ALBERT

Description: 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.

Description: 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.

Description: 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.

Description: Gene Expression Profiling through RNA arrays has provided new clues to Multiple Myeloma pathogenesis and prognostic pattern evaluation. Recently, ZHX2, CHC1L & RAN expression have been highlighted as key elements in MM. In the present paper, we have evaluated theses genes by real time quantitative RT-PCR (RQ-PCR) in purified plasma cells from 74 patients with plasma cell discrasias. MATERIAL AND METHODS: Purified Bone Marrow cells were obtained from the following patients: 6 MGUS, 7 smoldering MM, 59 newly diagnosed symptomatic MM patients and 2 Plasma cell leukemia (PCL). After RNA extraction, RQ-PCR of CHC1L(C), RAN(R) and ZHX2 (Z) genes was carried out using the standard protocol from TaqMan® gene expression Assays-on-Demand in an ABI-PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Expression levels were normalized with ABL gene and expressed in n-fold times compared to the expression in a pool of RNA from mononuclear cells from healthy donors. The expression level of the different genes was evaluated for correlation with the diagnosis, clinical characteristics and prognosis of the patients. RESULT AND CONCLUSIONS: None of these genes displayed a clear relationship with the different stages of disease pathogenesis, although ZHX2 gene was slightly more expressed in the indolent forms of the proliferative disorders (MGUS and SMM). Within symptomatic MM patients, several interesting associations were observed. Thus, in hyperdiploid MM cases, CHC1L expressions observed were fewer than in those with a normal DNA index, confirming the participation of the gene product in chromosomal condensation during the mitosis. No other important associations were observed for this gene, although patients with the lowest expression displayed a very good prognosis, but without reaching statistically significant differences. As expected, RAN expression was related to S-Phase PC, since patients with high S phase values (〉1.8 %) displayed higher levels of RAN transcripts. This, however, only resulted in a marginal impact on survival. ZHX2 provided the most interesting results, whereby decreased levels of ZHX2 were related to unfavorable prognostic indicators such as B2 microglobulin 〉4 mg/L and Hemoglobin levels

Description: 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.