Aplicació de la citogenètica convencional i la hibridació in situ a l'estudi de síndromes limfoproliferatives cròniques B

Descripció del recurs: 26 febrer 2002Titól obtingut de la portada digitalitzadaLes síndromes limfoproliferatives cròniques B (SLPC-B) són un grup divers de malalties que tenen en comú la proliferació neoplàsica i acumulació de cèl·lules limfoides de tipus B de tipus madur. Dins d'aquest grup, distingim bàsicament les leucèmies i els limfomes. El diagnòstic de les SLPC-B amb expressió leucèmica es basa en l'estudi integrat de la morfologia i l'immunofenotip de les cèl·lules leucèmiques, així com de les característiques citogenètiques i moleculars. Existeixen una sèrie d'entitats que poden ser confoses fàcilment i requereixen un estudi exhaustiu que permeti la seva classificació correcta. En el present treball s'analitzen tres d'aquestes entitats, la Leucèmia Limfàtica Crònica B atípica (LLC-Ba), el Limfoma de Cèl·lules del Mantell (LCM) i el Limfoma Esplènic de la Zona Marginal (LEZM) amb la intenció de trobar marcadors citogenètics que permetin separar-les, de manera que sigui possible establir un diagnòstic diferencial amb certesa. També es fa esment de la limfocitosi B Policlonal Persistent (LBPP). S'han estudiat 27 LLC-Ba, 13 LCM, 19 LEZM i 1 LBPP amb tècniques de citogenètica convencional i hibridació in situ, amb sondes centromèriques pels cromosomes 3, 12, 17 i 18, de locus específic per les regions 13q14 i 17p13 i de pintat cromosòmic. Entre les LLC-Ba, un 37% han presentat alteracions citogenètiques i l'alteració citogenètica més freqüent ha estat la trisomia 12 (63%), mentre que les delecions de 13q14 i 17p13 s'han detectat en % molt baix. Respecte els LCM, el 77% han presentat alteracions citogenètiques i dins d'aquests, en un 70% s'ha detectat la t(11;14)(q13;q32), alteració citogenètica característica d'aquesta entitat. Altres cromosomes implicats han estat: 1, 2, 9, 13 i 17 en forma d'alteracions estructurals principalment. No s'ha detectat la presència de trisomia 12 en cap pacient. En els LEZM, un 58% han presentat un cariotip alterat. Els cromosomes més freqüentment implicats han estat: 1, 3, 7, 8 i 14. Dels estudis inclosos en la tesi i d'altres posteriors s'han pogut definir la trisomia parcial del braç llarg del cromosoma 3 (+3q) i la delecioó del braç llarg del cromosoma 7 (del(7q)) com a alteracions més freqüents. No s'ha detectat presència de t(11;14) en cap dels casos, i la trisomia 12, així com la deleció de 17p13 només s'ha detectat en un pacient. D'aquest estudi es conclueix que les alteracions citogenètiques dels tres grups són clarament diferents i que el seu coneixement permet establir un millor diagnòstic diferencial d'aquestes malalties. La LBPP és una entitat que encara no se sap si és una neoplàsia de progressió lenta, o un fenòmen reactiu. S'ha inclòs en l'estudi amb la intenció de fer esment de la seva existència i de la possibilitat de ser confosa amb alguna de les altres entitats.B-cell lymphoproliferative disorders (BCLPD) constitute a group of pathologies in which mature B cells proliferate and accumulate in different tissues. Among BCLPD, leukaemic and lymphomatous diseases can be distinguished. In the present work, BCLPD with leukaemic expression are studied, taking into account at the same time morphologic, immunophenotypic, cytogenetic and molecular data. It is remarkable that the diagnosis of some BCLPD with leukaemic expression can be difficult because of some similar characteristics. In these cases, an exhaustive study will be required to a correct classification. In this thesis, three different entities would be analyzed: atypical B-cell lymphocytic leukaemia (aCLL), mantle cell lymphoma (MCL) and splenic marginal zone B-cell lymphoma (SMZBCL). The aim of the present study was to study the above mentioned entities by means of conventional cytogenetics and in situ hybridization techniques to analyze if different cytogenetic markers could be identified to establish a differential diagnosis among them. In addition, persistent polyclonal B-cell lymphocytosis (PPBL) was included in the study. Twenty seven aCLL, 13 MCL, 19 SMZBCL and 1 (PPBL) were studied cytogenetically and using in situ hybridization probes for centromeric regions of chromosomes 3, 12, 17 and 18, and for specific locus 13q14 (Rb) and 17p13 (p53). Among aCLL, 37% presented cytogenetic aberrations, being trisomy 12 the most frequent abnormality (63%), besides, 13q14 and 17p13 deletions were found in low percentage. Regarding MCL, chromosomal aberrations were found in 77% of patients, and among them, 70% presented a t(11;14)(q13;q32). Other affected chromosomes were 1, 2, 9, 13 and 17. No patient presented trisomy 12. In SMZBCL group, an abnormal karyotype was found in 58% of patients. The most frequently involved chromosomes were 1, 3, 7, 8 and 14. The most frequent structural abnormalities associated to SMZBCL were +3q and del(7q). No case presented +12, nor t(11;14)(q13;q32), and only one case showed 17p13 deletion. In conclusion, cytogenetic abnormalities seem to be different in each studied group, and its knowledge could be useful to establish a differential diagnosis among these pathologies. PPBL still is a controversial entity, because it is not clear if it represents a neoplastic disease with a very slow progression rate or if it represents a reactive phenomena

Absence of MALT1 traslocation in primary cutaneous marginal zone B-cell lymphoma

The implication of MALT1 gene in the pathogenesis of primary cutaneous marginal zone B-cell lymphomas (PCMZL) has been a matter of controversy. We examined the presence of MALT1 translocations in a series of 23 PCMZL. FISH assay with a MALT1 dual color break apart translocation probe revealed the absence of MALT1 translocations in all cases

Comparative Analysis of TCR-gamma Gene Rearrangements by Genescan and Polyacrylamide Gel-electrophoresis in Cutaneous T-cell Lymphoma

Demonstrating T-cell clonality has become an important approach supporting a diagnosis of malignant T-cell neoplasms. A comparative study between Genescan analysis, polyacrylamide gel and agarose gel electrophoresis in visualizing X-cell receptor gamma gene rearrangement was performed on 25 biopsy specimens from 18 patients with different forms of cutaneous T-cell lymphomas. Clonality was detected in 17 biopsy specimens when PCR products were evaluated by Genescan analysis. Seventeen showed discrete bands when visualized in polyacrylamide gel and 14 cases were clonal when visualized with agarose gel. In five cases, a clonal population was seen in the gels, but not with Genescan. On sequencing the PCR products we demonstrated nonclonality of these five samples. Our results confirm that PCR-Genescan is a useful, reliable and specific screening method for detecting dominant clones in patients with T-cell lymphoma

MicroRNA expression profiling and DNA methylation signature for deregulated microRNA in cutaneous T-cell lymphoma

MicroRNAs usually regulate gene expression negatively, and aberrant expression has been involved in the development of several types of cancers. Microarray profiling of microRNA expression was performed to define a microRNA signature in a series of mycosis fungoides tumor stage (MFt, n=21) and CD30+ primary cutaneous anaplastic large cell lymphoma (CD30+ cALCL, n=11) samples in comparison with inflammatory dermatoses (ID, n=5). Supervised clustering confirmed a distinctive microRNA profile for cutaneous T-cell lymphoma (CTCL) with respect to ID. A 40 microRNA signature was found in MFt including upregulated onco-microRNAs (miR-146a, miR-142-3p/5p, miR-21, miR-181a/b, and miR-155) and downregulated tumor-suppressor microRNAs (miR-200ab/429 cluster, miR-10b, miR-193b, miR-141/200c, and miR-23b/27b). Regarding CD30+ cALCL, 39 differentially expressed microRNAs were identified. Particularly, overexpression of miR-155, miR-21, or miR-142-3p/5p and downregulation of the miR-141/200c clusters were observed. DNA methylation in microRNA gene promoters, as expression regulatory mechanism for deregulated microRNAs, was analyzed using Infinium 450K array and approximately one-third of the differentially expressed microRNAs showed significant DNA methylation differences. Two different microRNA methylation signatures for MFt and CD30+ cALCL were found. Correlation analysis showed an inverse relationship for microRNA promoter methylation and microRNA expression. These results reveal a subgroup-specific epigenetically regulated microRNA signatures for MFt and CD30+ cALCL patients

In situ mantle cell lymphoma: clinical implications of an incidental finding with indolent clinical behavior

Background Cyclin D1-positive B cells are occasionally found in the mantle zones of reactive lymphoid follicles, a condition that has been called 'in situ mantle cell lymphoma'. The clinical significance of this lesion remains uncertain. Design and Methods The clinical and pathological characteristics, including SOX11 expression, of 23 cases initially diagnosed as in situ mantle cell lymphoma were studied. Results Seventeen of the 23 cases fulfilled the criteria for in situ mantle cell lymphoma. In most cases, the lesions were incidental findings in reactive lymph nodes. The t(11; 14) was detected in all eight cases examined. SOX11 was positive in seven of 16 cases (44%). Five cases were associated with other small B-cell lymphomas. In two cases, both SOX11-positive, the in situ mantle cell lymphoma lesions were discovered after the diagnosis of overt lymphoma; one 4 years earlier, and one 3 years later. Twelve of the remaining 15 patients had a follow-up of at least 1 year (median 2 years; range, 1-19.5), of whom 11 showed no evidence of progression, including seven who were not treated. Only one of 12 patients with an in situ mantle cell lymphoma lesion and no diagnosis of mantle cell lymphoma at the time developed an overt lymphoma, 4 years later; this case was also SOX11-positive. The six remaining cases were diagnosed as mantle cell lymphoma with a mantle zone pattern. Five were SOX11-positive and four of them were associated with lymphoma without a mantle zone pattern. Conclusions In situ mantle cell lymphoma lesions are usually an incidental finding with a very indolent behavior. These cases must be distinguished from mantle cell lymphoma with a mantle zone pattern and overt mantle cell lymphoma because they may not require therapeutic intervention

Trisomy 8, A Cytogenetic Abnormality In Myelodysplastic Syndromes, Is Constitutional Or Not?

Isolated trisomy 8 is not considered presumptive evidence of myelodysplastic syndrome (MDS) in cases without minimal morphological criteria. One reason given is that trisomy 8 (+8) can be found as a constitutional mosaicism (cT8M). We tried to clarify the incidence of cT8M in myeloid neoplasms, specifically in MDS, and the diagnostic value of isolated +8 in MDS. Twenty-two MDS and 10 other myeloid neoplasms carrying +8 were studied. Trisomy 8 was determined in peripheral blood by conventional cytogenetics (CC) and on granulocytes, CD3+ lymphocytes and oral mucosa cells by fluorescence in situ hybridization (FISH). In peripheral blood CC, +8 was seen in 4/32 patients. By FISH, only one patient with chronic myelomonocytic leukemia showed +8 in all cell samples and was interpreted as a cT8M. In our series +8 was acquired in all MDS. Probably, once discarded cT8M by FISH from CD3+ lymphocytes and non-hematological cells, +8 should be considered with enough evidence to MDS

FOXP1 molecular cytogenetics and protein expression analyses in primary cutaneous large B cell lymphoma, leg-type

FOXP1 protein is expressed in normal activated B cells and overexpressed in a subset of diffuse large B-cell lymphomas, including primary cutaneous large B-cell lymphomas (PCLBCL), leg type. High expression of FOXP1 has been associated to an unfavourable prognosis with independent survival significance. However, little is known regarding the mechanisms underlying the overexpression of FOXP1 in PCLBCL, leg type. Our aims were to analyze FOXP1 cytogenetic status and protein expression in a series of PCLBCL, leg type. Finally, we compared the observed results with those obtained in a group of patients with primary cutaneous follicle centre lymphoma (PCFCL). Fifteen patients with PCLBCL, leg type and nine patients with primary cutaneous follicle centre lymphoma (PCFCL) were included in the study. For each biopsy specimen, FOXP1 translocation and copy number changes were evaluated by fluorescence in situ hybridization (FISH) and protein expression by immunohistochemistry (IHC). Immunohistochemistry showed FOXP1 staining in 13 PCLBCL, leg type, whereas all PCFCL were negative. FISH analysis disclosed no translocations involving FOXP1 gene in any of the cases. However, FOXP1 gene gains (3 to 4 copies) were observed in 82% of samples of PCLBCL, leg type and in 37% of PCFCL. FOXP1 expression was independent from FOXP1 translocation. Our results confirm that overexpression of FOXP1 is present in a considerable proportion of PCLBCL, leg type and might indicate an unfavourable prognosis. Mechanisms not related to translocation seem to be responsible for this overexpression

Oligonucleotide array-CGH identifies genomic subgroups and prognostic markers for tumor stage mycosis fungoides

Mycosis fungoide (MF) patients who develop tumors or extracutaneous involvement usually have a poor prognosis with no curative therapy available so far. In the present European Organization for Research and Treatment of Cancer (EORTC) multicenter study, the genomic profile of 41 skin biopsies from tumor stage MF (MFt) was analyzed using a high-resolution oligo-array comparative genomic hybridization platform. Seventy-six percent of cases showed genomic aberrations. The most common imbalances were gains of 7q33.3q35 followed by 17q21.1, 8q24.21, 9q34qter, and 10p14 and losses of 9p21.3 followed by 9q31.2, 17p13.1, 13q14.11, 6q21.3, 10p11.22, 16q23.2, and 16q24.3. Three specific chromosomal regions, 9p21.3, 8q24.21, and 10q26qter, were defined as prognostic markers showing a significant correlation with overall survival (OS) (P=0.042, 0.017, and 0.022, respectively). Moreover, we have established two MFt genomic subgroups distinguishing a stable group (0-5 DNA aberrations) and an unstable group (>5 DNA aberrations), showing that the genomic unstable group had a shorter OS (P=0.05). We therefore conclude that specific chromosomal abnormalities, such as gains of 8q24.21 (MYC) and losses of 9p21.3 (CDKN2A, CDKN2B, and MTAP) and 10q26qter (MGMT and EBF3) may have an important role in prognosis. In addition, we describe the MFt genomic instability profile, which, to our knowledge, has not been reported earlier

Identification of gene mutations and fusion genes in patients with Sézary Syndrome

Sézary syndrome is a leukemic form of cutaneous T-cell lymphoma with an aggressive clinical course. The genetic etiology of the disease is poorly understood, with chromosomal abnormalities and mutations in some genes being involved in the disease. The goal of our study was to understand the genetic basis of the disease by looking for driver gene mutations and fusion genes in 15 erythrodermic patients with circulating Sézary cells, 14 of them fulfilling the diagnostic criteria of Sézary syndrome. We have discovered genes that could be involved in the pathogenesis of Sézary syndrome. Some of the genes that are affected by somatic point mutations include ITPR1, ITPR2, DSC1, RIPK2, IL6, and RAG2, with some of them mutated in more than one patient. We observed several somatic copy number variations shared between patients, including deletions and duplications of large segments of chromosome 17. Genes with potential function in the T-cell receptor signaling pathway and tumorigenesis were disrupted in Sézary syndrome patients, for example, CBLB, RASA2, BCL7C, RAMP3, TBRG4, and DAD1. Furthermore, we discovered several fusion events of interest involving RASA2, NFKB2, BCR, FASN, ZEB1, TYK2, and SGMS1. Our work has implications for the development of potential therapeutic approaches for this aggressive disease

BCL3-rearrangements in B-cell lymphoid neoplasms occur in two breakpoint clusters associated with different diseases

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5' (n=9) and 3' (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5' breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3' breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively