normalized ratio of the target gene (WT1) related to a reference gene (GUS) and using cell line K562 as calibrator. Inter-laboratories methodological standardization was accomplished through a pilot study with 20 BM donor samples, 20 BM patient samples and commercial WT1 plasmids (ProfileQuant Kit, Ipsogen-Qiagen). Results: No significant differences in WT1 gene expression (cycle threshold, Ct) were observed between different laboratories in the pilot study (kappa coefficient 〉0.7). The cut-off value of WT1 over-expression in BM was established for each laboratory (median+2SD values from healthy donors, data not shown). Median WT1 expression in patient samples at diagnosis was 38% (range, 2-850). Differential expression at diagnosis did not correlate with age, gender, leukocytes, karyotype (risk). However a significantly higher expression in patients with AML-M1 and AML-M2 subtypes as well as patients with mutant NPM1 and/or ITD-FLT3 was observed (p