Ana Rasillo

Increased frequency (12%) of circulating chronic lymphocytic leukemia-like B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach.

Monoclonal B-cell lymphocytosis (MBL) indicates the presence of less than 5 x 10(9)/L circulating monoclonal B cells in otherwise healthy subjects. Recently, it has been reported that circulating chronic lymphocytic leukemia (CLL)-like B cells can be detected using 4- or 5-multicolor flow cytometry in 5% to 7% of adults with normal lymphocyte counts. We investigated the frequency of circulating monoclonal B cells in 608 healthy subjects older than 40 years with normal blood counts, using a highly sensitive 8-color flow cytometry approach and systematic screening for total PB leukocyte count higher than 5 x 10(6). We show that the frequency of PB monoclonal B cells is markedly higher than previously reported (12% for CLL-like B cells, found at frequencies of 0.17 +/- 0.13 x 10(9) cells/L), the incidence progressively increasing with age. Most cases (62%) showed clonal B-cell levels below the maximum sensitivity of the techniques described by others (< 0.01%), supporting the notion that detection of MBL may largely depend on the sensitivity of the flow cytometry approach used.

Utility of Flow Cytometry Immunophenotyping in Multiple Myeloma and Other Clonal Plasma Cell-Related Disorders

In recent years, multiparameter flow cytometry (MFC) immunophenotyping has become mandatory in the clinical management of hematological malignancies, both for diagnostic and monitoring purposes. Multiple myeloma (MM) and other clonal plasma cell‐related (PC) disorders should be no exception to this paradigm, but incorporation of immunophenotypic studies in the management of patients with PC disorders is still far from being routinely established in many diagnostic flow cytometry laboratories. For clonal PC disorders, MFC is of clear and established clinical relevance in: (1) the differential diagnosis between MM and other PC‐related disorders; (2) the identification of high‐risk MGUS and smoldering MM; (3) minimal residual disease investigation after therapy; additionally it may also be useful for (4) the definition of prognosis‐associated antigenic profiles; and (5) the identification of new therapeutic targets. In this article, we review the clinical value of MFC in the study of PC disorders, with specific emphasis in those areas where consensus exists on the need to incorporate MFC into routine evaluation of MM and other clonal PC‐related disorders.

The immunophenotype of different immature, myeloid and B-cell lineage-committed CD34+ hematopoietic cells allows discrimination between normal/reactive and myelodysplastic syndrome precursors

Occurrence of phenotypic abnormalities in CD34+ hematopoietic progenitor and precursor cells (HPC) and their major B-cell and nonlymphoid compartments has been frequently reported in myelodysplastic syndromes (MDS). Here, we analyze for the first time the numerical and phenotypic abnormalities of different maturation-associated subsets of bone marrow (BM) CD34+ HPC from 50 newly diagnosed MDS patients in comparison to normal/reactive BM (n=29). Our results confirm the existence of heterogeneously altered phenotypes among CD34+ HPC from MDS and indicate that such variability depends both on the relative distribution of the different subsets of CD34+ HPC committed into the different myeloid and B-lymphoid compartments, and their immunophenotype (for example, higher reactivity for CD117 and CD13 and lower expression of CyMPO, CD64 and CD65 on CD34+ immature and neutrophil precursors), a clear association existing between the accumulation of CD34+ HPC and that of immature CD34+ HPC. Interestingly, expansion of erythroid- and neutrophil-lineage CD34+ cells is detected in low-grade MDS at the expense of CD34+ plasmacytoid dendritic cell and B-cell precursors, while expansion of immature CD34+ precursors occurs in high-grade MDS. On the basis of the number and severity of the phenotypic abnormalities detected, a scoring system is proposed that efficiently discriminates between normal/reactive and MDS CD34+ HPC, the mean score significantly increasing from low- to high-grade MDS.

Genetic abnormalities and patterns of antigenic expression in multiple myeloma.

Myelomatous plasma cells show a high heterogeneity both in their immunophenotypic characteristics as well as in their cytogenetic features. Thus far, no extensive studies have been carried out to explore whether such antigenic diversity is associated with specific genetic characteristics. We have investigated the relationship between the immunophenotypic profile at plasma cell and both their DNA ploidy status (evaluated by flow cytometry) and specific genetic features (ascertained by fluorescence in situ hybridization) in a large series of 915 patients with newly diagnosed multiple myeloma. The non-hyperdiploid multiple myeloma group (n = 454, 52%) was associated with a significantly higher frequency of positivity for CD28 and CD20 as well as a higher incidence of CD56− and CD117− cases (P < 0.001). Remarkably, 13q deletion and immunoglobulin heavy chain (IGH) gene rearrangements, which were significantly more common in non-hyperdiploid multiple myeloma, showed a strong association with CD117− cases. IGH translocation to 11q13 was associated with reactivity for CD20 (P < 0.001), down-regulation of CD56 (P < 0.001), and lack of expression of CD117 (P = 0.001). By contrast, IGH translocations to other chromosome partners were almost exclusively found among CD20− and CD117− cases (P < 0.001). These results suggest that genetic categories in multiple myeloma exhibit particular immunophenotypic profiles which in turn are strongly associated with the DNA ploidy status.

Adult precursor B-ALL with BCR/ABL gene rearrangements displays a unique immunophenotype based on the pattern of CD10, CD34, CD13 and CD38 expression

The Philadelphia chromosome (Ph+) reflects a balanced reciprocal translocation between the long arms of chromosomes 9 and 22 [t(9;22)(q34;q11.2] involving the BCRand ABL genes. At present, detection of BCR/ABL gene rearrangements is mandatory in precursor-B-ALL patients at diagnosis for prognostic stratification and treatment decision. In spite of the clinical impact, no screening method, displaying a high sensitive and specificity, is available for the identification of BCR/ABL+precursor-B-ALL cases. The aim of the present study was to explore the immunophenotypic characteristics of precursor B-ALL cases displaying BCR/ABL gene rearrangements using multiple stainings analyzed by quantitative flow cytometry in order to rapidly (<1 h) identify unique phenotypes associated with this translocation. From the 82 precursor-B-ALL cases included in the study 12 displayed BCR/ABL gene rearragements, all corresponding to adult patients, four of which also displayed DNA aneuploidy. Our results show that BCR/ABL+ precursor B-ALL cases constantly displayed a homogeneous expression of CD10 and CD34 but low and relatively heterogeneous CD38 expression, together with an aberrant reactivity for CD13. In contrast, this unique phenotype was only detected in three out of 70 BCR/ABL− cases. Therefore, the combined use of staining patterns for CD34, CD38 and CD13 expression within CD10-positive blast cells is highly suggestive of BCR/ABL gene rearrangements in adults with precursor B-ALL.

The Progression from MGUS to Smoldering Myeloma and Eventually to Multiple Myeloma Involves a Clonal Expansion of Genetically Abnormal Plasma Cells

Purpose: Genetic aberrations detected in multiple myeloma (MM) have also been reported in the premalignant conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Our aim was to investigate in depth the level of clonal heterogeneity of recurrent genetic abnormalities in these conditions. Experimental Design: Immunoglobulin heavy chain (IGH) translocations, 13q14 and 17p13 deletions, and 1q21 gains using FISH were evaluated in 90 MGUS, 102 high-risk SMM, and 373 MM. To this end, we not only purified plasma cells (PC) for the FISH analysis (purity > 90%), but subsequently, we examined the correlation between the proportion of PC with cytogenetic changes and the number of clonal PC present in the same sample, as measured by multiparametric flow cytometry. Results: We observed a significant difference between the proportion of clonal PC with specific genetic abnormalities in MGUS compared with SMM and in SMM compared with MM. Thus, the median proportion of PC with IGH translocations globally considered, t(11;14) and 13q deletions was significantly lower in MGUS than in SMM, and in SMM than in MM [IGH translocations: 34% vs. 57% vs. 76%; t(11;14): 38% vs. 61% vs. 81%; and 13q deletion: 37% vs. 61% vs. 74% in MGUS, SMM, and MM, respectively]. For t(4;14), the difference was significant in the comparison between MGUS/SMM and MM and for 1q between MGUS and SMM/MM. Conclusions: This study demonstrates that the progression from MGUS to SMM, and eventually to MM, involves a clonal expansion of genetically abnormal PC. Clin Cancer Res; 17(7); 1692–700. ©2011 AACR.

Patterns of BCR/ABL gene rearrangements by interphase fluorescence in situ hybridization (FISH) in BCR/ABL+ leukemias: incidence and underlying genetic abnormalities.

Interphase fluorescence in situ hybridization (iFISH) is increasingly used for the identification of BCR/ABL gene rearrangements in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). In the present study, we have explored the incidence of both typical and atypical iFISH patterns of BCR/ABL gene rearrangements in a series of 168 consecutive BCR/ABL+ patients – 135 CML, 31 precursor B-ALL and two acute myeloblastic leukemia (AML) cases – and established their underlying genetic alterations through further molecular and chromosome analyses. Two different FISH probes (Vysis Inc., Downers Grove, IL, USA) were used: the LSI BCR/ABL dual color extra signal (ES) and the dual color dual fusion BCR/ABL probe (D-FISH). Our results show that most BCR/ABL+ patients (83%, including 88% of all CML, 61% of ALL and one of two AML) displayed typical iFISH patterns of either Major (M) BCR/ABL (87% of CML, 13% of ALL and one of the two AML) or minor (m) BCR/ABL gene rearrangements (1% of all CML and 48% of ALL cases) with the two probes. Further molecular and cytogenetic studies confirmed the presence of such typical rearrangements in all except one of these ALL cases who had coexistence of an MBCR/ABL and an mBCR/ABL gene rearrangement together with monosomy 9. In the remaining 29 cases (17%), up to five different atypical iFISH patterns were detected with the ES probe. Atypical iFISH patterns were most frequently due to additional numerical changes – most often supernumerary Philadelphia (Ph) chromosome (7%) but also gain or loss of chromosome 9 (1%) or 22 (1%). Deletion of 9q sequences proximal to the breakpoint were also frequently observed with the ES probe (8%). Application of the D-FISH probe showed that in most of these latter cases (5%) deletion of 22q sequences distal to the breakpoint also occurred. The remaining cases with atypical iFISH had cryptic insertion of BCR in 9q34 (1%). Exact interpretation of each iFISH pattern was supported by FISH on metaphases and molecular determination of the BCR breakpoint. In summary, our results indicate that despite the high incidence of typical iFISH patterns of BCR/ABL gene rearrangements, atypical patterns are also found in BCR/ABL+ acute leukemias; the precise definition of the alteration present in individual cases is dependent on metaphase studies and molecular definition of the breakpoint.

Bone marrow cells from myelodysplastic syndromes show altered immunophenotypic profiles that may contribute to the diagnosis and prognostic stratification of the disease: A pilot study on a series of 56 patients

A heterogeneous spectrum of immunophenotypic abnormalities have been reported in myelodysplastic syndromes (MDS). However, most studies are restricted to the analysis of CD34+ cells and/or other major subsets of CD34− cells, frequently not exploring the diagnostic and prognostic impact of immunophenotyping.

Impact of trisomy 12, del(13q), del(17p), and del(11q) on the immunophenotype, DNA ploidy status, and proliferative rate of leukemic B‐cells in chronic lymphocytic leukemia

B‐cell chronic lymphocytic leukemia (B‐CLL) is a well‐defined clinical entity with heterogeneous molecular and cytogenetic features. Here, we analyze the impact of trisomy 12, del(13q), del(17p), and del(11q) as determined by interphase fluorescence in situ hybridization analysis of purified neoplastic B‐CLL cells on their immunophenotype, DNA ploidy status and proliferative rate.

Flow cytometry immunophenotyping of fine-needle aspiration specimens: utility in the diagnosis and classification of non-Hodgkin lymphomas

Barrena S, Almeida J, García‐Macias M D C, López A, Rasillo A, Sayagués J M, Rivas R A, Gutiérrez M L, Ciudad J, Flores T, Balanzategui A, Caballero M D & Orfao A
(2011) Histopathology 58, 906–918
Flow cytometry immunophenotyping of fine‐needle aspiration specimens: utility in the diagnosis and classification of non‐Hodgkin lymphomas