ERIC recommendations on IGHV gene mutational status analysis in chronic lymphocytic leukemia.
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Europe↗
Biomedical subjects
F Davi
Publications and source records attributed to F Davi.
Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 Concerted Action BHM4-CT98-3936.
The diagnosis of malignant lymphoma is a recognized difficult area in histopathology. Therefore, detection of clonality in a suspected lymphoproliferation is a valuable diagnostic criterion. We have developed primer sets for the detection of rearrangements in the B- and T-cell receptor genes as reliable tools for clonality assessment in lymphoproliferations suspected for lymphoma. In this issue of Leukemia, the participants of the BIOMED-2 Concerted Action CT98-3936 report on the validation of the newly developed clonality assays in various disease entities. Clonality was detected in 99% of all B-cell malignancies and in 94% of all T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The combined BIOMED-2 results are summarized in a guideline, which can now be implemented in routine lymphoma diagnostics. The use of this standardized approach in patients with a suspect lymphoproliferation will result in improved diagnosis of malignant lymphoma.
False Negative Reactions↗
Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936.
Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.
Chromosomes, Human, Pair 11↗
Powerful strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of the BIOMED-2 Concerted Action BHM4 CT98-3936.
Polymerase chain reaction (PCR) assessment of clonal T-cell receptor (TCR) and immunoglobulin (Ig) gene rearrangements is an important diagnostic tool in mature T-cell neoplasms. However, lack of standardized primers and PCR protocols has hampered comparability of data in previous clonality studies. To obtain reference values for Ig/TCR rearrangement patterns, 19 European laboratories investigated 188 T-cell malignancies belonging to five World Health Organization-defined entities. The TCR/Ig spectrum of each sample was analyzed in duplicate in two different laboratories using the standardized BIOMED-2 PCR multiplex tubes accompanied by international pathology panel review. TCR clonality was detected in 99% (143/145) of all definite cases of T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, peripheral T-cell lymphoma (unspecified) and angioimmunoblastic T-cell lymphoma (AILT), whereas nine of 43 anaplastic large cell lymphomas did not show clonal TCR rearrangements. Combined use of TCRB and TCRG genes revealed two or more clonal signals in 95% of all TCR clonal cases. Ig clonality was mostly restricted to AILT. Our study indicates that the BIOMED-2 multiplex PCR tubes provide a powerful strategy for clonality assessment in T-cell malignancies assisting the firm diagnosis of T-cell neoplasms. The detected TCR gene rearrangements can also be used as PCR targets for monitoring of minimal residual disease.
Gene Amplification↗
Prospective multicentric molecular study for poor prognosis fusion transcripts at diagnosis in adult B-lineage ALL patients: the LALA 94 experience.
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Adolescent↗
High Mda-7 expression promotes malignant cell survival and p38 MAP kinase activation in chronic lymphocytic leukemia.
Chronic lymphocytic leukemia (CLL)-B-cells are quiescent differentiated cells that produce interleukin (IL)-10 and accumulate due to resistance to apoptosis. The mechanisms underlying such resistance are poorly understood. Herein we show that all CLL B-cells tested (30/30) display high mRNA and protein expression of the tumor suppressor Mda-7/IL-24, an IL-10 family member, in comparison to normal B cells. A downstream Mda-7 signaling target, p38 mitogen-activated protein kinase (MAPK) was highly phosphorylated in all CLL cells but not in normal B-cells. Mda-7 expression and p38 MAPK phosphorylation diminished in culture and the latter could be reinduced by recombinant (r)-IL-24 or LPS and Mda-7 transfection. Mda-7/IL-24 siRNA specifically inhibited p38 MAPK phosphorylation in CLL without affecting p38 MAPK, bcl2, or Lyn expression, further demonstrating the direct role of Mda-7/IL-24 in p38 MAPK activation. Both pharmacological inhibition of p38 MAPK and Mda-7 silencing augmented spontaneous apoptosis by three-fold in CLL cells cultured in autologous serum, which was reversed by LPS and r-IL-24. We established the role of p38 MAPK in CLL cell survival and demonstrated a paradoxical effect, whereby Mda-7 and IL-24, inducers of apoptosis in diverse cancer cells, promote the survival of CLL B-cells through p38 MAPK activation.
Aged↗
Prediction of relapse by day 100 BCR-ABL quantification after allogeneic stem cell transplantation for chronic myeloid leukemia.
Chronic myeloid leukemia (CML) relapse after allogeneic stem cell transplantation (SCT) is a relatively frequent situation, which is correlated to disease status, time from diagnosis to transplant and T-cell depletion. We evaluated the potential for early minimal residual disease (MRD) BCR-ABL quantification to predict relapse of CML patients receiving allogeneic SCT. Minimal residual disease was analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction (RQ-PCR) at day 100 (d100) in 38 patients with >1 year follow-up after conventional non-T-cell-depleted SCT. Normal ABL control values from 1724 follow-up blood samples were used to define an RQ-PCR amplifiability index and the limits of reliable use of BCR-ABL ratios. We then compared the 14 patients with a high-level d100 BCR-ABL/ABL ratio (> or = 10(-4)) to that of the 24 patients with a negative/low-level ratio (<10(-4)). Despite being comparable for all classical parameters, the incidence of relapse was significantly higher in the high MRD group (11/14 (79%)) compared to that of the low/negative MRD group (7/24 (29%)) (P = 0.009), with d100 MRD values representing an independent risk factor of relapse and disease-free survival, but not of overall survival, in multivariate analysis. These data should facilitate risk-adapted post-transplant immunosuppression and/or tyrosine kinase inhibitor therapy based on an early evaluation of MRD.
Adolescent↗
Genetic heterogeneity of the hypervariable region I of Hepatitis C virus and lymphoproliferative disorders.
B-cell lymphoproliferative disorders (BCLD) have been associated with chronic hepatitis C virus (HCV) infection. The HCV glycoprotein E2 (gpE2) hypervariable region I (HVR-I) may be a potential antigenic candidate to promote B-cell proliferation. The purpose of this study was to analyze the influence of HVR-I sequence variability in the development of BCLD. HVR-I sequences were studied in 29 chronically HCV-infected patients with (n=15) or without (n=14) BCLD. After PCR amplification of the gpE2 region, analysis of the 81 bp HVR-I encoding fragment was performed on 7-18 clones per patient. HVR-I sequence complexity was slightly lower in patients with BCLD (mean 0.347) than without (0.468) (P=0.2), though, sequence diversities were similar (0.0370 vs 0.0954, P=0.239). Phylogenetic analysis did not reveal any BCLD-associated clustering. In our population, neither the recently described insertion between positions 1 and 2 of HVR-I nor residues at positions 4 and 13 were particularly linked to BCLD. As previously described, we confirm the high degree of conservation of HVR-I residues T-2, G-6 and G-23 in our patients. Contrary to recent findings, our analysis based on multiple clones per patient analysis did not reveal any particular motif associated with BCLD.
Adult↗
Gene expression profiling of chronic lymphocytic leukemia can discriminate cases with stable disease and mutated Ig genes from those with progressive disease and unmutated Ig genes.
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B-Lymphocytes↗
hTERT expression and prognosis in B-chronic lymphocytic leukemia.
BACKGROUND: In B-chronic lymphocytic leukemia (B-CLL), there is a need for molecular markers to predict the evolution of this heterogeneous disease in individual patients. The level of expression of the human telomerase reverse transcriptase (hTERT) gene has been associated with disease aggressiveness in human cancers. The purpose of the present study was to examine the prognostic significance of hTERT expression in B-CLL. PATIENTS AND METHODS: We used real-time reverse transcription-PCR to quantitate the amount of hTERT transcripts in mononuclear blood cells from 90 B-CLL patients. In addition, samples were analyzed for somatic mutations in the immunoglobulin V (IgV) genes. RESULTS: The expression of hTERT gene was detected in 59% of patients. The level of expression increased with advancing B-CLL stage (P=0.0064). Patients expressing hTERT showed significantly shorter survival than hTERT-negative patients (P=0.000034), irrespective of the disease stage. On average, the level hTERT mRNA expression was seven-fold higher in the poor-prognosis B-CLL group with unmutated IgV than in the Ig-mutated group (P<10(-7)). The level of hTERT expression discriminated the Ig-unmutated from Ig-mutated B-CLL in 89% of cases. CONCLUSION: Our data indicate that hTERT expression in B-CLL may serve as a molecular prognostic marker.
Adult↗
Predictive value of serum thymidine kinase level for Ig-V mutational status in B-CLL.
In B-CLL IgV(H) genes mutational status is a major prognostic factor. Since sequencing of IgV(H) genes is not available in most laboratories, an easily performed surrogate assay is desirable. To identify the best surrogate assay, and to better discriminate prognostic subgroups we analyzed clinical and biological data from 58 typical CLL cases. A higher serum thymidine kinase level (>15 U/l) proved to be a strong predictor of mutational status, and the only independent one among the studied parameters. To further identify prognostic subgroups, cluster analysis was employed on 38 cases on which all data were available, which segregated two groups including 25 and 13 patients, respectively. These two clusters differed by their proliferative potential and appeared to discriminate patients with very different clinical course and outcome. s-TK was strikingly different among these two clusters, suggesting that s-TK level could be used routinely to identify patients at risk of progression.
Aged↗
Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.
In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.
Chromosome Aberrations↗
Agranular CD4+/CD56+ cutaneous neoplasm.
CD4+ CD56+ cutaneous neoplasm with hematological relapse is a rare malignant disease and has been described recently in the literature as blastic or agranular NK-cell leukemia/lymphoma. The origin of this neoplasm is uncertain. We describe a 75-year-old patient with a primary cutaneous neoplasm CD4+ CD56+ who evolved to leukemic phase despite standard lymphoma chemotherapy. Morphologically, the cells were undifferentiated without granules in the cytoplasm. The immunophenotype showed the expression of CD4, CD56, CD68, CD33, CD7, CD2, CD45RA, and CD38. Histological analysis revealed a cell infiltration mainly located in the dermis. T-cell receptor and immunoglobulin heavy chain genes were in germline configuration. Cytogenetic study showed complex structural abnormalities with a deletion of the chromosome 5 del(5q). The clinical course was aggressive with an early hematological relapse.
Aged↗
Identification of prognostic factors in 61 patients with posttransplantation lymphoproliferative disorders.
PURPOSE: Prognostic studies of posttransplantation lymphoproliferative disorders (PTLDs) are hindered by the small number of cases at each transplant center. We analyzed prognostic factors and long-term outcome according to clinical manifestations, pathologic features, and treatment and investigated the prognostic value of the non-Hodgkin's lymphoma International Prognostic Index (IPI) in 61 patients with PTLD. PATIENTS AND METHODS: We studied 61 patients in two institutions who developed PTLD and analyzed factors influencing the complete remission and survival rates. RESULTS: In univariate analysis, factors predictive of failure to achieve complete remission were performance status (PS) > or = (P =.0001) and nondetection of Epstein-Barr virus (EBV) in the tumor (P =.01). Only a negative link with PS > or = 2 was observed in multivariate analysis. In univariate analysis, factors predictive of lower survival were PS > or = 2, the number of sites (one v > one), primary CNS localization, T-cell origin, monoclonality, nondetection of EBV, and treatment with chemotherapy. The IPI failed to identify a patient subgroup with better survival and was less predictive of the response rate than was a specific index using two risk factors (PS and number of involved sites), which defined three groups of patients: low-risk patients whose median survival time has not yet been reached, intermediate-risk patients with a median survival time of 34 months, and high-risk patients with a median survival time of 1 month. CONCLUSION: PS and the number of involved sites defined three risk groups in our population. The value of these prognostic factors needs to be confirmed in larger cohorts of patients treated in prospective multicenter studies.
Adolescent↗
The incidence of clonal T-cell receptor rearrangements in B-cell precursor acute lymphoblastic leukemia varies with age and genotype.
B-cell precursor acute lymphoblastic leukemias (BCP-ALLs) are increasingly treated on risk-adapted protocols based on presenting clinical and biological features. Residual molecular positivity of clonal immunoglobulin (IG) and T-cell receptor (TCR) rearrangements allows detection of patients at an increased risk of relapse. If these rearrangements are to be used for universal follow-up, it is important to determine the extent to which they are informative in different BCP-ALL subsets. We show that IGH V-D-J rearrangements occur in 89% of 163 BCP-ALL, with no significant variation according to age or genotype (BCR-ABL, TEL-AML1, MLL-AF4, and E2A-PBX1). In contrast, TCRG rearrangements, which occur in 60% of patients overall, are frequent in BCR-ABL and TEL-AML1, are less so in MLL-AF4, and are virtually absent in infants aged predominantly from 1 to 2 years and in E2A-PBX1 ALLs. Incidence of the predominant TCRD Vdelta2-Ddelta3 rearrangement decreases with age but is independent of genotype. These differences are not due to differential recombination activating gene activity, nor can they be explained adequately by stage of maturation arrest. Analysis of MLL-AF4 BCP-ALL is in keeping with transformation of a precursor at an early stage of ontogenic development, despite the adult onset of the cases analyzed. We postulate that the complete absence of TCRG rearrangement in E2A-PBX1 cases may result from deregulated E2A function. These data also have practical consequences for the use of TCR clonality for the molecular follow-up of BCP-ALL.
Adolescent↗
Expression of unmutated VH genes is a detrimental prognostic factor in chronic lymphocytic leukemia.
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Female↗
Expression of p16/INK4a in posttransplantation lymphoproliferative disorders.
It was recently demonstrated that classification of posttransplantation lymphoproliferative disorders (PT-LPDs) into morphological and molecular categories is clinically relevant. It was also reported that PT-LPD not associated with Epstein-Barr virus (EBV) had a more aggressive course than most lesions associated with EBV. Because the cyclin-dependent kinase inhibitor p16/INK4a has been reported to be frequently inactivated in high-grade lymphomas, we evaluated 17 PT-LPD to determine whether p16/INK4a expression could be correlated to morphology, EBV detection, and a Ki-67 labeling index. We demonstrated that tumors with no p16/INK4a expression (n = 8) had a predominantly monomorphic appearance, and most were EBV negative (respectively, 7/8 and 5/8), whereas lesions with p16/INK4a expression (n = 9) were mostly polymorphic PT-LPD (6/9) (P = 0.049) and associated with EBV (9/9) (P = 0.015). In particular, strong p16/INK4a expression was observed in atypical immunoblasts and Reed-Sternberg-like cells. Furthermore, the proliferation index was significantly higher in tumors lacking p16/INK4a expression than in other lesions (P = 0.0008). In conclusion, down-regulation of p16/INK4a was mostly observed in PT-LPD lesions known to follow more aggressive courses: monomorphic tumors and EBV-negative PT-neoplasms. Conversely, overexpression of p16/INK4a was associated with EBV-positive PT-LPD. While p16/INK4a might play a role in the proliferative rate of LP-LPD, further investigations are needed to assess the clinical relevance of p16/INK4a expression in predicting the evolution of tumors and to explain how EBV could favor p16/INK4a protein accumulation in lesions.
Adult↗
Epstein-Barr virus and gastric carcinoma in Western patients: comparison of pathological parameters and p53 expression in EBV-positive and negative tumours.
AIMS: The presence of Epstein-Barr virus (EBV) was studied in 56 gastric carcinomas from Western patients by in-situ hybridization for EBV-encoded RNAs (EBER). EBV-positive and negative carcinomas were compared for various pathological parameters including p53 overexpression. METHODS AND RESULTS: EBERs transcripts were detected in seven cases overall: four cases of 52 conventional carcinomas (7. 7%) and three cases of four gastric carcinomas with lymphoid stroma (75%). EBER positivity was diffuse in five cases and restricted to a localized area of the tumour in two cases of conventional carcinoma. A monoclonal EBV genomic pattern was demonstrated in the case tested by Southern blot analysis. By immunohistochemical analysis, neither EBV latent or lytic cycle proteins nor C3d/EBV receptor were expressed by neoplastic cells. EBER positivity was significantly correlated with prominent lymphoid reaction (P = 0.0002) which was associated with numerous PS100-positive dendritic cells and with HLA-DR expression by tumour cells (P = 0.03). p53 immunoreactivity in more than 30% of tumour cells was detected in 25 out 49 EBV-negative cases and was absent in EBV-positive cases except in one case with focal EBER-positivity. CONCLUSIONS: Focal staining for EBER is an unusual finding in the setting of gastric carcinoma and these results suggest that there might be two types of EBV-associated gastric carcinoma in which the viral infection will play a different role. The presence of a stromal lymphoid reaction which is strongly correlated with EBV positivity, is associated with antigen-presenting ability by HLA-DR-positive tumour cells or abundant dendritic cells. The function of p53 appears preserved in all EBV-associated carcinomas except in one case with focal EBER expression whereas the immunohistochemical pattern of p53 is suggestive of a mutational phenomenon in 51% of EBV-negative cases.
Female↗