Posttransplant monomorphic Burkitt's lymphoma: clinical characteristics and outcome of a multicenter series.

Burkitt's monomorphic posttransplant lymphoproliferative disorder (B-PTLD) is an uncommon subtype of PTLD. Owing to the paucity of this complication, clinical characteristics and outcome has not been fully described. Clinical characteristics and outcomes of 20 patients diagnosed with B-PTLD from 10 transplant centers belonging to the GEL/TAMO group were reviewed. Median time from transplant to B-PTLD was 7.2 years. All the cases fulfill the morphologic and genetic criteria of B-PTLD, whereas Epstein-Barr virus (EBV) was detected in 70% of cases. Patients were treated with different chemotherapy combinations, and three patients received upfront rituximab monotherapy. The great majority of patients receiving CHOP-like regimens attained a complete response (CR) (73%), similar to that obtained with dose-intensive chemotherapy (83% CR). In contrast, patients receiving upfront rituximab monotherapy required subsequent chemotherapy. Two patients (10%) died during treatment due to infection. The median progression-free survival and overall survival (OS) were 16 months and 139 months, respectively. When analyzing variables predicting for OS, we found that patients with bone marrow involvement had an adverse prognosis, with a median OS of 6 months (p = 0.008). In conclusion, B-PTLD is an uncommon complication usually associated with EBV infection and with an aggressive clinical course, particularly in patients with bone marrow involvement. High-dose chemoimmunotherapy obtained similar responses to R-CHOP, suggesting that R-CHOP could be an adequate alternative for these patients. In contrast, rituximab monotherapy does not seem to be effective enough to control the disease.

Quantitative PCR: an alternative approach to detect common copy number alterations in multiple myeloma.

Chromosome 1q gains and 13q deletions are common cytogenetic aberrations in multiple myeloma (MM) that confer a poor prognosis. There are several techniques for the targeted study of these alterations, but interphase fluorescence in situ hybridization (FISH) is the current gold standard. The aim of the present study was to validate quantitative PCR (qPCR) as an alternative to FISH studies in CD138+-enriched plasma cells (PCs) from MM patients at diagnosis. We analyzed 1q gains and 13q deletions by qPCR in 57 and 60 MM patients, respectively. qPCR applicability was 84 and 88% for 1q and 13q, respectively. The qPCR and FISH methods had a sensitivity and specificity of 88 and 71% for 1q gains, and 79 and 100% for 13q deletions. A second qPCR assay for each region was carried out to confirm the previous results. Paired qPCR (two assays) and FISH results were available from 53 MM patients: 26 for 1q amplification and 27 for 13q deletion. qPCR assays gave concordant results (qPCR-consistent) in 20 of the 26 (77%) 1q gains and 25 of the 27 (93%) 13q deletions. Considering only the consistent data, the overall concordance among qPCR and FISH was 85 and 100% for 1q gains and 13q deletions, respectively. Our results show a substantial agreement between qPCR and the gold standard FISH technique, indicating the potential of qPCR as an alternative approach, particularly when the starting material is too scarce or cells are too damaged to obtain accurate results from FISH studies.

Extranodal and nodal diffuse large B cell lymphoma of the head and neck: two different entities?

This study analyzes patients with head and neck diffuse large B cell lymphoma (HN-DLBCL), focusing on the differences in the biological characteristics and prognosis of lymphomas of nodal and extranodal origin. We have included 72 patients with stage I-II HN-DLBCL who had updated survival information and diagnostic paraffin-embedded tissue blocks available for review. Non-germinal center phenotype (73.7 vs. 32.4 %, P = 0.001) and high level of Bcl-2 expression (78.9 vs. 52.9 %, P = 0.025) were more frequent in nodal than extranodal lymphomas. Univariate analyses indicated that bulky disease, Ann Arbor stage II, high level of Ki-67 expression, and primary nodal disease had adverse effects on complete remission (CR), but these effects were confirmed in a multivariate analysis for primary nodal disease and bulky disease. Patients with primary extranodal lymphoma also had better overall survival (OS) (87.7 vs. 72.5 %, P = 0.04) and event-free survival (EFS) (84 vs. 58.5 %, P = 0.046) than patients with nodal disease, although in the multivariate analysis, only Ann Arbor stage II continued to predict worse OS and EFS, whereas bulky disease was an independent prognostic factor only for EFS. We found significant differences in the biological characteristics and prognosis between primary nodal and extranodal HN-DLBCL.

Frequency of HLA-A, -B and -DRB1 specificities and haplotypic associations in the population of Castilla y León (northwest-central Spain).

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 León 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 León 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.

Primary cutaneous T-cell lymphoproliferative disorder of donor origin after allogeneic haematopoietic stem-cell transplantation.

A 56-year-old male patient had a history of mantle-cell lymphoma, which was treated with polychemotherapy and reduced-intensity conditioning allogeneic haematopoietic stem cell transplantation (ASCT) from his healthy sister with an identical human leucocyte antigen profile. Six years after transplantation, the patient developed asymptomatic eczema-like cutaneous lesions. Histologically the lesions contained a dense superficial lichenoid infiltrate, mainly consisting of CD4+ atypical medium to large lymphocytes showing indented hyperchromatic nuclei. In situ hybridization for Epstein-Barr virus was negative. PCR amplification of the T-cell receptor-gamma chain gene from several lesions revealed a monoclonal rearrangement without clonal variation. Two-colour fluorescence in situ hybridization (X and Y chromosomes) and microsatellite genotyping were used to compare samples from the patient (transplant recipient), his sister (donor) and the skin biopsy sample, which confirmed that the origin of the neoplastic cells was the donor graft. To our knowledge, this is the first case of post-transplant primary cutaneous T-cell lymphoproliferative disorder after ASCT.

Low expression of ZHX2, but not RCBTB2 or RAN, is associated with poor outcome in multiple myeloma.

RAN, ZHX2 and RCBTB2 (CHC1L) expression was evaluated by quantitative real time reverse transcription polymerase chain reaction in plasma cells from 85 monoclonal gammopathies: 58 symptomatic multiple myeloma (MM) (52 untreated, six relapsed), eight smouldering MM, five monoclonal gammopathy of undetermined significance, four plasma cell leukaemias and 10 myeloid cell lines. ZHX2 was weakly expressed in high-risk/proliferative disease compared to low-risk or indolent disease. High ZHX2 expression was associated with better response and longer survival after high-dose therapy. RCBTB2 expression was weaker in hyperdiploid versus non-hyperdiploid cases while RAN was more expressed in symptomatic MM and cell lines.

Clinical impact of the subclonal architecture and mutational complexity in chronic lymphocytic leukemia.

Genome studies of chronic lymphocytic leukemia (CLL) have revealed the remarkable subclonal heterogeneity of the tumors, but the clinical implications of this phenomenon are not well known. We assessed the mutational status of 28 CLL driver genes by deep-targeted next-generation sequencing and copy number alterations (CNA) in 406 previously untreated patients and 48 sequential samples. We detected small subclonal mutations (0.6-25% of cells) in nearly all genes (26/28), and they were the sole alteration in 22% of the mutated cases. CNA tended to be acquired early in the evolution of the disease and remained stable, whereas the mutational heterogeneity increased in a subset of tumors. The prognostic impact of different genes was related to the size of the mutated clone. Combining mutations and CNA, we observed that the accumulation of driver alterations (mutational complexity) gradually shortened the time to first treatment independently of the clonal architecture, IGHV status and Binet stage. Conversely, the overall survival was associated with the increasing subclonal diversity of the tumors but it was related to the age of patients, IGHV and TP53 status of the tumors. In conclusion, our study reveals that both the mutational complexity and subclonal diversity influence the evolution of CLL.

Integrating genomic alterations in diffuse large B-cell lymphoma identifies new relevant pathways and potential therapeutic targets.

Genome studies of diffuse large B-cell lymphoma (DLBCL) have revealed a large number of somatic mutations and structural alterations. However, the clinical significance of these alterations is still not well defined. In this study, we have integrated the analysis of targeted next-generation sequencing of 106 genes and genomic copy number alterations (CNA) in 150 DLBCL. The clinically significant findings were validated in an independent cohort of 111 patients. Germinal center B-cell and activated B-cell DLBCL had a differential profile of mutations, altered pathogenic pathways and CNA. Mutations in genes of the NOTCH pathway and tumor suppressor genes (TP53/CDKN2A), but not individual genes, conferred an unfavorable prognosis, confirmed in the independent validation cohort. A gene expression profiling analysis showed that tumors with NOTCH pathway mutations had a significant modulation of downstream target genes, emphasizing the relevance of this pathway in DLBCL. An in silico drug discovery analysis recognized 69 (46%) cases carrying at least one genomic alteration considered a potential target of drug response according to early clinical trials or preclinical assays in DLBCL or other lymphomas. In conclusion, this study identifies relevant pathways and mutated genes in DLBCL and recognizes potential targets for new intervention strategies.

From Waldenström's macroglobulinemia to aggressive diffuse large B-cell lymphoma: a whole-exome analysis of abnormalities leading to transformation.

Transformation of Waldenström's macroglobulinemia (WM) to diffuse large B-cell lymphoma (DLBCL) occurs in up to 10% of patients and is associated with an adverse outcome. Here we performed the first whole-exome sequencing study of WM patients who evolved to DLBCL and report the genetic alterations that may drive this process. Our results demonstrate that transformation depends on the frequency and specificity of acquired variants, rather than on the duration of its evolution. We did not find a common pattern of mutations at diagnosis or transformation; however, there were certain abnormalities that were present in a high proportion of clonal tumor cells and conserved during this transition, suggesting that they have a key role as early drivers. In addition, recurrent mutations gained in some genes at transformation (for example, PIM1, FRYL and HNF1B) represent cooperating events in the selection of the clones responsible for disease progression. Detailed comparison reveals the gene abnormalities at diagnosis and transformation to be consistent with a branching model of evolution. Finally, the frequent mutation observed in the CD79B gene in this specific subset of patients implies that it is a potential biomarker predicting transformation in WM.

Critical evaluation of ASO RQ-PCR for minimal residual disease evaluation in multiple myeloma. A comparative analysis with flow cytometry.

We have analyzed the applicability, sensitivity and prognostic value of allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) as a method for minimal residual disease (MRD) assessment in patients with multiple myeloma (MM), comparing the results with those of multiparameter flow cytometry (MFC). A total of 170 patients enrolled in three consecutive Spanish trials achieving at least partial response after treatment were included. Lack of clonality detection (n=31), unsuccessful sequencing (n=17) and suboptimal ASO performance (n=51) limited the applicability of PCR to 42% of cases. MRD was finally investigated in 103 patients (including 32 previously studied) with persistent disease identified by PCR and MFC in 54% and 46% of cases, respectively. A significant correlation in MRD quantitation by both the techniques was noted (r=0.881, P<0.001), being reflective of treatment intensity. Patients with <10(-4) residual tumor cells showed longer progression-free survival (PFS) compared with the rest (not reached (NR) vs 31 months, P=0.002), with similar results observed with MFC. Among complete responders (n=62), PCR discriminated two risk groups with different PFS (49 vs 26 months, P=0.001) and overall survival (NR vs 60 months, P=0.008). Thus, although less applicable than MFC, ASO RQ-PCR is a powerful technique to assess treatment efficacy and risk stratification in MM.

Mutation status and immunoglobulin gene rearrangements in patients from northwest and central region of Spain with chronic lymphocytic leukemia.

The aim of this study was to investigate the frequency and mutation status of the immunoglobulin heavy variable chain (IGHV) in a cohort of 224 patients from northwest and central region of Spain diagnosed with chronic lymphocytic leukemia (CLL), and to correlate it with cytogenetic abnormalities, overall survival (OS) and time to first treatment (TTFT). 125 patients had mutated IGHV, while 99 had unmutated IGHV. The most frequently used IGHV family was IGHV3, followed by IGHV1 and IGHV4. The regions IGHV3-30, IGHV1-69, IGHV3-23, and IGHV4-34 were the most commonly used. Only 3.1% of the patients belonged to the subfamily IGHV3-21 and we failed to demonstrate a worse clinical outcome in this subgroup. The IGHV4 family appeared more frequently with mutated pattern, similar to IGHV3-23 and IGHV3-74. By contrast, IGHV1-69 was expressed at a higher frequency in unmutated CLL patients. All the cases from IGHV3-11 and almost all from IGHV5-51 subfamily belonged to the group of unmutated CLL.

MYD88 L265P is a marker highly characteristic of, but not restricted to, Waldenström's macroglobulinemia.

We evaluated the MYD88 L265P mutation in Waldenström's macroglobulinemia (WM) and B-cell lymphoproliferative disorders by specific polymerase chain reaction (PCR) (sensitivity ∼10(-3)). No mutation was seen in normal donors, while it was present in 101/117 (86%) WM patients, 27/31 (87%) IgM monoclonal gammapathies of uncertain significance (MGUS), 3/14 (21%) splenic marginal zone lymphomas and 9/48 (19%) non-germinal center (GC) diffuse large B-cell lymphomas (DLBCLs). The mutation was absent in all 28 GC-DLBCLs, 13 DLBCLs not subclassified, 35 hairy cell leukemias, 39 chronic lymphocytic leukemias (16 with M-component), 25 IgA or IgG-MGUS, 24 multiple myeloma (3 with an IgM isotype), 6 amyloidosis, 9 lymphoplasmacytic lymphomas and 1 IgM-related neuropathy. Among WM and IgM-MGUS, MYD88 L265P mutation was associated with some differences in clinical and biological characteristics, although usually minor; wild-type MYD88 cases had smaller M-component (1.77 vs 2.72 g/dl, P=0.022), more lymphocytosis (24 vs 5%, P=0.006), higher lactate dehydrogenase level (371 vs 265 UI/L, P=0.002), atypical immunophenotype (CD23-CD27+ +FMC7+ +), less Immunoglobulin Heavy Chain Variable gene (IGHV) somatic hypermutation (57 vs 97%, P=0.012) and less IGHV3-23 gene selection (9 vs 27%, P=0.014). These small differences did not lead to different time to first therapy, response to treatment or progression-free or overall survival.

Prognostic significance of FLT3 mutational status and expression levels in MLL-AF4+ and MLL-germline acute lymphoblastic leukemia.

There is barely any information about the prognostic significance of FLT3 expression and mutational status in cytogenetically distinct subgroups of acute lymphoblastic leukemia (ALL). We analyzed the presence of FLT3-tyrosine kinase domain (TKD) and FLT3-internal tandem duplication (ITD) mutations as well as FLT3 expression levels in 54 newly diagnosed patients with B-ALL (n=49) or T-ALL (n=5). All B/T-ALL samples tested negative for the presence of FLT3-TKD or FLT3-ITD. None of the T-ALL and E2A-PBX1+ B-ALL overexpressed FLT3. In contrast, mainly MLL-AF4+ B-ALL but also ETV6-RUNX1+, BCR-ABL+ or B-ALL displaying normal cytogenetics exhibited significantly higher FLT3 expression levels than normal bone marrow, supporting that aberrantly increased transcription of FLT3, rather than activating FLT3 mutations, contributes to the pathogenesis of these B-ALL. Using the median FLT3 expression as cut-off value we found that high-level FLT3 expression is associated with an extremely poor 1-year overall survival (OS; 0 vs 71%; P=0.002) and disease-free survival (DFS; 0 vs 43%; P=0.03) in MLL-AF4+ B-ALL but not in MLL-germline B-ALL. Cox regression analysis with OS/DFS as end points showed that age>14 years and high-level FLT3 expression were independent prognostic factors when all ALL patients were analyzed together. Importantly, when the MLL-AF4+ B-ALL subgroup was analyzed separately, high-level FLT3 expression was the only independent prognostic factor for OS and treatment outcome. These findings indicate that high FLT3 expression identifies MLL-AF4+ ALL patients at very high risk of treatment failure and poor survival, emphasizing the value of ongoing/future clinical trials for FLT3 inhibitors.

The EuroChimerism concept for a standardized approach to chimerism analysis after allogeneic stem cell transplantation.

Hematopoietic stem cell transplantation is becoming an increasingly important approach to treatment of different malignant and non-malignant disorders. There is thus growing demand for diagnostic assays permitting the surveillance of donor/recipient chimerism posttransplant. Current techniques are heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from 10 European countries have therefore performed a collaborative study supported by a European grant, the EuroChimerism Concerted Action, with the aim to develop a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. Following extensive analysis of a large set of microsatellite/short tandem repeat (STR) loci, the EuroChimerism (EUC) panel comprising 13 STR markers was established with the aim to optimally meet the specific requirements of quantitative chimerism analysis. Based on highly stringent selection criteria, the EUC panel provides multiple informative markers in any transplant setting. The standardized STR-PCR tests permit detection of donor- or recipient-derived cells at a sensitivity ranging between 0.8 and 1.6%. Moreover, the EUC assay facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells. Wide use of the European-harmonized protocol for chimerism analysis presented will provide a basis for optimal diagnostic support and timely treatment decisions.

Standardization of DNA isolation from low cell numbers for chimerism analysis by PCR of short tandem repeats.

Analysis of short tandem repeats (STR) by PCR analysis is routinely used in chimerism diagnostics to monitor donor engraftment and to diagnose relapse. Some applications require chimerism analysis of low cell numbers, but no standardized protocol is available for DNA isolation from 1000 to 30,000 cells. The EU-supported EuroChimerism Consortium (project QLRT-2001-01485) selected four different protocols for 'small-scale' DNA isolation, which were tested by six laboratories for their ability to recover reproducible amounts of good quality DNA, suited for PCR-based STR analysis. The protocols included two direct lysis methods with and without detergents and proteinase K, and two commercial column-based kits. The direct lysis method using detergents and proteinase K showed the highest DNA recovery and the best performance in the multiplex PowerPlex16 STR assay. DNA isolated with this method also showed the highest sensitivity in chimerism analysis using singleplex PCR reactions of EuroChimerism STR markers. Sensitivity was reached ranging from 1 to 20% of recipient cells in a donor background. In conclusion, the direct lysis method using detergents and proteinase K is a standardized DNA isolation method well suited for chimerism studies on low cell numbers.

The presence of DRB1*01 allele in multiple myeloma patients is associated with an indolent disease.

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.