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The B-cell compartment in which multiple myeloma stem cells reside remains unclear. We investigated the potential presence of mature, surface-membrane immunoglobulin-positive B lymphocytes clonally related to the tumor bone marrow plasma cells among different subsets of peripheral blood B cells from ten patients (7 with multiple myeloma and 3 with monoclonal gammopathies of undetermined significance). The presence of clonotypic immunoglobulin heavy chain gene rearrangements was determined in multiple highly-purified fractions of peripheral blood B-lymphocytes including surface-membrane IgM+ CD27− naïve B-lymphocytes, plus surface-membrane IgG+, IgA+ and IgM+ memory CD27+ B cells, and normal circulating plasma cells, in addition to (mono)clonal plasma cells, by a highly-specific and sensitive allele-specific oligonucleotide polymerase chain reaction directed to the CDR3 sequence of the rearranged IGH gene of tumor plasma cells from individual patients. Our results showed systematic absence of clonotypic rearrangements in all the different B-cell subsets analyzed, including M-component isotype-matched memory B-lymphocytes, at frequencies

Background Internal tandem duplications of the FLT3 gene (FLT3-ITDs) are frequent in patients with acute promyelocytic leukemia (APL), however its clinical impact remains controversial.Design and Methods We analyzed the prognostic significance of FLT3-ITD mutant level and size, as well as FLT3-D835 point mutations, PML-RARα expression and other predictive factors in 129 APL patients at diagnosis enrolled on the Spanish LPA96 (n=43) or LPA99 (n=86) PETHEMA trials.Results FLT3-ITDs and D835 mutations were detected in 21% and 9% of patients, respectively. Patients with increased ITD mutant/wild-type ratio or longer ITD size displayed shorter 5-year relapse-free survival (RFS) (P=0.048 and P

Background The gene for preferentially expressed antigen of melanoma (PRAME) has been shown to be over-expressed in acute promyelocytic leukemia, but its actual incidence and clinical impact are still unknown.Design and Methods We studied PRAME expression at diagnosis using real-time quantitative polymerase chain reaction in 125 patients with acute promyelocytic leukemia enrolled in the Spanish PETHEMA-96 (n=45) and PETHEMA-99 (n=80) clinical trials. In addition, PRAME expression was evaluated as a marker of disease activity in 225 follow-up samples from 67 patients with acute promyelocytic leukemia.Results At diagnosis, PRAME expression in patients with acute promyelocytic leukemia was significantly higher (p100-fold PRAME expression (86% vs. 74%; p=0.03), and this cut-off established two sub-groups with different relapse-free survival rates among patients with a white cell count 109/L, although differences were not statistically significant. In multivariate analysis, white cell count >109/L (p90% (p=0.001), and PRAME expression 10-fold increase in PRAME expression levels.Conclusions Low PRAME expression defines a subgroup of patients with acute promyelocytic leukemia with a short relapse-free survival. This marker could be useful as a secondary marker for monitoring patients with acute promyelocytic leukemia.

Background and Objectives Waldenström macroglobulinemia (WM) and monoclonal gammopathy of undetermined significance (MGUS) are IgM-related disorders in which monoclonal B cells harbor a unique clonotypic rearrangement of the immunoglobulin heavy chain gene (IgH). The aim of this study was to characterize IgH rearrangements in a larger series of IgM-related disorders than any previously described.Design and Methods Seventy-two patients with monoclonal IgM disorders (64 with WM and eight with IgM-MGUS) were studied to amplify and sequence both VDJH and DJH rearrangements. Twenty-nine of them were also tested for the existence of class switch recombination (CSR).Results VDJH and DJH rearrangements were detected in 91% and 42% of WM patients and in 100% and 13% of IgM-MGUS patients, respectively. In WM, the most frequently observed VH family and single segment were VH3 and VH3-23 (76% and 29%, respectively), with their frequencies differing markedly from those that would occur if the rearrangements were random. The VH3-23 segment was never selected in IgM-MGUS. The distribution of both DH and JH families in WM did not differ from that in normal B-lymphocytes. Somatic hypermutation with >2% deviation was seen in 90% of cases of WM and in 71% of IgM-MGUS. DJH rearrangements were more frequent in WM than in MGUS (42% and 13%, respectively). All DJH rearrangements were unmutated, which makes them an attractive target for minimal residual disease investigation. IgM clonotypic transcripts were observed in all cases and IgD in 83%. IgA and/or IgG monoclonal isotypes were seen in three WM cases (14%) but in none of the IgM-MGUS patients.Interpretation and Conclusions WM and IgM-MGUS exhibit dissimilarities in VDJH and DJH rearrangements that could suggest different differentiation processes. There is evidence that WM cells are able to undergo CSR in vivo, a fact that was initially thought to be impossible in this disease.

Background and Objectives The detection of PML-RARα by real-time polymerase chain reaction (RQ-PCR) is becoming an important tool for monitoring minimal residual disease (MRD) in patients with acute promyelocytic leukemia (APL). However, its clinical value remains to be determined. Our aim was to analyze any associations between the risk of relapse and RQ-PCR results in different phases of treatment, comparing these data with those yielded by conventional qualitative reverse transcriptase-PCR.Design and Methods Follow-up samples from 145 APL patients treated with the PETHEMA protocols were evaluated by the RQ-PCR protocol (Europe Against Cancer program) and by the RT-PCR method (BIOMED-1 Concerted Action). Hematologic and molecular relapses and relapse-free survival were recorded. We then looked for associations between relapse risk and RQ-PCR results.Results After induction therapy, no association was found between positive RQ-PCR results and relapse. The PCR result here did not imply any change in the scheduled therapy. After the third consolidation course, two out of three cases with positive RQ-PCR relapsed in contrast to 16 out of 119 (13%) patients with negative RQ-PCR. During maintenance therapy and out-of treatment, all patients with >10 PML-RARα normalized copy number (NCN) (n=19) relapsed while all patients with

The B-cell compartment in which multiple myeloma stem cells reside remains unclear. We investigated the potential presence of mature, surface-membrane immunoglobulin-positive B lymphocytes clonally related to the tumor bone marrow plasma cells among different subsets of peripheral blood B cells from ten patients (7 with multiple myeloma and 3 with monoclonal gammopathies of undetermined significance). The presence of clonotypic immunoglobulin heavy chain gene rearrangements was determined in multiple highly-purified fractions of peripheral blood B-lymphocytes including surface-membrane IgM(+) CD27(-) naïve B-lymphocytes, plus surface-membrane IgG(+), IgA(+) and IgM(+) memory CD27(+) B cells, and normal circulating plasma cells, in addition to (mono)clonal plasma cells, by a highly-specific and sensitive allele-specific oligonucleotide polymerase chain reaction directed to the CDR3 sequence of the rearranged IGH gene of tumor plasma cells from individual patients. Our results showed systematic absence of clonotypic rearrangements in all the different B-cell subsets analyzed, including M-component isotype-matched memory B-lymphocytes, at frequencies <0.03 cells/μL (range: 0.0003-0.08 cells/μL); the only exception were the myeloma plasma cells detected and purified from the peripheral blood of four of the seven myeloma patients. These results indicate that circulating B cells from patients with multiple myeloma and monoclonal gammopathies of undetermined significance are usually devoid of clonotypic B cells while the presence of immunophenotypically aberrant myeloma plasma cells in peripheral blood of myeloma patients is a relatively frequent finding.

p14/p16 and p15 gene expression was assessed by quantitative polymerase chain reaction in purified plasma cells (PC) from 52 patients with symptomatic multiple myeloma (MM) and seven with smoldering MM in order to clarify the impact of these genes on the proliferative activity of tumor cells and patients' outcome. p15 expression was lower in symptomatic MM than in smoldering SMM (-1.80 vs.1.51,p=0.026); similar results were observed for p14/p16. MM patients whose PC displayed high p15 and/or p14/p16 expression had a lower percentage of S-phase PC than the remaining cases (1.79%+/-1.35 vs. 3.04%+/-1.42, p=0.028), favorable prognostic factors and longer survival (100% vs. 49%at 2.5 years; p=0.007).

Background and Objectives: B-cell chronic lymphoproliferative disorders (B-CLPD) are usually monoclonal expansions of a single B-cell clone. However in some cases, two unrelated B-cell clones co-exist. Additionally, cases with two B-cell subpopulations displaying a similar phenotype but distinct DNA contents exist, the exact nature of these cases remaining unknown. In order to gain insight into the characteristics of these complex B-CLPD we examined two cohorts of patients., Material and Methods: One cohort had two phenotypically distinct B-cell populations (group A; n= 9) and the other, two B-cell subsets showing different DNA contents and/or light scatter properties, but a similar immunophenotype (group B; n= 7)., Results: Fluorescent in situ hybridization studies revealed the presence of genetic abnormalities in six cases from group A, either in one (n=5) or the two co-existing B-cell populations (n=1); in all these cases the two B-cell populations showed unrelated IgH gene rearrangements. In all seven cases from group B, the B-cell population showing higher DNA contents had additional chromosomal abnormalities as compared to the other subset; molecular analysis confirmed the monoclonal nature of these cases., Interpretation and Conclusions: In summary, we show that in group A, two phenotypically/cytogenetically distinct, unrelated B-cell clones co-exist, while the two B-cell populations from group B appear to represent different stages of evolution of a single clone.

Background and Objectives: Analysis of IgH rearrangements in B-cell malignancies has provided clinical researchers with a wide range of information during the last few years. However, only a few studies have contributed to the characterization of these features in multiple myeloma (MM), and they have been focused on the analysis of the expressed IgH allele only. Comparison between the expressed and the non-functional IgH alleles allows further characterizion of the selection processes to which pre-myeloma cells are submitted., Design and Methods: We analyzed a cohort of 84 untreated MM patients in order to characterize their functional VDJH and non-functional DJH rearrangements. The pattern of mutations and gene segment usage for both types of rearrangements was analyzed by polymerase chain reaction and sequencing., Results: VH3 and VH1 family members were over- and under-represented, respectively. VH3-30 and VH3-15 segments were the most frequently used, whereas VH4-34 was found only in non-functional or heavily mutated VDJH rearrangements. DH2 and DH3 family members were over-represented in both VDJH and DJH repertoires, while the DH1 family was under-represented only in the productive VDJH rearrangements. Finally, DH3-22 and DH2-21 gene segments were found to be over-represented in the functional repertoire while segments commonly used by less mature B-cell malignancies, such as DH6-19 or DH3-3, were under-represented., Interpretation and Conclusions: Data reported here help to identify the clonogenic MM cell as a post-germinal center B cell that has undergone selection processes during the germinal center reaction.

Background and Objectives: The exact immunophenotypic criteria for the identification of Sézary cells in the blood are still poorly defined., Design and Methods: We analyzed the immunophenotype and DNA cell content of blood T cells in a series of 18 consecutive cases of Sézary's syndrome (SS), 21 normal individuals and 10 patients with reactive erythroderma, and correlated them with molecular and morphological findings., Results: Phenotypically abnormal CD3+/TCRalphabeta+/CD4+ T cells were found in all SS patients but in none of the reactive erythroderma cases; small diploid, or less frequently hypodiploid Sézary's cells coexisted with large nearly tetraploid Sézary's cells in some cases. The most frequent phenotypic aberrations consisted in decreased expression of CD3/TCRalphabeta (94%), CD4 (94%), CD7 (100%) and/or CD2 (83%). In addition, Sézary's cells were constantly CD28+ and CD5+ and they did not express natural-killer associated (NKa) antigens. Phenotypic heterogeneity was a common finding and phenotypic changes over time were frequently observed. In contrast to what was found in patients with reactive erythroderma, flow cytometry analysis of the T-cell receptor (TCR) repertoire revealed a major TCR-Vbeta expansion in all SS cases., Interpretation and Conclusions: The presence of CD28+/CD5+/NKa-/CD4+ T cells expressing abnormally low levels of CD3, TCRalphabeta, CD4, CD7 and/or CD2 would support the diagnosis of SS in patients with erythroderma. Further analyses on larger series of patients are necessary in order to cover less frequent phenotypic patterns, establish the preferential usage of specific TCR-Vb families and investigate the specificity of these phenotypic abnormalities for diagnosing SS.

Background and Objectives: Molecular analysis has contributed to the identification of several non-random chromosomal translocations, such as t(15;17), t(8:21), inv(16)/t(16;16) and 11q23 abnormalities, typically associated with acute myeloid leukemia (AML). The identification of these chromosomal abnormalities helps not only to define different AML subtypes with distinct prognoses and treatments but also to monitor the disappearance of malignant cells after treatment. Recent reports suggest that the frequency of these alterations may differ according to geographic distribution. However, most of these reports focus on just one or two genetic alterations, which may lead to some selection bias. Appropriate epidemiological studies should be based on unselected consecutive series of patients in which all relevant genes are simultaneously analyzed. The aim of the present study was to explore whether or not the incidence of genetic lesions in Spanish AML patients differs from that reported in other countries., Design and Methods: In a series of 145 consecutive un-selected adult patients with AML we simultaneously analyzed the presence of 4 genetic abnormalities, PML/RARalpha for t(15;17), AML1/ETO for t(8;21), CBFbeta/MYH11 for inv(16)/t(16;16) and rearrangements of the MLL gene for 11q23 abnormalities. AML were classified using the new World Health Organization (WHO) classification for hematologic malignancies. The techniques used were standardized according to the recommendations of the European BIOMED-1 Concerted Action., Results: The PML/RARalpha transcript was present in 34 patients (23.4%) (23 were bcr1, 2 bcr2 and 9 bcr3). The AML1/ETO fusion transcript was detected in only 2 cases (1.4%) both with M2 morphology, but 29 other cases with M2 morphology were negative. CBFbeta/MYH11 transcript was present in 9 cases (6.2%) eight of them displaying M4Eo morphology. Finally, 5 cases (3.5%) showed rearrangements of theMLL gene. Our results differ from those reported from the United States and North/Central Europe, particularly regarding the incidence of t(15;17) and t(8;21) translocations. In Spain the frequency of t(15;17) is higher while that of t(8;21) is lower., Interpretation and Conclusions: These data add epidemiological information about geographic heterogeneity of such chromosome aberrations in AML and would contribute to the design of specific screening strategies adapted to the incidence in each country.

Background and Objective: Molecular analysis by PCR of monoclonally rearranged immunoglobulin (Ig) genes can be used for diagnosis in B-cell lymphoproliferative disorders (LPD), as well as for monitoring minimal residual disease (MRD) after treatment. This technique has the risk of false-positive results due to the "background" amplification of similar rearrangements derived from polyclonal B-cells. This problem can be resolved in advance by additional analyses that discern between polyclonal and monoclonal PCR products, such as the heteroduplex analysis. A second problem is that PCR frequently fails to amplify the junction regions, mainly due to somatic mutations frequently present in mature (post-follicular) B-cell lymphoproliferations. The use of additional targets (e.g. Ig light chain genes) can avoid this problem., Design and Methods: We studied the specificity of heteroduplex PCR analysis of several Ig junction regions to detect monoclonal products in samples from 84 MM patients and 24 patients with B cell polyclonal disorders., Results: Using two distinct VH consensus primers (FR3 and FR2) in combination with one JH primer, 79% of the MM displayed monoclonal products. The percentage of positive cases was increased by amplification of the Vlamda-Jlamda junction regions or kappa(de) rearrangements, using two or five pairs of consensus primers, respectively. After including these targets in the heteroduplex PCR analysis, 93% of MM cases displayed monoclonal products. None of the polyclonal samples analyzed resulted in monoclonal products. Dilution experiments showed that monoclonal rearrangements could be detected with a sensitivity of at least 10(-2) in a background with >30% polyclonal B-cells, the sensitivity increasing up to 10(-3) when the polyclonal background was <1% of polyclonal B-cells., Interpretation and Conclusions: Heteroduplex analysis of PCR amplified products is a simple and quick alternative for detecting monoclonally rearranged Ig genes in MM. This can be applied for diagnosis of B cell LPD and as a previous step in MRD strategies.