José María Sayagués

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.

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.

Early recurrences in histologically benign/grade I meningiomas are associated with large tumors and coexistence of monosomy 14 and del(1p36) in the ancestral tumor cell clone

Tumor recurrence is the major clinical complication in meningiomas, and its prediction in histologically benign/grade I tumors remains a challenge. In this study, we analyzed the prognostic value of specific chromosomal abnormalities and the genetic heterogeneity of the tumor, together with other clinicobiological disease features, for predicting early relapses in histologically benign/grade I meningiomas. A total of 149 consecutive histologically benign/grade I meningiomas in patients who underwent complete tumor resection were prospectively analyzed. Using interphase fluorescence in situ hybridization, we studied the prognostic impact of the abnormalities detected for 11 different chromosomes, together with other relevant clinicobiological and histopathological characteristics of the disease, on recurrence-free survival (RFS) at 2.5, 5, and 10 years. From the prognostic point of view, losses of chromosomes 9, 10, 14, and 18 and del(1p36) were associated with a shorter RFS at 2.5, 5, and 10 years. Similarly, histologically benign/grade I meningiomas showing coexistence of monosomy 14 and del(1p36) in the ancestral tumor cell clone displayed a higher frequency of early relapses. In fact, coexistence of -14 and del(1p36) in the ancestral tumor cell clone, together with tumor size, represented the best combination of independent prognostic factors for the identification of those patients with a high risk of an early relapse. Our results indicate that patients with large histologically benign/grade I meningiomas carrying monosomy 14 and del(1p36) in their ancestral tumor cell clone have a high probability of relapsing early after diagnostic surgery. These findings suggest the need for closer follow-up in this small group of patients.

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

Intratumoral Patterns of Clonal Evolution in Meningiomas as Defined by Multicolor Interphase Fluorescence in Situ Hybridization (FISH) : Is There a Relationship between Histopathologically Benign and Atypical/Anaplastic Lesions?

Meningiomas are cytogenetically heterogeneous tumors in which chromosome gains and losses frequently occur. Based on the intertumoral cytogenetic heterogeneity of meningiomas, hypothetical models of clonal evolution have been proposed in these tumors which have never been confirmed at the intratumoral cell level. The aim of this study was to establish the intratumoral patterns of clonal evolution associated with chromosomal instability in individual patients as a way to establish tumor progression pathways in meningiomas and their relationship with tumor histopathology and behavior. A total of 125 meningioma patients were analyzed at diagnosis. In all cases, multicolor interphase fluorescence in situ hybridization (iFISH) studies were performed on fresh tumor samples for the detection of quantitative abnormalities for 11 different chromosomes. In addition, overall tumor cell DNA content was measured in parallel by flow cytometry. iFISH studies were also performed in parallel on tissue sections in a subset of 30 patients. FISH studies showed that 56 (45%) of the 125 cases analyzed had a single tumor cell clone, all these cases corresponding to histologically benign grade I tumors. In the remaining cases (55%) more than one tumor cell clone was identified: two in 45 cases (36%), three in 19 (15%), and four or more clones in five cases (4%). Overall, flow cytometric analysis of cell DNA contents showed the presence of DNA aneuploidy in 44 of these cases (35%), 30% corresponding to DNA hyperdiploid and 5% to hypodiploid cases; from the DNA aneuploid cases, 35 (28%) showed two clones and 9 (7%) had three or more clones. A high degree of correlation (r >/= 0.89; P < 0.001) was found between FISH and flow cytometry as regards the overall quantitative DNA changes detected with both techniques, the former being more sensitive. Among the cases with chromosome abnormalities, the earliest tumor cell clone observed was frequently characterized by the loss of one or more chromosomes (64% of all meningiomas); loss of either a single chromosome 22 or, less frequently, of a sex chromosome (X or Y) and del (1p) was commonly found as the single initial cytogenetic aberration (30%, 5%, and 5% of the cases, respectively). Interestingly, an isolated loss of chromosome 22 was only found as the initial abnormality in one out of 14 atypical/anaplastic meningiomas, while the same cytogenetic pattern was present in the ancestral tumor cell clone of 32% of the benign tumors. Cytogenetic patterns based on chromosome gains were found in the ancestral tumor cell clone in 4% of the patients, 2% corresponding to tetraploid tumors. Overall, cytogenetic evolution of the earliest tumor cell clones was frequently associated with tetraploidization (31%). Our results show that meningiomas are genetically heterogeneous tumors that display different patterns of numerical chromosome changes, with the presence of more than one tumor cell clone detected in almost half of the cases including all atypical/anaplastic cases. Interestingly, the pathways of intratumoral clonal evolution observed in the benign tumors were different from those observed in atypical/anaplastic meningiomas, suggesting that the latter tumors might not always represent a more advanced stage of histologically benign meningiomas.

The Cytogenetic Relationship between Primary and Recurrent Meningiomas Points to the Need for New Treatment Strategies in Cases at High Risk of Relapse

Purpose: Recurrence is the major factor influencing the clinical outcome of meningioma patients although the exact relationship between primary and recurrent tumors still needs to be clarified. The aim of the present study is to analyze the cytogenetic relationship between primary and subsequent recurrent meningiomas developed within the same individual. Experimental Design: Multicolor interphase fluorescence in situ hybridization was done for the identification of numerical abnormalities of 12 chromosomes in single-cell suspensions from 59 tumor samples corresponding to 25 recurrent meningioma patients. In 47 of these tumors, the distribution of different tumor cell clones was also analyzed in paraffin-embedded tissue sections. In parallel, 132 nonrecurrent cases were also studied. Results: Most recurrent meningiomas showed complex cytogenetic aberrations associated with two or more tumor cell clones in the first tumor analyzed. Interestingly, in most individuals (74%), exactly the same tumor cell clones identified in the initial lesion were also detected in the subsequent recurrent tumor samples. In the recurrent tumor samples of the remaining cases (26%), we observed tumor cell clones related to those detected in the initial lesion but which had acquired one or more additional chromosome aberrations associated with either the emergence of new clones with more complex karyotypes or the disappearance of the most representative clones from the primary lesions. Multivariate analysis of prognostic factors showed that the Maillo et al. prognostic score, based on age of patient, tumor grade, and monosomy 14, together with tumor size was the best combination of independent variables for predicting tumor recurrence at diagnosis. Conclusion: Overall, our results indicate that the development of recurrent meningiomas after complete tumor resection is usually due to regrowth of the primary tumor and rarely to the emergence of an unrelated meningioma, underlining the need for alternative treatment strategies in cases at high risk of relapse, particularly those with a high Maillo et al. prognostic score and larger tumors.

Cytogenetic profiles in multiple myeloma and monoclonal gammopathy of undetermined significance: a study in highly purified aberrant plasma cells

Cytogenetic studies in clonal plasma cell disorders have mainly been done in whole bone marrow or CD138+ microbead-enriched plasma cells and suggest that recurrent immunoglobulin heavy chain translocations - e.g. t(4;14) -are primary oncogenetic events. The aim of this study was to determine cytogenetic patterns of highly purified aberrant plasma cells (median purity ≥98%) in different clonal plasma cell disorders. We analyzed aberrant plasma cells from 208 patients with multiple myeloma (n=148) and monoclonal gammopathy of undetermined significance (n=60) for the presence of del(13q14), del(17p13) and t(14q32) using multicolor interphase fluorescence in situ hybridization. Additionally, immunoglobulin heavy chain gene arrangements were analyzed and complementarity determining region 3 was sequenced in a subset of patients and combined multicolor interphase fluorescence in situ hybridization/immunofluorescent protein staining analyses were performed in selected cases to confirm clonality and cytogenetic findings. At diagnosis, 96% of cases with multiple myeloma versus 77% of monoclonal gammopathy of undetermined significance cases showed at least one cytogenetic alteration and/or hyperdiploidy. The cytogenetic heterogeneity of individual cases reflected coexistence of cytogenetically-defined aberrant plasma cell clones, and led to the assumption that karyotypic alterations were acquired stepwise. Cases of multiple myeloma and monoclonal gammopathy of undetermined significance frequently showed different but related cytogenetic profiles when other cytogenetic alterations such as deletions/gains of the immunoglobulin heavy chain or the fibroblast growth factor receptor 3 were additionally considered. Interestingly, in 24% of multiple myeloma versus 62% of monoclonal gammopathy of undetermined significance patients with an immunoglobulin heavy chain translocation, aberrant plasma cells with and without t(14q32) coexisted in the same patient. Our data suggest that recurrent immunoglobulin heavy chain translocations might be absent in the primordial plasma cell clone in a significant proportion of patients with clonal plasma cell disorders carrying these cytogenetic alterations.

Intratumoural cytogenetic heterogeneity of sporadic colorectal carcinomas suggests several pathways to liver metastasis

Much has been learned about the chromosomal abnormalities of colorectal carcinomas but the cytogenetic relationship between the neoplastic clones present in primary versus metastatic tumour samples remains unclear. We analyse the frequency of abnormalities for 47 chromosome regions using the interphase fluorescence in situ hybridization technique in a group of 48 tumours, including 24 primary colorectal tumours and 24 paired liver metastases. All tumours showed complex karyotypes with numerical/structural abnormalities for seven or more different chromosomes/chromosome regions both in the primary tumours and in their paired metastases. Chromosome 8 was the most frequently altered (22/24 primary tumours), consistently showing del(8p22) and/or gains/amplification of 8q24, followed by abnormalities of the entire chromosome 7 (21/24 primary tumours) and of chromosomes 17p and 20q (20/24 primary tumours). Simultaneous staining for multiple chromosome probes revealed the presence of two or more tumour cell clones in 23/24 cases (46/48 tumour samples). Interestingly, the liver metastases typically contained tumour cell clones similar to those found in the primary tumours, suggesting the absence of selective selection of specific tumour clones. Despite this, additional chromosomal abnormalities were detected in 23/24 metastatic tumours, which preferentially consisted of del(17p13) and gains/amplification of 11q13 and 20q13; moreover, compared to primary tumours, metastases showed an increased number of abnormalities of chromosomes 1p, 7q, 8q, 13q, and 18q, and new chromosomal abnormalities involving chromosomes 6, 10q23, 14q32, 15q22, and 19q13. Owing to the high frequency of numerical abnormalities of the entire chromosome 7 and loss and/or gain/amplification of specific regions of chromosome 8, eg del(8p22) and/or gains/amplification of 8q24 in primary colorectal tumours with associated metastases, it is suggested that their assessment at diagnosis could be of great clinical utility for the identification of colorectal cancer patients at higher risk of developing liver metastases. Copyright

Association between the proliferative rate of neoplastic B cells, their maturation stage, and underlying cytogenetic abnormalities in B-cell chronic lymphoproliferative disorders: analysis of a series of 432 patients.

Limited knowledge exists about the impact of specific genetic abnormalities on the proliferation of neoplastic B cells from chronic lymphoproliferative disorders (B-CLPDs). Here we analyze the impact of cytogenetic abnormalities on the proliferation of neoplastic B cells in 432 B-CLPD patients, grouped according to diagnosis and site of sampling, versus their normal counterparts. Overall, proliferation of neoplastic B cells highly varied among the different B-CLPD subtypes, the greatest numbers of proliferating cells being identified in diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Compared with normal B cells, neoplastic B-CLPD cells showed significantly increased S + G(2)/M-phase values in mantle cell lymphoma (MCL), B-chronic lymphocytic leukemia (B-CLL), BL, and some DLBCL cases. Conversely, decreased proliferation was observed in follicular lymphoma, lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM), and some DLBCL patients; hairy cell leukemia, splenic marginal zone, and MALT-lymphoma patients showed S + G(2)/M phase values similar to normal mature B lymphocytes from LN. Interestingly, in B-CLL and MCL significantly higher percentages of S + G(2)/M cells were detected in BM versus PB and in LN versus BM and PB samples, respectively. In turn, presence of 14q32.3 gene rearrangements and DNA aneuploidy, was associated with a higher percentage of S + G(2)/M-phase cells among LPL/WM and B-CLL cases, respectively.