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A halotolerant phenylacetate-degrading fungus Penicillium CLONA2, previously isolated from a salt mine at Algarve (Portugal), was identified as a variant of P. chrysogenum using the ITS-5,8S rDNA and the D1/D2 domain of 28S rDNA sequences. The metabolic features and genetic characteristics suggest that this strain belongs to a subgroup of P. chrysogenum, named var. halophenolicum. The presence of the penicillin biosynthetic cluster was proven by Southern hybridizations using probes internal to the pcbAB and penDE genes and sequencing of the pcbAB-pcbC intergenic region. However the pcbAB-pcbC divergent promoter region contained 20 point modifications with respect to that of the wild type P. chrysogenum NRRL1951. The CLONA2 strain produced non-aromatic natural penicillins rather than benzylpenicillin in a medium containing potassium phenylacetate (the precursor of benzylpenicillin) and was able to grow well on phenylacetatic acid using it as sole carbon source. Due to the ability of P. chrysogenum CLONA2 to degrade aromatic compounds, this strain may be an interesting organism for aromatic compounds remediation in high salinity environments.

The human leukocyte antigen (HLA) system could play an essential role in multiple myeloma (MM) disease control. This report describes the results comparing HLA-DRB1 phenotypic frequencies in 181 MM patients (53 smoldering/indolent MM and 128 symptomatic MM patients) and healthy individuals. Higher DRB1*01 phenotypic frequencies were found in the smoldering patients compared with symptomatic MM patients (38% vs 14%, P = 0.001) and with the healthy individuals (38% vs 22%, P = 0.01). Additionally, higher DRB1*07 phenotypic frequencies were found in symptomatic MM compared with control population (38% vs 28%, P = 0.01). The present data suggest that HLA-DRB1*01 individuals may have a better ability to efficiently present myeloma-related antigens to immunocompetent cells, which could favor a better immune response against the tumor. This would translate into a more appropriate disease control associated with more indolent disease and prolonged survival.

The frequencies of human leukocyte antigen (HLA) class I and class II specificities and haplotypic associations were determined in 1940 unrelated donors from Castilla y Leon and compared with other Iberian, Mediterranean and European populations. Specificities were determined using polymerase chain reaction reverse sequence-specific oligonucleotide or polymerase chain reaction sequence-specific primer techniques. In the analysis, 19, 29 and 13 specificities were found for HLA-A, -B and -DRB1, respectively, with HLA-A*02 (26%), -A*01 (11%), -B*44 (16%), -B*35 (10%), -DRB1*07 (16%) and -DRB1*13 (14%) showing the highest frequencies. In addition, 10 common HLA-A-B-DRB1 haplotypic associations were observed, A*01-B*08-DRB1*03 (3%) and A*29-B*44-DRB1*07 (3%) being the most frequent ones. These findings indicate that the population of Castilla y Leon is genetically equidistant from the Portuguese and other Spanish populations and shares a common origin with other Iberian populations, in which European, Mediterranean and North African genetic components are present; this is in agreement with the historical and genetic background of the population. These data contribute to a better understanding of the genetic structure of the Iberian Peninsula and provide a healthy control population from our region that should be useful for the study of disease associations.

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.

INTRODUCTION: Several studies have reported the presence of expanded T-cell clones in patients with multiple myeloma (MM), which could be involved in an anti-tumour response and extended survival. The Human Leukocyte Antigen (HLA) system seems to play an essential role in MM control and could influence disease control. This feature has been poorly studied and there are only few data favouring higher incidence of some HLA specifities such as B18 and B5 in myeloma patients. AIM: To compare HLA-DRB1 phenotypic frequencies in smoldering MM versus symptomatic MM patients and control individuals. PATIENTS: A total of 181 patients with a diagnosis of MM were analysed. According to their behaviour patients were classified into two subsets: 128 symptomatic MM who were homogeneously treated according to the GEM-2000 protocol (Spanish Myeloma Group/PETHEMA protocol) and 53 patients with the diagnosis of smoldering MM according to the criteria of the International Myeloma Working Group and who were free of therapy for at least 1 year following diagnosis. Additionally, 1818 healthy donor individuals from the Castilla y Leon registry for hematopoietic stem cell-transplantation were included as control population. All three populations involved Caucasian individuals. METHODS: After genomic DNA extraction, HLA-DRB1 typing at low-resolution level (two digits) was carried out using the PCR-rSSO methodology according to the standards of the European Federation of Immunogenetics. Allele frequencies were estimated by direct counting. Comparisons of allele and phenotype frequencies between populations were performed with the two-sided Fisher’s exact test using GraphPad Prism 4.0 Software. The strength of associations was estimated by the odds ratio (OR), and their 95% confidence intervals (CI) were calculated by Cornfield methods (values of p < 0.05 were considered statistically significant). P-value was corrected (Pc) for the number of valid comparisons made (Bonferroni correction). RESULTS: DRB1 phenotypic frequencies were not significantly different among MM patients and healthy control individual. In contrast, when the two MM cohorts were analyzed, DRB1*01 phenotypic frequencies were significantly higher in the smoldering patients as compared to symptomatic MM patients (37.7% vs. 14.1, p=0.0011, Pc=0.0143, OR: 3.7, 95% CI: 1.76–7.81). Furthermore, DRB1*01 phenotypic frequencies were significantly higher in the smoldering patients as compared to the healthy control individuals (37.7% vs. 21.7%, p=0.0106, Pc>0.05, OR: 2.19, 95% CI: 1.24–3.86). In addition, symptomatic MM patients showed a higher incidence in DRB1*07 phenotypic frequencies as compared to control population (38.3% vs. 27.6%, p=0.0111, Pc>0.05, OR: 1.63, 95% CI: 1.12–2.36). CONCLUSIONS: The present data suggest that HLA-DRB1*01 phenotype is associated with indolent MM and this may reflect a better ability to efficiently present myeloma-related antigens to immunocompetent cells.

Waldenström Macroglobulinemia (WM) is characterized by monoclonal IgM paraprotein and bone marrow (BM) infiltration by lymphoplasmacytic lymphoma. The normal counterpart of WM malignant cell seems to be a post germinal centre IgM B-cell, which tumoral transformation occurs after cessation of somatic mutation (SM) but prior to Class switch recombination (CSR). However, recently has been reported that CSR can be possible “ex-vivo”, since clonotypic transcripts encoding post-switch isotypes have been observed in some WM cells cultured with CD40L/IL-40. However, this process has not been shown to occur “in vivo” until now. #3754, a 51-year-old woman, was diagnosed in 2001 of WM with a M-IgM spike (46 g/L), anemia (Hb 9·6 g/dL), 76% lymphoplasmacytic monoclonal B-cells in BM and normal cytogenetics. In April 2005, an important IgG increase was observed (23 g/L). Immunofixation demonstrated an IgG-k paraprotein in the mid g-region and a monoclonal IgM-k paraprotein at the b-region corresponding to the two monoclonal peaks detected on serum electrophoresis. After 6-mercaptoethanol treatment, a single band was seen at the line stained with kappa, suggesting the presence of a single clone. Other causes of IgG monoclonal components were excluded considering clinical factors, immunophenotype analyses (San Miguel et al, 2003), quantity of DNA and cell cycle analyses (Ocio et al, 2005). However, the definitive proof for a unique monoclonal population was provided through molecular analysis. A single clonotypic rearrangement was detected by amplifying the complete VDJH fragment at diagnosis moment, according to the protocol describes in Biomed II (Leukemia2003; 17.2257–2317). Method describes from Billadeu et al (Billadeau et al, 1993) was used for isotype identification. So, cDNA monoclonal amplification was observed at tubes corresponding to Cm, Cd and Cg. All monoclonal PCR products were directly sequenced in an automated ABI 377 DNA sequencer. VH, DH & JH segments identification, as well as SH recognition was made using the V-BASE sequence directory alignment program, and the CH regions were compared at BLAST. All sequences obtained showed the same clonotypic CDR3 sequence (VH4-59/JH6) as well as the same SH (10,75%) pattern that monoclonal amplification at diagnosis, indicating the presence of the same clone at that moment (Figure 1). In conclusion, we report for the first time a WM case in which tumor cells were able to carry out CSR, showing IgG and IgM clonotypic amplification, as well as producing both paraprotein components. This constitutes the first in vivo demonstration that CSR is possible in WM cells, and are able to develop a fully functional isotype class switch recombination not only in vitro but also in vivo. Figure 1: Deduced amino acid sequence of tumor-derived VDJH gene with the three heavy chain isotypes (A: Cμ, B: Cδ C: Cγ). Sequences indicates the somatic mulation pattern. Comparison for WM are made with the closest germline VH gone; uppercase, replacement (R) mutation; lower case, silent (S) mutation. Each mutation was defined by nuclieotide exchanges in a single codon, with successive mutations leading in some cases to 2 or 3 distinct R or S events. These are shown as aligned amino acid changes at specific sites. Figure 1:. Deduced amino acid sequence of tumor-derived VDJH gene with the three heavy chain isotypes (A: Cμ, B: Cδ C: Cγ). Sequences indicates the somatic mulation pattern. Comparison for WM are made with the closest germline VH gone; uppercase, replacement (R) mutation; lower case, silent (S) mutation. Each mutation was defined by nuclieotide exchanges in a single codon, with successive mutations leading in some cases to 2 or 3 distinct R or S events. These are shown as aligned amino acid changes at specific sites.

The detection of PML-RARa by real-time PCR (RQ-PCR) is becoming an important tool for monitoring minimal residual disease (MRD) in acute promyelocytic leukemia (APL) patients; however, the clinical value of using a RQ-PCR approach remains to be determined. Our aim was to analyze the correlation between relapse risk and MRD levels assessed by RQ-PCR at different phases of treatment, and to compare these data with conventional qualitative RT-PCR. Follow-up samples from 145 APL patients treated with the PETHEMA protocols were analyzed by RQ-PCR protocol from Europe Against Cancer program and by RT-PCR method from European BIOMED-1 Concerted Action. After induction therapy, no association was found between the presence of a RQ-PCR positive result and relapse. After the third consolidation course, three cases remained positive by RQ-PCR assays and two of them (67%) relapsed. By contrast, only 16 out of 119 (13%) patients with negative RQ-PCR relapsed. During maintenance therapy and out-of treatment, a significant correlation between a RQ-PCR positive result and a poor relapse-free survival was observed (p < 0.0001). All patients with > 10 PML-RARa normalized number copies (NCN) at maintenance or out-of therapy relapsed (n=19), while all patients with < 1 NCN within out-of treatment group remained in hematological remission (n=75). 8 patients with < 1 NCN during maintenance therapy finally relapsed, but all of them appeared beyond the end of treatment. In addition, in the intermediate group of patients (NCN 1–10) (n=18) relapse probability of 40% at 5 years was observed. All hematological relapses were predicted if a follow-up sample had been obtained within the previous 4 months. Based on the information provided by RQ-PCR in samples obtained after the end of consolidation and subsequently, a relapse risk stratification could be established for APL patients.

p15 and p14/p16 tumor suppressor genes, have been reported to be frequently inactivated by various mechanisms in haematological malignancies such us MM. Alterations of these cell cycle inhibitors in MM display a close correlation with the cell cycle and clinical outcome. We have evaluated by real time quantitative RT-PCR (RQ-PCR) the expression of the p14/p16 and p15 genes in purified bone marrow plasma cells (PBMPC) from MM patients in order to evaluate the possible clinical, biological and prognostic significance of these cell cycle regulators. RNA extracted from purified BMPC from 53 untreated symptomatic MM and a pool of buffy coat from healthy donors (reference value) was analyzed by RQ-PCR using Assays-on-Demand gene expression mixes specific for p14/p16 and p15 genes in an ABI PRISM 7700 SDS (Applied Biosystems, Foster City, CA, USA). Values were corrected with a control gene (ABL). The relative quantification of gene expression was performed through the cycle threshold (CT) increment method. Patients were classified into different groups depending on gene expression values. Thus, according to p15 expression, 29% of patients (n=14) showed higher levels than the control and this group was characterized by the presence of good prognostic markers such us low Lactato dehidrogenase levels (LDH), low b2-microglobulin (B2M) and C-Reactive Protein (CRP) serum levels and absence of monoclonal proteinuria. Similar results were found for p14/p16 expression. Fifteen patients (28%) displayed a high p14/p16 expression and the group was also characterized by good prognostic features: low CRP, B2M and LDH levels. When p14/p16 and p15 genes were simultaneously analyzed, clinical and biological parameters showed a statistically significant correlation with gene expression. Thus patients with low gene expression had a high B2M (≥3 mg/dl) and high C-reactive protein (≥3 mg/dl). As far as survival was concerned, patients with a high p15 expression had a longer overall survival of 100% vs. 58% at 30 months (p=0,01), with the additional value that no deaths have been observed in any such patients. Similar results were observed for the group of patients displaying a high p14/p16 expression since they displayed a much better OS (100% vs. 63% at 30 months, p=0,02). Again, we should note that no deaths have been presented in this group. All these findings were much more evident when the three genes were simultaneously considered. Thus, within the group of 21 patients with at least one of the two genes overexpressed there have been no deaths vs. 11 among the 27 patients with low levels. This resulted in quite different OS curves for the two groups of patients (Figure 1) of 100% vs. 49% at 30 months (p=0,00). In conclusion, these genes significantly determine patients’ outcome thanks to their ability to classify them into different groups with different clinical, biological and outcome characteristics.

Introduction: Waldenström’s Macroglobulinemia (WM) is an uncommon lymphoproliferative disorder characterized by bone marrow infiltration of the lymphoplasmocytic cells and IgM monoclonal gammopathy. The normal counterpart of the WM malignant cell is believed to be a post germinal-center B cell. However, the low frequency of WM has hampered investigation into Immunoglobulin Heavy Chain (IgH) rearrangements in large series of patients and whether preferential use of specific genetic segments or non-functional rearrangements exits. Aim: Molecular characterization in a large cohort of WM patients analyzing their functional, complete VDJH, and non-functional, incomplete DJH, IgH rearrangements, the pattern of somatic hypermutation (SHM) and gene segment usage. Patients and methods: 47 patients with unequivocal diagnosis of WM were included in the study. Bone Marrow samples (always with more than 10% of tumor cell) were used for amplification of clonal rearrangements employing the Biomed-2 strategy (Leukemia2003; 17; 2257), followed by direct automated sequencing in ABI 377 DNA sequencer using Big-Dye terminators (Applied Biosystems, Foster City, CA). Results: All patients showed a monoclonal amplification of at least one VDJH or DJH rearrangement. VDJH monoclonal rearrangements were detected in 42/47 (89.3%) patients while DJH were observed in 20 (42.5%) patients. VH, DH and JH gene segment usage: VH3 gene segment was the most commonly represented family (76,2%) while the other gene segments were scarcely detectable, and VH5 was totally absent. In the complete VDJH rearrangements, DH6 was the family most commonly represented while DH2 (45%) and DH4 (30%) were the most common in the incomplete DJH rearrangement. Regarding JH elements, JH4 (38%) was the most represented in the complete rearrangements and JH5 (46%) in the incomplete rearrangements. Somatic hypermutation: SHM were observed in all except two of the cases (94%, 34 of 36 cases), in which the study could be performed. (median 9,38; range2.9–16.6). In the remaining clonal cases (6 cases, 14.3%) no amplification of FR1 region was obtained, also suggesting the presence of SHM. Conclusions: In this series of patients, we observed the presence of incomplete rearrangements with an incidence similar (44%) to that described in multiple myeloma. The preferential usage for determined families of genetic segments, in VDJH and in DJH, suggests the presence of positive and negative selection processes. Finally, the majority, (94 %) of the cases, presented SHM, although the presence of two patients lacking in SHM suggests a pre-follicular origin for some WM cases.

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.

Introduction: Currently, the origin of the HCL tumor clone is believed to be marginal zone B-cell that has previously contacted the antigen, and has therefore undergone somatic hypermutation (SHM). However, the low frequency of HCL has hamper the investigation of IgH rearrangements in large series of patients and whether preferential uses of specific VDJ segments or non-functional rearrangements exist. Aims: To characterize IgH gene rearrangements in 25 HCL patients in order to ascertain the frequency and characteristics of both incomplete DJH and complete VDJH rearrangements and to determine their somatic mutations. Patients and methods: 25 patients with unequivocal diagnosis of HCL (CD19+CD5-CD22+FMC7+CD23-CD103+) were included in the study. Amplification of clonal rearrangements was carried out using the Biomed-2 strategy (Leukemia2003; 17:2257), followed by direct automated sequencing. Results: All 25 patients displayed a clonal rearrangement, including 21 VDJH rearrangements (84%) and 14 DJH rearrangements (56%). Families VH3 & VH1 were over-represented, while families VH5, VH6 and VH7 were completely absent. The most frequently used VH specific segment was VH3–30 (3/21), by contrast segments used in normal mature B-cells were not found in any of the HLC cases (i.e. VH3–20 & VH3–49). As far as the DH segments is concerned, DH2 family was the most common in both complete and incomplete rearrangements, followed by DH3 in the complete and DH5 in incomplete fusions. Finally, JH6 and JH4 segments were the most frequently observed JH segments in both complete (52 & 42%, respectively) and incomplete (30 & 46%, respectively) rearrangements. Interestingly, JH3 was over-represented in DJH rearrangements three times more frequent than in VDJH rearrangements. Sequence analysis showed that HLC cases displaying a complete VDJH rearrangement were mutated (76%, average deviation to germline sequence of 4.12%). By contrast, none of the 14 cases with incomplete rearrangements showed SHM, (11 exactly matched the germline sequence and three had some deviation, but always below the 2%, which is the consensus cut-off point to define the presence of SHM). Conclusions: Although HCL seems to be a homogeneous disease, molecular analysis of IgH rearrangements of tumor cells shows an important biological heterogeneity. Thus, incomplete rearrangements are frequent, and there is a preferential usage in some VH, DH and JH segments. Furthermore, although the majority of cases seem to have a post-follicular origin, a quarter of them may have originated in cells that have not undergone the SHM process. This is only possible if they have not crossed the germinal center or if their SHM machinery is damaged. These observations suggest that the post-follicular origin (marginal zone B-cell) of HCL should be reviewed.

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).

Multiple myeloma (MM) remains as an incurable disease although new therapies can achieve a high rate of complete remissions (CR). Unfortunately, most patients ultimately relapse due to the persistence of minimal residual disease (MRD), and only a minority could be cured. Detection and quantification of these cells is an important tool for monitoring these patients and predicting a potential relapse. Here we analyze by RQ-PCR the MRD in MM patients achieving CR in order to classify them into different risk categories. MATERIAL AND METHODS: 38 MM patients uniformly treated according to the GEM-2000 (Spanish group for Myeloma) protocol, and that have achieved CR following PBSCT were included in the study. 22 were IgG, 9 IgA, 6 B-J and 1 non-secretory (κ/λ 21/16). 27 were male & 11 female with a median age of 58 (range 48–65). Bone marrow samples obtained at diagnosis and 3 months after transplant were analyzed. Complete (VDJH) and incomplete (DJH) Ig rearrangements were amplified with the Biomed-2 strategy (Leukemia2003;17:2257). PCR clonal products were sequenced on an ABI Prism 377 Sequence detector. VH, DH and JH segments were identified by comparing with germinal sequences on V-Base and BLAST databases. An ASO primer at the N-region was designed for each patient with the OLIGO 6.0 software. RQ-PCR was then performed on an ABI Prism 7700 using the ASO specific forward primer, a JH reverse intronic primer (JH1–6) and a TaqMan probe (JH1,2,4,5, JH3 or JH6) to amplify the patient specific rearrangement. Sample quality and quantity was controlled evaluating the standard curve of the albumin gene amplification. MRD was calculated according to ΔCT method. RESULTS: In 14 out of the cases included in the study, MRD investigation was not possible because the N-region was not longer enough to design the ASO primer (n=3), poor quality in the diagnostic sample to obtain the standard curve (n=8) or low plasma cell infiltration at diagnosis to obtain correct dilutions (n=3). The remaining 24 patients were classified into different risk groups according to the MRD level obtained 3 months after transplantation with a cut-off point of 0.01% tumor cells. Thus, progression free survival (PFS) was longer in those patients with MRD< 10−4 (p=0.03, figure 1A). By contrast, upon comparing the impact on PFS of immofixation (IFX) in these 24 patients that were in CR (defined by conventional electrophoresis criteria), it was observed that patients with IFX (−) didn’t showed a different outcome from those IFX (+) (figure 1B). CONCLUSION: In summary, although RQ-PCR is a labor and time-consuming technique, it is an useful tool for monitoring MRD in MM. The level of 10−4 can contribute to classify patients into 2 groups (high and low MRD) with different risk of relapse that can be used to design specific therapies.