Francisca I. Camacho

Genomic and Gene Expression Profiling Defines Indolent Forms of Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is typically a very aggressive disease with poor outcomes, but some cases display an indolent behavior that might not necessitate treatment at diagnosis. To define molecular criteria that might permit recognition of such cases, we compared the clinicopathologic features, gene expression, and genomic profile of patients who had indolent or conventional disease (iMCL or cMCL). Patients with iMCL displayed nonnodal leukemic disease with predominantly hypermutated IGVH and noncomplex karyotypes. iMCL and cMCL shared a common gene expression profile that differed from other leukemic lymphoid neoplasms. However, we identified a signature of 13 genes that was highly expressed in cMCL but underexpressed in iMCL. SOX11 was notable in this signature and we confirmed a restriction of SOX11 protein expression to cMCL. To validate the potential use of SOX11 as a biomarker for cMCL, we evaluated SOX11 protein expression in an independent series of 112 cases of MCL. Fifteen patients with SOX11-negative tumors exhibited more frequent nonnodal presentation and better survival compared with 97 patients with SOX11-positive MCL (5-year overall survival of 78% versus 36%, respectively; P = 0.001). In conclusion, we defined nonnodal presentation, predominantly hypermutated IGVH, lack of genomic complexity, and absence of SOX11 expression as qualities of a specific subtype of iMCL with excellent outcomes that might be managed more conservatively than cMCL.

Primary cutaneous CD4+ small/medium-sized pleomorphic T-cell lymphoma expresses follicular T-cell markers.

Cutaneous CD4+ small/medium-sized pleomorphic T-cell lymphoma (CSTCL) is a cutaneous T-cell lymphoma defined by a predominance of small-to-medium–sized CD4+ pleomorphic T cells, with a favorable clinical course. Cases are also characterized by the presence of a rich infiltrate of reactive B cells. Recently, it has been reported that follicular helper T cells (TFH cells) display a distinct gene expression profile, positive for PD-1, CXCL13, and BCL-6. We report for the first time the expression of PD-1 and other TFH cell markers in CSTCLs and discuss its biologic significance. Sixteen CSTCLs were included in this study, and also 20 reactive inflammatory conditions, 10 primary cutaneous marginal zone, 10 follicular center lymphomas, and 5 primary CD30+ cutaneous lymphomas. They were immunohistochemically analyzed for a large panel of markers. Double immunoperoxidase labeling of paraffin sections was performed for PD-1, OCT-2, and BCL-6. Clonal Ig and T-cell receptor rearrangements and Epstein-Barr virus-encoded RNA expression were also evaluated. Morphologic and clinical data were reviewed. Histologic examination showed a dense polymorphic lymphoid infiltrate throughout the dermis. Atypical large CD4+ cells were positive for PD-1, CXCL13, and BCL-6 in all cases, and were attached in small clusters, or formed rosettes around CD30/OCT-2+ B blast cells. Epstein-Barr virus was not apparent in any of the cases. A dominant T-cell clone was identified in 14 cases, whereas polymerase chain reaction IgH gene rearrangement studies showed that all cases were polyclonal. None of the patients had lymphadenopathy or showed any evidence of systemic disease, nor did they have any previous history of mycosis fungoides or drug reactions. FTH cell markers are not exclusive to angioimmunoblastic lymphadenopathy but may also be seen in neoplastic cells of CSTCLs. Moreover, these findings suggest that B-cell stimulation by FTH could also take place in some cutaneous T-cell lymphomas.

Expression of the NF‐κB targets BCL2 and BIRC5/Survivin characterizes small B‐cell and aggressive B‐cell lymphomas, respectively

Nuclear factor kappa B (NF‐κB) activation has been proposed as a cardinal feature of tumourigenesis, although the precise mechanism, frequency, relevance, and extent of NF‐κB activation in lymphomas remain to be fully elucidated. In this study, expression profiling and tissue microarray studies of 209 and 323 non‐Hodgkins lymphomas (NHLs) respectively, including the most frequent sub‐types of NHL, were employed to generate a hypothesis concerning the most common NF‐κB targets in NHL. These analyses showed that NF‐κB activation is a common phenomenon in NHL, resulting in the expression of distinct sets of NF‐κB target genes, depending on the cell context. BCL2 and BIRC5/Survivin were identified as key NF‐κB targets and their expression distinguished small and aggressive B‐cell lymphomas, respectively. Interestingly, in the vast majority of B‐cell lymphomas, the expression of these markers was mutually exclusive. A set of genes was identified whose expression correlates either with BIRC5/Survivin or with BCL2. BIRC5/Survivin expression, in contrast to BCL2, was associated with a signature of cell proliferation (overexpression of cell cycle control, DNA repair, and polymerase genes), which may contribute to the aggressive phenotype and poor prognosis of these lymphomas. Strikingly, mantle cell lymphoma and chronic lymphocytic leukaemia expressed highly elevated levels of BCL2 protein and mRNA, higher than that observed in reactive mantle zone cells or even in follicular lymphomas, where BCL2 expression is deregulated through the t(14;18) translocation. In parallel with this observation, BIRC5/Survivin expression was higher in Burkitts lymphoma and diffuse large B‐cell lymphoma than in non‐tumoural germinal centre cells. In vitro studies confirmed that NF‐κB activation contributes to the expression of both markers. In cell lines representing aggressive lymphomas, NF‐κB inhibition resulted in a decrease in BIRC5/Survivin expression. Meanwhile, in chronic lymphocytic leukaemia (CLL)‐derived lymphocytes, NF‐κB inhibition resulted in a marked decrease in BCL2 expression. Copyright

Identification of novel candidate target genes in amplicons of Glioblastoma multiforme tumors detected by expression and CGH microarray profiling

BackgroundConventional cytogenetic and comparative genomic hybridization (CGH) studies in brain malignancies have shown that glioblastoma multiforme (GBM) is characterized by complex structural and numerical alterations. However, the limited resolution of these techniques has precluded the precise identification of detailed specific gene copy number alterations.ResultsWe performed a genome-wide survey of gene copy number changes in 20 primary GBMs by CGH on cDNA microarrays. A novel amplicon at 4p15, and previously uncharacterized amplicons at 13q32-34 and 1q32 were detected and are analyzed here. These amplicons contained amplified genes not previously reported. Other amplified regions containg well-known oncogenes in GBMs were also detected at 7p12 (EGFR), 7q21 (CDK6), 4q12 (PDGFRA), and 12q13-15 (MDM2 and CDK4). In order to identify the putative target genes of the amplifications, and to determine the changes in gene expression levels associated with copy number change events, we carried out parallel gene expression profiling analyses using the same cDNA microarrays. We detected overexpression of the novel amplified genes SLA/LP and STIM2 (4p15), and TNFSF13B and COL4A2 (13q32-34). Some of the candidate target genes of amplification (EGFR, CDK6, MDM2, CDK4, and TNFSF13B) were tested in an independent set of 111 primary GBMs by using FISH and immunohistological assays. The novel candidate 13q-amplification target TNFSF13B was amplified in 8% of the tumors, and showed protein expression in 20% of the GBMs.ConclusionThis high-resolution analysis allowed us to propose novel candidate target genes such as STIM2 at 4p15, and TNFSF13B or COL4A2 at 13q32-34 that could potentially contribute to the pathogenesis of these tumors and which would require futher investigations. We showed that overexpression of the amplified genes could be attributable to gene dosage and speculate that deregulation of those genes could be important in the development and progression of GBM. Our findings highlight the important influence in GBM of signaling pathways such as the PI3K/AKT, consistent with the invasive features of this tumor.

Progression to large B-cell lymphoma in splenic marginal zone lymphoma: a description of a series of 12 cases.

Splenic marginal zone lymphoma (SMZL) is considered to be an indolent extranodal B-cell lymphoma. Despite its low aggressivity, histologic progression has been described in sporadic reports, although the frequency, characteristics, and underlying molecular abnormalities of this phenomenon are largely unknown. We review here the clinical, morphologic, immunohistochemical, and molecular features of a series of 12 SMZL cases that showed progression to large B-cell lymphoma (LBCL). The most frequent location of secondary LBCL was in peripheral lymph node. This occurred between 12 and 85 months after diagnosis of SMZL. However, in two cases LBCL was diagnosed at the initial stage of the disease (one spleen tumoral nodule and one hilar lymph node). The histologic and immunophenotypic features of these cases were similar to those of transformed LBCL at other sites. In four cases the immunoglobulin heavy chain gene polymerase chain study revealed the same rearrangement pattern in both primary and secondary tumors, thereby confirming their identity and excluding the possibility of a second malignancy. As is the case with other low-grade lymphoproliferative disorders, SMZL may undergo high-grade transformation. These 12 cases represent 13% of our series of SMZL with adequate follow-up. The incidence of large cell transformation in SMZL seems to be lower than in follicular lymphoma (25–60%) and mantle cell lymphoma (11–39%), although it is similar to the frequency of transformation in B-chronic lymphocytic lymphoma/small lymphocytic lymphoma (1–10%). The mean proliferative index (MIB1 staining) in initial SMZL specimens of cases with LBCL transformation was 28.6%, higher than that of MIB1 staining in the overall SMZL series (21.8%), although not statistically significantly so. p53 or p16 INK4a inactivation in this series was observed in only one case, in contrast with the situation observed in chronic lymphocytic leukemia, follicular lymphoma, and mantle cell lymphoma. It seems that progression in SMZL is mainly independent of p53 or p16 INK4a inactivation. The frequency of the 7q deletion in this series was 3 of 7 (42%). 7q loss may play an alternative role in the inactivation of the p53 and p16 INK4a pathway, thereby favoring tumoral progression.

EBV-positive diffuse large B-cell lymphoma of the elderly is an aggressive post-germinal center B-cell neoplasm characterized by prominent nuclear factor-kB activation.

Here, we report a retrospective series of 47 EBV-positive diffuse large B-cell lymphoma associated with advanced age. Histopathology allowed to the identification of different histological patterns: cases with polymorphic diffuse large B-cell lymphoma (29 cases), Hodgkin-like (8 cases) and polymorphic lymphoproliferative disorder-like (9 cases) patterns. One case was purely monomorphic diffuse large B-cell lymphoma. We show that this lymphoma type is a neoplasm with prominent classical and alternative nuclear factor-kB pathway activation in neoplastic cells (79% of the cases showed nuclear staining for p105/p50, 74% for p100/p52 and 63% for both proteins), with higher frequency than that observed in a control series of EBV-negative diffuse large B-cell lymphoma (χ2 <0.001). Most cases showed an activated phenotype (95% non-germinal center (Hans algorithm); 78% activated B cell (Choi algorithm)). Clonality testing demonstrated IgH and/or K/Kde/L monoclonal rearrangements in 64% of cases and clonal T-cell populations in 24% of cases. C-MYC (1 case), BCL6 (2 cases) or IgH (3 cases) translocations were detected by FISH in 18% cases. These tumors had a poor overall survival and progression-free survival (the estimated 2-year overall survival was 40±10% and the estimated 2-year progression-free survival was 36±9%). Thus, alternative therapies, based on the tumor biology, need to be tested in patients with EBV-positive diffuse large B-cell lymphoma of the elderly.

Peripheral T-cell Lymphoma With Follicular T-cell Markers

Introduction Peripheral T-cell lymphomas (PTCLs) in western countries are uncommon tumors with unfavorable prognosis. They may be subclassified as anaplastic large-cell lymphomas (ALCLs), angioimmunoblastic-T-cell lymphomas (AITLs), or unspecified peripheral T-cell lymphomas (PTCLs-U). It has recently been demonstrated that AITLs originate from germinal center follicular helper T cells (TFH), whereas the normal counterparts of other PTCLs remain essentially unknown. The aim of this study was to establish whether other PTCL subgroups also express TFH cell markers. Materials and Methods One hundred forty-six PTCLs were analyzed for programmed death-1 (PD-1) expression in tissue microarrays using a new monoclonal antibody called NAT-105. PD-1–positive cases, which did not fulfill all the criteria for AITL, were further evaluated in whole-tissue sections for another 12 immunohistochemical markers, including the TFH cell markers CXCL13, CD10, and BCL6. Clonal Ig and T-cell receptor rearrangements and Epstein-Barr virus-encoded RNA expression were also evaluated. Morphologic, clinical, and follow-up data were reviewed. Results Twenty-five out of 87 non-AITL cases (28.75%) showed PD-1 immunostaining. CXCL13, BCL6, and CD10 expression was found in 24/25 (96%), 16/25 (64%), and 6/25 (24%) cases, respectively. All cases expressed at least 2 TFH cell markers. Moreover, 5 cases were positive for all 4 markers. Most cases (17/25, 68%) displayed some AITL-like features. Of the remainder, 1 was considered to be early AITL, 1 was diagnosed as ALCL–anaplastic lymphoma kinase-negative, and 4 of the other 6 PTCLs-U had morphology consistent with lymphoepithelioid (Lennerts) lymphoma. Three AITL-like cases showed IgH clonal rearrangement, 2 of which were associated with Epstein-Barr virus expression. Our series of patients did not differ significantly in their clinical presentation from most reported PTCL cases in the literature: 55% of them were alive and 35% were in complete remission after a median follow-up of 15 months after cyclophosphamide, dexorubicin, vincristine, and prednisone-based chemotherapy. Conclusions TFH cell markers, especially PD-1, were expressed in a subset of PTCLs not classified as AITL, although most of them shared some morphologic features with AITL. This suggests that the spectrum of AITL may be wider than previously thought, possibly including cases of lymphoepithelioid (Lennerts) lymphoma. Additionally, the results suggest that a subgroup of PTCLs-U, distinct from AITL and including some cases denominated as ALCL, may also be derived from TFH cells, although they develop along a distinct pathogenic pathway.

Nodal Marginal Zone Lymphoma: A Heterogeneous Tumor: A Comprehensive Analysis of a Series of 27 Cases

We have reviewed the clinical, morphologic, immunophenotypical, and molecular features of a series of 27 cases of nodal marginal zone lymphoma with the aim of defining this entity more precisely. The series was characterized by a relatively favorable clinical course, with a low clinical stage at diagnosis (59% patients with clinical stage I–II) and a 5-year overall survival probability of 79%. However, the disease persisted in a relatively large fraction of the patients, thus yielding a 5-year failure-free survival probability of 22%. Molecular and immunohistochemical analyses of the series revealed heterogeneity in the frequency of IgVH somatic mutation and in the expression of IgD, CD43, MUM1, and CD38. Apart from the absence of nuclear Bcl10, no clear distinction could be made from the expression profiling of other B-cell lymphomas claimed to be derived from marginal zone B cells. Additionally, the immunophenotype of the tumoral cells in all cases but one differed from that described in monocytoid B cells. It was characterized by a Bcl2−, p21+, cyclin E+ profile. The analysis of apoptosis-regulator proteins disclosed abnormalities in the expression of survivin and active caspase 3, which could partially explain the abnormal regulation of apoptosis observed in these tumors. Molecular and immunohistochemical data obtained in this study strongly imply that there is significant heterogeneity among the cases included in the category termed nodal marginal zone lymphoma.

Overall Survival in Aggressive B-Cell Lymphomas Is Dependent on the Accumulation of Alterations in p53, p16, and p27

Different studies have already shown that the isolated inactivation of p21, p16, or p27 cyclin-dependent kinase inhibitors (CKIs) is associated with increased growth fraction, tumor progression, or decreased overall survival in cases of non-Hodgkins lymphoma. In this study we linked molecular study of the p53 and p16 genes with immunohistochemical analysis of p27 expression in a group of aggressive B-cell lymphomas [large B-cell lymphomas (LBCLs) and Burkitts lymphomas]. This was done to analyze the relationship between p53 and p16 silencing, p27 anomalous overexpression, and clinical follow-up, testing the hypothesis that the accumulation of CKI alterations could confer to the tumors a higher aggressivity. In a group of 62 patients, p53 inactivation as a result of mutation was observed in 11 cases (18%) and p16 silencing was seen in 27 cases (43.5%) as a result of methylation (20 of 62), 9p21 deletion (7 of 44), or p16 mutation (2 of 62). The simultaneous inactivation of p53 and p16 was detected exclusively in five LBCL cases. Anomalous expression of p27, which has been proven to be associated with the absence of p27/CDK2 complexes and the formation of p27/cyclin D3 complexes where p27 is inactivated, was detected in 19 of 61 cases (31%). Cases characterized by p27 anomalous expression display concurrent inactivation of p21 (provided by p53 mutations) and/or p16 CKIs in 11 of 14 LBCL cases (P = 0.040). When the relationship between the association of inactivated CKIs and overall survival was considered, a significant relationship was found between a lower overall survival probability and an increased number of inactivated CKIs in LBCL cases, with the worst prognosis for the cases displaying concurrent p53, p16, and p27 alterations. This proves that simultaneous inactivation of different tumor suppressor pathways does indeed take place, and that tumor aggressiveness takes advantage of this CKI-concerted silencing. In this same series of data, Burkitts lymphoma patients seem to behave in a different way than LBCLs, with p53 and p16 alteration being mutually exclusive and the association with p27 anomalous expression not being clinically significant. These facts seem to support that the additive effect of the inactivation of different CKIs could be dependent of the histological type.

Implication of Polycomb Members Bmi-1, Mel-18, and Hpc-2 in the Regulation of p16INK4a, p14ARF, h-TERT, and c-Myc Expression in Primary Breast Carcinomas

Purpose: Deregulation of mammalian Polycomb group (PcG) members may contribute to human carcinogenesis. p16INK4a and p14ARF tumor suppressors, human telomerase reverse transcriptase (h-TERT), and oncoprotein c-Myc have been implicated in the regulation of the cell cycle and proliferation mediated by PcG proteins, mainly Bmi-1, in mice and in cell culture experiments. Here, we examine whether these in vitro findings can be extrapolated to the in vivo situation. Experimental Design: We measure the expression of PcG members Bmi-1, Mel-18, and Hpc-2 and their potential targets by reverse transcription-PCR, immunostaining, and Western blotting in a series of 134 breast carcinomas and correlate the data with several clinical-pathologic variables of the tumors. Results: Expression of PcG genes was variably detected, but overexpression of Bmi-1 was the most frequent PcG alteration observed. In addition, statistical direct correlation in expression level of the three PcG members was detected. A correlation between c-Myc and Bmi-1 expression levels was observed; however, there was no correlation between expression of Bmi-1 and p16INK4a, p14ARF, or h-TERT. However, expression of the other PcG members Mel-18 and Hpc-2 correlated with the cell cycle regulators. Moreover, PcG mRNA–altered expression correlated significantly with certain clinical-pathologic variables associated with poor prognosis. Conclusions: Our data suggest that the oncogenic role of Bmi-1 in human primary breast carcinomas is not determined by its capacity to inhibit INK4a/ARF proteins or to induce telomerase activity.