JUAN F GARCIA

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Abstract: The hedgehog signaling pathway has been shown to play a pathogenic role in diffuse large B-cell lymphoma and anaplastic large cell lymphoma, but has not been assessed in classical Hodgkin lymphoma. Glioma-associated oncogene homologues 1, 2, and 3 are transcriptional effectors of the hedgehog pathway. In this study, we first used real-time quantitative polymerase chain reaction to investigate the expressions of GLI1, GLI2, and GLI3 in 3 classical Hodgkin lymphoma cell lines. GLI1 and GLI2 were variably expressed, but GLI3 was highly expressed in all cell lines. We then used immunohistochemistry to assess glioma-associated oncogene homologues 1, 2, and 3 in 39 classical Hodgkin lymphoma patient samples. Glioma-associated oncogene homologues 1 and 2 were weakly to variably expressed in a subset of classical Hodgkin lymphoma patient samples. In contrast, glioma-associated oncogene homologue 3 showed strong, uniform nuclear expression in virtually all Hodgkin/Reed-Stenberg cells. We then performed an immunohistochemical survey of glioma-associated oncogene homologue 3 expression in 13 cases of nodular lymphocyte predominant Hodgkin lymphoma and 218 non-Hodgkin lymphomas. Most other lymphoma types showed variable or no expression of glioma-associated oncogene homologue 3, with a minor subset of cases of nodular lymphocyte predominant Hodgkin lymphoma, ALK-positive and ALK-negative anaplastic large cell lymphoma, and B-cell lymphoma, unclassifiable with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma showing a glioma-associated oncogene homologue 3 staining pattern indistinguishable from classical Hodgkin lymphoma. Our data provide a rationale to further investigate the biologic significance of glioma-associated oncogene homologue 3 in classical Hodgkin lymphoma biology.

Abstract: DLBCL prognostication requires additional biological markers. MicroRNAs (miRNAs) may constitute markers for cancer diagnosis, outcome or therapy response. Here we have analyzed the miRNA expression profile in a retrospective multicenter series of DLBCL patients (258 cases) uniformly treated with chemoimmunotherapy. Findings were correlated with OS and PFS. miRNA and gene expression profiles were studied using microarrays in an initial set of 36 cases. A selection of miRNAs associated with either DLBCL molecular subtypes (GCB/ABC) or clinical outcome were studied by multiplex RT-PCR in a test group of 240 cases with available FFPE diagnostic sample, divided into a training set (123 patients), which was used to derive miRNA-based and combined (with IPI score) Cox regression models, and an independent validation series (117 patients). A model based on miRNA expression predicts OS and PFS. This model improves the prediction based on clinical variables. Combined models with IPI score identify a high-risk group of patients with a 2-year OS and PFS probability of less than 50%. In summary, a precise miRNA signature is associated with poor clinical outcome in chemoimmunotherapy-treated DLBCL patients. This information improves IPI-based prediction and identifies a subgroup of candidate patients for alternative therapeutic regimens to those of standard therapies.

Abstract: The majority of meningiomas are probably benign but a number of tumors display considerable histological and/or clinical aggressivity, sometimes with unexpectedly high recurrence rates after radical removal. Understanding the potential behavior of these tumors in individual patients is critical for rational therapeutic decision-making. This study aimed to identify gene expression profiles and candidate markers associated with original and recurrent meningiomas. Unsupervised hierarchical clustering of the samples confirmed 2 main groups of meningiomas with distinct clinical behaviors. The gene expression profiling study identified genes and pathways potentially associated with meningioma recurrence, revealing an overall lower level of gene expression. The differential gene expression profiling analyses of original and recurrent meningiomas identified 425 known genes and expressed sequence tags related to meningioma recurrence, with SFRP1 (8p12), TMEM30B (14q23), and CTGF (6q23) showing the most disparate expression. Most of the differentially expressed genes were located at 1p, 6q, and 14q and were underexpressed in recurrences. Loss of such chromosomal regions has previously been associated with a higher risk of meningioma recurrence or malignant progression. Thus, at these locations, we propose the existence of novel candidate genes that could be involved in meningioma recurrence. In addition, the overexpression of genes of histone cluster 1 (6p) in recurrent meningiomas is reported here for the first time. Finally, the altered genes related to meningioma recurrence are involved in pathways such as Notch, TGFbeta, and Wnt, as described previously, and in other pathways such as cell cycle, oxidative phosphorylation, PPAR, and PDGF, not related before to meningioma recurrence.

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Abstract: Despite improvement in the treatment of advanced classical Hodgkin Lymphoma, around 30% relapse or die as result of the disease. Current predictive systems, based on clinical and analytical parameters, fail to identify these high risk patients accurately. A multistep approach was used to design a quantitative RT-PCR assay to be applied to routine formalin-fixed paraffin-embedded samples, integrating genes expressed either by the tumor cells and their microenvironment. The significance of 30 genes chosen on the basis of previously published data was evaluated in a set of 282 samples (divided into estimation and validation sets) to build a molecular risk score to predict failure. Adequate RT-PCR profiles were obtained from 262 of 282 cases (92.9%). Best predictor genes were integrated into an 11-gene model including four functional pathways (Cell Cycle, Apoptosis, Macrophage Activation and IRF4) able to identify low- and high-risk patients with different rates of 5-year FFS: 74% versus 44.1% in the estimation set (p < 0.0001); and 67.5% vs. 45.0% in the validation set (p = 0.0217). Moreover, this biological model can be combined with stage IV into a final predictive model able to identify a group of patients with very bad outcome (5-year FFS probability 25.2%).

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Abstract: Surgery for rectal cancer continues to develop towards improving local control and overall survival, maintaining quality of life and preserving sphincter, genitourinary and sexual function. The multidisciplinary approach integrated in a team of different specialists ensures an individualised treatment for each patient with rectal cancer. Thus, the role of the pathologist has acquired an important relevance, not only in diagnosis, management and evaluation of the surgical specimen, but also for selection of the best adjuvant treatment. Parameters such as macroscopic quality of the mesorectum, status of the circumferential margin and lymph node harvest are considered basic criteria by current guidelines. Additionally, consistency in reporting based on the histologic classification proposed by the World Health Organization (WHO) is mandatory, along with inclusion into the pathologic report of current criteria for tumour node metastasis (TNM) staging, assessment of response to neoadjuvant chemoradiation therapy and clinically relevant molecular studies. Detection of defects in mismatch repair genes and mutational analysis of specific genes should be included as predictive markers for therapy.

Abstract: Plasmablastic lymphoma (PBL) has recently come to be considered a distinct entity among mature B cell neoplasms, although the limits with Diffuse Large B Cell Lymphoma (DLBCL) need to be more accurately defined. Here we show the results of an immunohistochemical study of 35 cases of PBL compared with a set of 111 conventional DLBCLs . Our results demonstrate that the use of a limited combination of immunohistochemical markers (PAX5&CD20, PRDM1/BLIMP1 and XBP1s) enables the identification of a plasmablastic immunophenotype that is highly characteristic of plasmablastic lymphoma cases and associated with an aggressive clinical behavior. Additionally, the study shows that the acquisition of a partial plasmablastic phenotype (PRDM1/BLIMP1 expression) in DLBCL is associated with shorter survival in R-CHOP-treated patients.

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Abstract: The existence, diagnostic features, and the biological and clinical relevance of lymphocyte-rich classical Hodgkin's lymphoma remain controversial. A comparative marker analysis of lymphocyte-rich classical Hodgkin's lymphoma, nodular lymphocyte-predominance Hodgkin's lymphoma, and of other subtypes of classical Hodgkin's lymphoma was carried out. Markers were selected focusing on B-cell lineage and transcription program (OCT.1, OCT.2, BOB.1, BCL6, PAX-5, GCET1, KLHL6, and BLIMP1), the NF-kappaB signaling pathway (REL-B, C-REL, TRAF-1, p-50, and MUM-1) and the T-cell microenvironment (CD3, CD57, PD-1, CXCL-13, and CD10, BCL-6, CD23). Lymphocyte-rich classical Hodgkin's lymphoma cases displayed features intermediate between those of classical Hodgkin's lymphoma and nodular lymphocyte-predominance Hodgkin's lymphoma. The expression of B-cell transcription factors such as OCT.1, OCT.2, BOB.1, and BCL6 was more frequent in lymphocyte-rich classical Hodgkin's lymphoma than in classical Hodgkin's lymphoma. A follicular T-cell microenvironment was also identified in 50% of lymphocyte-rich classical Hodgkin's lymphoma cases. NF-kB markers were expressed at frequencies comparable with those observed in classical Hodgkin's lymphoma. The neoplastic cell immunophenotype and microenvironment in lymphocyte-rich classical Hodgkin's lymphoma closely mimic that which are observed in the outer zone of the germinal center, where B-cell blasts with germinal-center markers co-express CD30 and the B-cell transcription program, surrounded by follicular T-cell rosettes. Lymphocyte-rich classical Hodgkin's lymphoma seems to be characterized by a stronger expression of the B-cell transcription program by the neoplastic cells and by a follicular T-cell background, occupying an intermediate position between classical Hodgkin's lymphoma and nodular lymphocyte-predominance Hodgkin's lymphoma.Modern Pathology advance online publication, 22 May 2009; doi:10.1038/modpathol.2009.54.

Abstract: 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.

Abstract: PURPOSE: Despite major advances in the treatment of classic Hodgkin's lymphoma (cHL), approximately 30% of patients in advanced stages may eventually die as result of the disease, and current methods to predict prognosis are rather unreliable. Thus, the application of robust techniques for the identification of biomarkers associated with treatment response is essential if new predictive tools are to be developed. EXPERIMENTAL DESIGN: We used gene expression data from advanced cHL patients to identify transcriptional patterns from the tumoral cells and their nonneoplastic microenvironment, associated with lack of maintained treatment response. Gene-Set Enrichment Analysis was used to identify functional pathways associated with unfavorable outcome that were significantly enriched in either the Hodgkin's and Reed-Sternberg cells (regulation of the G2-M checkpoint, chaperones, histone modification, and signaling pathways) or the reactive cell microenvironment (mainly represented by specific T-cell populations and macrophage activation markers). RESULTS: To explore the pathways identified previously, we used a series of 52 formalin-fixed paraffin-embedded advanced cHL samples and designed a real-time PCR-based low-density array that included the most relevant genes. A large majority of the samples (82.7%) and all selected genes were analyzed successfully with this approach. CONCLUSIONS: The results of this assay can be combined in a single risk score integrating these biological pathways associated with treatment response and eventually used in a larger series to develop a new molecular outcome predictor for advanced cHL.

Abstract: Combined 1p/19q deletions are very prevalent in oligodendrogliomas (OGs) and, to a lesser extent, in oligoastrocytomas (OAs). These losses are associated with responsiveness to therapy. Using array-based comparative genomic hybridization, we screened for recurrent genomic alterations in OG and oligoastrocytoma subtypes on chromosome 19. Concomitant 1p/19q loss was detected in most of the tumors with allelic loss, but array-based comparative genomic hybridization revealed some tumors to have unrelated 1p/19q arm losses, suggesting alternative mechanisms of loss to that related to the reported t(1;19) translocation. Analyses of 1p/19q loss by fluorescence in situ hybridization and loss of heterozygosity assays and correlations of genomic data with the Ki-67 proliferation marker were also performed. Four 1q (or 19p) and 2 1p (or 19q) fluorescence in situ hybridization probe signals together with homozygosity of the 1p/19q microsatellites suggested a hypothetical mechanism of genome duplication consecutive to the loss of the derivative chromosome der(1p;19q) from the t(1;19)(1q;19p) translocation. This genome duplication was frequent in high-grade OGs and was strongly correlated with Ki-67 expression; thus, it could be related to tumor progression. Finally, in addition to the frequent 1p/19q loss, we report a novel 17q amplified region in OGs with BIRC5 as one of the possible candidate target genes of the amplicon.

Abstract: This article review the current situation of the exenterative procedures as part of the treatment of recurrent cervical cancer after radiation. Pelvic exenteration has been proven the only curative choice of treatment in selected cases of this clinical situation. A review of historical and recent published series have shown an increase of 5-y survival from 30 to 42 %. Almost one out of two patients will suffer complications of some kind, and one out of three will have a severe complication with pelvic exenteration. During the past sixty years, a number of outstanding improvements have been achieved - not only in surgical outcomes, but also in quality of life - owing to new reconstructive approaches. Women facing an exenterative procedure must be counseled carefully about the risks and long-term concerns related to the procedure. Each should undergo a comprehensive evaluation to make sure there is no evidence of unresectable or metastatic disease that would make her an unsuitable candidate for exenteration.

Abstract: Lymphomas originating from the lymphatic system comprise about 30 entities classified according to the World Health Organization (WHO). The histopathological diagnosis is generally considered difficult and prone to mistakes. Since non-random chromosomal translocations are specifically involved in different lymphoma entities, their detection will be increasingly important. Hence, a split-signal fluorescence in situ hybridisation (FISH) procedure would be helpful in discriminating the most difficult classifications. The Euro-FISH programme, a concerted action of nine European laboratories, has validated a robust, standardised protocol to improve the diagnostic approach on lymphoma entities. Therefore, 16 fluorescent probes and 10 WHO entities, supplemented with reactive cases, were selected. The results of the Euro-FISH programme show that all probes were correctly cytogenetically located, that the standardised protocol is robust, resulting in reliable results in approximately 90% of cases, and that the procedure could be implemented in every laboratory, bringing the relatively easy interpretation of split-signal probes within the reach of many pathology laboratories.

Abstract: The objective of this review is to recognize the characteristics of endometrial adenocarcinoma in young patients and to evaluate the published experience with conservative approach in patients with endometrial adenocarcinoma. We searched MEDLINE articles describing patients with endometrial adenocarcinoma who were treated with hormonal therapy. The search included articles published between January 1966 and January 2007. Endometrial carcinoma in patients under 45 years of age is an unusual condition that shows a more favorable pattern than in older patients. One hundred thirty three patients were found in the search. The average duration of hormonal therapy was approximately 6 months. The average response time was 12 weeks. Seventy six percent of patients treated with hormonal therapy had a complete response and the other 24% never responded to treatment. Of those who initially responded, 66% didn't show recurrence of disease. The other 34% had a relapse. There have been published 4 deaths of patients conservatively managed. A conservative approach in these patients can offer reasonable oncological security and the opportunity of fulfilling their maternal desires in selected cases. However, consideration should be taken regarding the potential adverse outcomes that have been recently published in the literature.

Abstract: BACKGROUND: Immunoglobulin gene somatic hypermutation is a biologically relevant and clinically useful prognostic factor in different types of low-grade B-cell lymphomas, including chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. DESIGN AND METHODS: With the aim of identifying surrogate markers of somatic hypermutation, a combined investigation of IgV(H) mutational status and expression profiles of 93 samples from patients with small B-cell lymphoma was performed. RESULTS: The analysis identified an somatic hypermutation signature of genes involved in the regulation of gene transcription, DNA repair and replication, and chromosome maintenance. Eight of these genes were subjected to protein analysis using tissue microarrays, for a set of 118 cases. We found a clear link between RAD51C and CDK7 protein expression and somatic hypermutation status, in that positive expression of either marker was significantly associated with a mutated status (p<0.003). We also found that positive expression of TFDP1 and POLA was significantly associated with ongoing somatic hypermutation (p<0.001). To assess the potential clinical applicability of these somatic hypermutation markers, we studied a series of cases of mantle cell lymphoma included in a tissue microarray. The expression of RCC1 and CDK7, separately and together, was found to be significantly associated with longer overall survival. CONCLUSIONS: An somatic hypermutation signature has been identified for different types of small B-cell lymphoma. This has a potential mechanistic and diagnostic value.

Abstract: The role of neoadjuvant chemotherapy (NACT) in cervical cancer has been a matter of investigation over the last 20 years. A systematic review and meta-analysis of individual patient data (IPD) demonstrated that NACT followed by surgery is superior to radiotherapy alone in terms of overall survival. However, in spite of the results of the meta-analysis, NACT has not been adopted as the new standard of care. In the present paper, we review the reasons why NACT is still considered an investigational approach in cervical cancer.

Abstract: PURPOSE: To analyze tumor-microenvironment relationships in Hodgkin lymphoma (HL) as potential determinants in the decision-making process related to the alterations in cell cycle and apoptotic pathways of Hodgkin/Reed-Sternberg (H/RS) cells. EXPERIMENTAL DESIGN: Based on a cohort of 257 classic HL patients, we carried out a global descriptive correlational analysis and logistic regression study to identify tumor-infiltrated immune cell rate in HL that could be interconnected with genes involved in the regulation of apoptotic/proliferative pathways in H/RS cells. RESULTS: Our results reveal the existence of a connection between the reactive microenvironment and molecular changes in apoptotic/proliferative pathways in H/RS cells. A lesser incidence of infiltrated cytotoxic cells in the tumor (CD8(+) T lymphocytes, CD57(+) natural killer, and granzyme B(+) cells) was associated with overexpression of antiapoptotic proteins (Bcl-X(L), survivin, caspase-3, and nuclear factor-kappaB) in tumoral cells. Increased incidence of general infiltrated immune cells, such as CD4(+) T lymphocytes, CD57(+) natural killer cells, activated CTL, and dendritic cells, in the microenvironment of the tumor was associated with increased growth fraction of tumoral cells, including G(1)-S checkpoint (cyclin D and cyclin E) and tumor suppressor pathways (p16 and SKP2), and with the presence of EBV (signal transducers and activators of transcription 1 and 3 expression; STAT1/STAT3). CONCLUSIONS: A lower level of cytotoxic cells correlated with an increase of antiapoptotic mechanisms in H/RS cells, whereas the global infiltrated immune population correlated with the growth fraction of the tumor. Our collective data suggest a causal relationship between infiltrated immune response and concurrent changes of the different proliferative checkpoints, tumor suppressor, and apoptotic pathways of H/RS cells in HL.

Abstract: BACKGROUND: Low-grade B-cell lymphomas are a very heterogeneous group of tumors, whose differential diagnosis is frequently compromised by the lack of specific cytogenetic or molecular features. Our objective was to search for genomic features that allow a better molecular identification of the different types of lymphoma studied. DESIGN AND METHODS: We selected a panel of 87 low-grade B-cell lymphoma tumor samples that were unambiguously diagnosed (clinically and cytogenetically) as: follicular, splenic marginal zone, nodal marginal zone, lymphoplasmacytic, mantle cell, extranodal marginal zone MALT-type lymphoma or B-cell chronic lymphocytic leukemia. All samples were subjected to the same high-resolution genomic DNA analysis (array-based comparative genomic hybridization): a whole genome platform that contained 44000 probes distributed across the genome. Genomic imbalances were recorded, compiled and analyzed. RESULTS: Eighty percent of analyzed cases showed genomic imbalances (deletions and gain/amplifications) but the frequency of these imbalances ranged from 100% in mantle cell lymphomas to 33% in MALT lymphomas. A total of 95 new genomic imbalances affecting all lymphoma subtypes, were defined. We evaluated the extension of the genomic instability, detecting distinct patterns of genomic instability within subtypes. Specific pathways, such as nuclear factor kB (gains of REL and BCL11A, and losses of COMMD3, BIRC1, IKK1 and NFKB2), Polycomb group proteins (gain of BMI1 and deletion of PCGF6), DNA repair checkpoint pathways (deletion of 16q24 involving CDT1), or miRNA with a role in B-cell lymphoma pathogenesis (MIRN15A, MIRN16-1), were targeted by this genomic instability. CONCLUSIONS: Although all subtypes of lymphomas showed gains and losses of DNA, the analysis of their genomic profiles indicated that there are specific aberrations in almost every subtype as well as frequent aberrations that are common to a large number of lymphoma types. These common aberrations target genes that are important in B-cell lymphomagenesis.

Abstract: The diagnosis of malignant lymphoma is a recognized difficult area in histopathology. Therefore, detection of clonality in a suspected lymphoproliferation is a valuable diagnostic criterion. We have developed primer sets for the detection of rearrangements in the B- and T-cell receptor genes as reliable tools for clonality assessment in lymphoproliferations suspected for lymphoma. In this issue of Leukemia, the participants of the BIOMED-2 Concerted Action CT98-3936 report on the validation of the newly developed clonality assays in various disease entities. Clonality was detected in 99% of all B-cell malignancies and in 94% of all T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The combined BIOMED-2 results are summarized in a guideline, which can now be implemented in routine lymphoma diagnostics. The use of this standardized approach in patients with a suspect lymphoproliferation will result in improved diagnosis of malignant lymphoma.

Abstract: Genetic immunization (GI), which is primarily used for vaccine purposes, is a method for producing antibodies to a protein by delivering the gene encoding the protein as a eukaryotic expression vector instead of the protein itself. The mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) is one of the most likely candidates for involvement in pathogenesis of MALT lymphoma and probably of multiple myelomas. In the present work we describe the production and characterization of a mouse monoclonal antibody (mAb) directed against MALT1 and the study of MALT1 protein expression in a large series of lymphomas and myeloma cell lines. The full-length coding sequence of human MALT1 was inserted into pcDNA3 vector and delivered into mouse skin using a helium gene gun. Six new mAbs against the MALT1 molecule were produced. In order to characterize and confirm the specificity of these mAbs, Western blot (WB) and immunoprecipitation (IP) analyses were performed. A new anti-MALT1 mAb was selected and tested in a large series of cell lines. These results confirm that GI is a reliable and effective alternative method for production of mAbs, allowing accurate and sensitive detection and screening of proteins by WB.

Abstract: Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Abstract: Integrative genomic and gene-expression analyses have identified amplified oncogenes in B-cell non-Hodgkin lymphoma (B-NHL), but the capability of such technologies to localize tumor suppressor genes within homozygous deletions remains unexplored. Array-based comparative genomic hybridization (CGH) and gene-expression microarray analysis of 48 cell lines derived from patients with different B-NHLs delineated 20 homozygous deletions at 7 chromosome areas, all of which contained tumor suppressor gene targets. Further investigation revealed that only a fraction of primary biopsies presented inactivation of these genes by point mutation or intragenic deletion, but instead some of them were frequently silenced by epigenetic mechanisms. Notably, the pattern of genetic and epigenetic inactivation differed among B-NHL subtypes. Thus, the P53-inducible PIG7/LITAF was silenced by homozygous deletion in primary mediastinal B-cell lymphoma and by promoter hypermethylation in germinal center lymphoma, the proapoptotic BIM gene presented homozygous deletion in mantle cell lymphoma and promoter hypermethylation in Burkitt lymphoma, the proapoptotic BH3-only NOXA was mutated and preferentially silenced in diffuse large B-cell lymphoma, and INK4c/P18 was silenced by biallelic mutation in mantle-cell lymphoma. Our microarray strategy has identified novel candidate tumor suppressor genes inactivated by genetic and epigenetic mechanisms that substantially vary among the B-NHL subtypes.

Abstract: Genetic immunization can be combined with hybridoma technology to generate high-affinity monoclonal antibodies (MAbs). A new anti-BCL-6 MAb (GI191E/A8) was produced by cloning full-length BCL-6 cDNA into a eukaryotic vector and delivering this into mouse epidermis using a helium gene gun. A comparative study was made of the specificity and the effects of formalin fixation on immunohistochemistry quality of GI191E/A8 and two other anti-BCL-6 MAbs. To evaluate its possible application to differential diagnosis of lymphomas, two tissue microarrays (89 diffuse large B-cell lymphomas and 24 B-cell chronic lymphocytic leukemia cases) were stained with GI191E/A8 and another anti-BCL-6 MAb produced by conventional means. Using GI191E/A8, the detection of BCL-6 protein was significantly increased, and its specificity was independent of formalin-fixation time. Using automatic quantified analysis, the correlation between the two anti-BCL-6 MAbs tested was identical in cases with overexpression or absence of BCL-6. In cases with intermediate BCL-6 protein expression, detection with GI191E/A8 was more sensitive. A significant association of higher BCL-6 expression and longer median overall survival times in diffuse large B-cell lymphomas was found. Using conventionally produced MAbs in the same patient group, the association was not significant.

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Abstract: The HDM2 oncoprotein is a cellular inhibitor of p53 and is frequently deregulated in human cancer. However, the HDM2 gene encodes alternatively spliced variants whose functional significance is poorly understood. We had previously reported the detection of alternative HDM2 forms in Hodgkin's lymphoma (HL)-derived cell lines. Here, we have cloned several of these transcripts, including the previously described HDM2-A, -B and -C (which encode the COOH terminus of HDM2), and two novel variants (HDM2-HL1 and -HL2) containing a complete p53 interaction domain. Real-time PCR assays demonstrated that HDM2-A and -B were selectively expressed by HL cell lines and primary tumors, compared with their non-neoplastic counterparts. In transient transfection experiments, alternatively spliced HDM2 isoforms were partially or totally localized within the cytoplasm. HDM2-HL2 was able to inhibit transactivation of a p53-inducible reporter construct and induced a partial relocalization of p53 to the cytoplasm. Expression of HDM2-A and -B caused the activation of p53/p21 and induced growth arrest in primary cells, but also increased the expression levels of cyclins D1 and E. Other possible genes regulated by HDM2-A and -B were identified using cDNA microarray technology. These results imply that HDM2 isoforms may have multiple effects on cell cycle control, and provide insight into the mechanisms through which these molecules contribute to tumorigenesis.

Abstract: Around 20% to 30% of patients with Hodgkin lymphoma (HL) do not benefit from standard therapies and finally succumb to their disease. The factors that influence the outcome of HL have not been elucidated, underscoring the demand for the identification of biologic risk factors and new therapeutic targets. We analyzed the gene expression profiles of samples from 29 patients with advanced classic HL treated with standard therapy and compared the expression profiles of patients with favorable and unfavorable clinical outcome. Using supervised methods, we identified 145 genes associated with outcome, which were grouped into 4 signatures representing genes expressed by either the tumoral cells (genes involved in the regulation of mitosis and cell growth/apoptosis) or the tumor microenvironment. The relationship between the expression of 8 representative genes and survival was successfully validated in an independent series of 235 patients by quantification of protein expression levels on tissue microarrays. Analysis of centrosomes and mitotic checkpoint confirmed the existence of an abnormal transition through mitosis in HL cells. Therefore, genes related to tumor microenvironment, cell growth/apoptosis, and regulation of mitosis are associated with treatment response and outcome of patients with HL.

Abstract: BACKGROUND AND OBJECTIVES: The positive regulatory domain I (PRDM1) protein or BLIMP-1, belonging to the PRDM gene family of transcriptional repressors, is a key regulator of terminal differentiation in B-lymphocytes and is critical for plasma cell differentiation. DESIGN AND METHODS: Here we document the expression of PRDM1 in normal and neoplastic lymphoid cells, through the use of a monoclonal antibody that recognizes the molecule in paraffin-embedded tissue sections. A large series of B and T-cell lymphomas (679 cases) was studied, using tissue microarrays. RESULTS: Multiple myeloma, plasmacytoma and lymphoplasmacytic lymphoma cases (n=19) were positive. Plasmablastic lymphoma, oral mucosa-type (n=15), were also found to be positive. PRDM1 protein was expressed in some cases of B-cell neoplasia, i.e. chronic lymphocytic leukemia/small lymphocytic lymphoma (15%), diffuse large B-cell lymphoma (43%), classical Hodgkin's lymphoma (41%) and also in T-cell lymphoma (23%). INTERPRETATION AND CONCLUSIONS: Most B-neoplastic cells showing plasmablastic differentiation were PRDM1-positive. Unexpectedly, a subset of diffuse large B-cell lymphoma expressed PRDM1, lacked detectable plasmablastic or immunoblastic changes and displayed more aggressive behavior, with a shorter failure-free survival. In contrast to normal B-cells, diffuse large B-cell lymphoma cases with increased PRDM1 expression co-expressed BCL-6 and MUM1/IRF4, confirming that PRDM1 expression in these tumors is insufficient to drive the full genetic program associated with plasmacytic differentiation.

Abstract: BACKGROUND AND OBJECTIVES: The presence of tumor-associated macrophages (TAM) is a prognostic factor for survival in follicular lymphoma (FL). Overexpression and/or activation of the signal transducer and activator of transcription 1 (STAT1) in these TAM have also been observed. The aim of this study was to determine the extent to which macrophages are present in FL and to investigate the expression of STAT1 in these cells. DESIGN AND METHODS: We retrospectively analyzed 211 patients with distinct stages and grades of FL. Expression of the CD68 proteins, chosen as a marker for macrophages, and STAT1 was quantified by immunohistochemistry and double immunofluorescence. RESULTS: Automated determinations revealed the presence of CD68-positive macrophages in all FL tissues studied (mean 57.6+/-45.1 cells/field), while STAT1 protein was expressed in 29.94% of cases. Double-fluorescence staining confirmed that STAT1 protein co-localized exclusively with CD68, indicating the presence of a subset of STAT1-expressing TAM localized principally in the vicinity of tumor cells. Multivariate analysis showed that, besides the Follicular Lymphoma International Prognostic Index (FLIPI) classification, expression of STAT1 was an important independent prognostic factor for shorter overall survival in FL. INTERPRETATION AND CONCLUSIONS: These results demonstrate the presence of STAT1-expressing TAM in FL and their association with an adverse outcome, thus emphasizing the relevance of non-tumor cells in the control of the growth and survival of lymphoma cells.

Abstract: Nuclear factor kappa B (NF-kappaB) activation has been proposed as a cardinal feature of tumourigenesis, although the precise mechanism, frequency, relevance, and extent of NF-kappaB activation in lymphomas remain to be fully elucidated. In this study, expression profiling and tissue microarray studies of 209 and 323 non-Hodgkin's lymphomas (NHLs) respectively, including the most frequent sub-types of NHL, were employed to generate a hypothesis concerning the most common NF-kappaB targets in NHL. These analyses showed that NF-kappaB activation is a common phenomenon in NHL, resulting in the expression of distinct sets of NF-kappaB target genes, depending on the cell context. BCL2 and BIRC5/Survivin were identified as key NF-kappaB 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 Burkitt's lymphoma and diffuse large B-cell lymphoma than in non-tumoural germinal centre cells. In vitro studies confirmed that NF-kappaB activation contributes to the expression of both markers. In cell lines representing aggressive lymphomas, NF-kappaB inhibition resulted in a decrease in BIRC5/Survivin expression. Meanwhile, in chronic lymphocytic leukaemia (CLL)-derived lymphocytes, NF-kappaB inhibition resulted in a marked decrease in BCL2 expression.

Abstract: Splenic marginal zone lymphoma (SMZL) and nodal marginal zone lymphoma (NMZL) are newly defined, separate clinicopathological entities. Both are rare lymphoma types, with low reproducibility in the diagnosis, although a conjunction of molecular and clinical studies seems to be now facilitating a more accurate diagnosis and understanding of the neoplastic process. SMZL is a disease involving the spleen, bone marrow and peripheral blood since the initial manifestations of the disease. The diagnosis has been until very recently based on the pathological study of the spleen with the conjunction of the clinical features, although the integration of the morphology in bone marrow and peripheral blood with the immunophenotype and molecular characteristics of the tumour makes a more accurate diagnosis now possible. The most frequent molecular alteration found in SMZL is allelic loss at the 7q chromosomal region. SMZL is an indolent lymphoma, although there is small subset of patients in which it follows an aggressive course. Molecular studies of SMZL are starting to reveal new diagnostic and prognostic markers, and to identify new potentially useful therapeutic targets. Nodal marginal zone lymphoma is a B-cell neoplasm originated in the lymph node, whose histology resembles the nodal infiltration by MALT- or Splenic-type marginal zone lymphoma, in the absence of clinical evidence of extranodal or spleen disease. The lack of characteristic phenotypic or molecular diagnostic findings is still hampering the reproducibility of this diagnosis. Here we review the main morphological and immunophenotypical markers, discussing the differential with other overlapping entities, singularly follicular lymphoma. Specific therapeutic protocols and prognostic factors are required to more precisely define this tumour.

Abstract: FOXP3 is a forkhead transcription factor family member, implicated in T-cell regulation, activation and differentiation. FOXP3 has been shown to be a master control gene for the development and function of CD4+/CD25+ regulatory T-cells (T(reg)). In this study, FOXP3 protein expression has been analysed using a new anti-FOXP3 monoclonal antibody in 172 paraffin-embedded lymphoma samples. FOXP3 expression in tumour cells was confined to adult T-cell leukaemia/lymphoma (ATLL) cases (17/25, 68%), with some variability in the intensity of the staining and the proportion of positive cells. No other lymphoma types studied exhibited FOXP3 expression in the malignant population. The selective expression of FOXP3 by tumour cells in ATLL makes this antibody a potentially useful diagnostic tool.

Abstract: AIMS: To describe the features of a series of nine cases of diffuse large B-cell lymphoma (DLBCL) showing morphological and immunophenotypic features that are intermediate with Hodgkin's lymphoma (HL). METHODS AND RESULTS: Most cases (6/9) presented as mediastinal tumours affecting young males, while the other three cases arose in extramediastinal locations. Histopathologically, tumours showed diffuse large cell areas in a polymorphous background, with pleomorphic cytology and the common presence of Hodgkin's and Reed-Sternberg cells. Immunophenotypically, tumours shared features of DLBCL and classical HL, with expression of CD30, CD15 (6/9), and a full B-cell profile including CD45RB, CD20, CD79a and OCT2. Epstein-Barr virus-latent membrane protein expression was found in 2/9 cases. The majority of tumours had immunohistochemical features consistent with activation of the NF-(kappa)B pathway, including nuclear location of the c-REL/p65 subunit, overexpression of phosphorylated I(kappa)B(alpha), and overexpression of NF-(kappa)B targets. Finally, 2/9 cases showed 3q27 (BCL6) rearrangement, and 1/9 had p53 gene mutations, both of which are rarely detected in classical HL. CONCLUSIONS: These findings suggest that DLBCLs with HL features constitute a distinctive subgroup of aggressive lymphomas whose neoplastic growth and peculiar characteristics could be facilitated by a particular microenvironment found in the mediastinum.

Abstract: PURPOSE: Recent studies of Hodgkin's lymphoma (HL) have suggested that the presence of regulatory T cells in the reactive background may explain the inhibition of the antitumoral host immune response observed in these patients. This study aimed to assess the relevance of regulatory T cells and CTLs present in the background of HL samples in the prognosis of a series of classic HL (cHL) patients. EXPERIMENTAL DESIGN: Expression of granzyme B and TIA-1 (markers for CTL) and FOXP3 (a marker for regulatory T cells) were evaluated independently by immunohistochemistry in tissue microarrays of 257 cHL patients and correlated with patient outcome. RESULTS: The combined influence of the presence of FOXP3(+) and TIA-1(+) cells distinguished three risk groups of patients with 5-year overall survival of 100%, 88%, and 73%. The presence of a small number of FOXP3(+) cells and a high proportion of TIA-1(+) cells in the infiltrate represent an independent prognostic factor that negatively influenced event-free survival and disease-free survival in cHL. Compared with the features at diagnosis, relapsed samples tended to have more TIA-1(+) cells and a lower proportion of FOXP3(+) cells in the reactive background. CONCLUSIONS: These data suggest that low infiltration of FOXP3(+) cells in conjunction with high infiltration of TIA-1(+) cells in cHL may represent biological markers predicting an unfavorable outcome. Moreover, the variation of these markers over the course of the disease implies a possible role for them in the progression of HL cases.

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Abstract: Mantle cell lymphoma is routinely considered as a Bcl6-negative B-cell lymphoma carrying the translocation t(11;14). Here we describe a series of five Bcl6-positive mantle cell lymphoma cases, including three classic and two blastoid variants. The proliferative index of these cases, measured with the Ki-67 antibody, was slightly higher than in Bcl6-negative mantle cell lymphoma cases (32.2 vs. 23.7%) Bcl6 expression was associated with translocations involving 3q27 in four of the five cases and an extra copy of the BCL6 gene in the fifth. A mutational study of the major mutational cluster in the BCL6 gene revealed no increased mutation rate, except in one case. One of the three cases displayed a high mutational index in the IgVH gene, suggesting exposure to a germinal center microenvironment. Chromosomal alterations involving 3q27 seem to be responsible for this increased Bcl6 expression, which needs to be considered when Bcl6 is used in lymphoma diagnosis.

Abstract: To study the oncogenic potential of Rgr in vivo, we have generated several transgenic Rgr mouse lines, which express the oncogene under the control of different promoters. These studies revealed that Rgr expression leads to the generation of various pathological alterations, including fibrosarcomas, when its transgenic expression is restricted to nonlymphoid tissues. Moreover, the overall incidence and latency of fibrosarcomas were substantially increased and shortened, respectively, in a p15INK4b-defective background. More importantly, we also have demonstrated that Rgr expression in thymocytes of transgenic mice induces severe alterations in the development of the thymocytes, which eventually lead to a high incidence of thymic lymphomas. This study demonstrates that oncogenic Rgr can induce expression of p15INK4b and, more importantly, that both Rgr and p15INK4b cooperate in the malignant phenotype in vivo. These findings provide new insights into the tumorigenic role of Rgr as a potent oncogene and show that p15INK4b can act as a tumor suppressor gene.

Abstract: Hodgkin's lymphoma can be considered in most cases a B-cell lymphoma due to the presence of potentially functional immunoglobulin (Ig) gene rearrangements in the neoplastic cells. In contrast to lymphocyte-predominant Hodgkin's lymphoma, Hodgkin/Reed-Sternberg (HRS) cells from classical Hodgkin's lymphoma have low frequency of B-cell marker expression and lack Ig light and Ig heavy messenger RNA. Recent studies have shown transcription machinery deficiency in Hodgkin's lymphoma caused by an absence of the transcription factors OCT.1, OCT.2 and/or BOB.1. By using the tissue microarray technique, we have performed an immunohistochemical study of OCT.1, OCT.2 and BOB.1 in 325 classical Hodgkin's lymphoma cases. The results have been correlated with the expression of the B-cell markers CD20, CD79a, B-cell-specific activator protein (BSAP) and MUM.1, the presence of Epstein-Barr virus and the histological subtype. The percentage of CD20 and CD79a positivity was low (18 and 18%, respectively), whereas MUM.1 and BSAP were positive in the majority of cases. Considering the positive cases with independence of the intensity of staining, 62% of them expressed OCT.2, 59% OCT.1 and 37% BOB.1. Nevertheless, when we considered only the strongly positive cases, the results were similar to those previously described by others. No statistical association was found between the transcription factor expression, histological subtype and Epstein-Barr virus presence. To our knowledge, this is the largest series of classical Hodgkin's lymphoma cases in which the expression of transcription factors has been studied. We have found a notorious percentage of cases displaying weak positivity for OCT.2 and BOB.1 factors in HRS cells. We propose that other mechanisms different from the absence of transcription factors OCT.2 and BOB.1 might be involved in the control of Ig transcription and B lineage in classical Hodgkin's lymphoma.

Abstract: The Polycomb group (PcG) of proteins comprises a family of repressors of homeobox genes that play key roles in body formation, haematopoiesis and cell cycle control. In this study, a large-scale analysis of PcG protein expression (BMI1, MEL18, PH1, RNF2, RING1, and RYBP) was performed in 321 Hodgkin's lymphoma (HL) biopsies and in reactive lymphoid tissues using tissue microarrays. The relevance of PcG proteins in HL was also investigated by the simultaneous analysis of PcG and other proteins involved in the control of cell cycle, transcription machinery and lymphoid differentiation. The analysis revealed increased expression of a set of PcG proteins (particularly RYBP and BMI1) in tumour cells in comparison with reactive lymphoid tissue. One of the most striking findings was anomalous RYBP expression in 55% of classical HL cases associated with an unfavourable response to treatment and shorter survival. The data obtained in this study also show an association of PcG proteins with E2F6 and NFkappaB transcription factors. The statistical relationship between PcG and NFkappaB activation was further explored in HL-derived cell lines treated with curcumin, an NFkappaB inhibitor, and TNFalpha. Up- or downregulation of MEL18 was paralleled by loss or gain of activated NFkappaB, which suggests that NFkappaB may regulate expression of this protein. Investigation of the relationship between E2F6 and RING1 by immunofluorescence and confocal analysis, in HL cell lines and paraffin sections, revealed co-expression of both proteins in the same tumour cells. These results allow us to propose that the formation of transcription complexes with E2F6 may modify the functional status of PcG proteins in HSR cells.

Abstract: PURPOSE: Based on previous preliminary observations, we hypothesize that the molecular and clinical variability of chronic lymphocytic leukemia (CLL) reflects differences in the degree of nuclear factor (NF)-kappaB activation, as determined by the expression of phosphorylated IkappaBalpha (p-IkappaBalpha). EXPERIMENTAL DESIGN: The expression profile (mRNA and protein expression) was analyzed with the Centro Nacional de Investigaciones Oncologicas Oncochip, a cDNA microarray containing 6386 cancer-related genes, and a tissue microarray (TMA). The results were correlated with the IgV(H) mutational status, ZAP-70 expression, cytogenetic alterations, and clinical outcome. RESULTS: We found correlations between the presence of p-IkappaBalpha, a surrogate marker of NF-kappaB activation, and changes in the expression profile (mRNA and protein expression) and clinical outcome in a series of CLL cases with lymph node involvement. Activation of NF-kappaB, as determined by the expression of p-IkappaBalpha, was associated with the expression of a set of genes comprising key genes involved in the control of B-cell receptor signaling, signal transduction, and apoptosis, including SYK, LYN, BCL2, CCR7, BTK, PIK3CD, and others. Cases with increased expression of p-IkappaBalpha showed longer overall survival than cases with lower expression. A Cox regression model was derived to estimate some parameters of prognostic interest: IgV(H) mutational status, ZAP-70, and p-IkappaBalpha expression. The multivariate analysis disclosed p-IkappaBalpha and ZAP-70 expression as independent prognostic factors of survival. CONCLUSIONS: A variable degree of activation of NF-kappaB, as determined by the expression of p-IkappaBalpha, is an identifiable event in CLL, and is correlated with changes in the expression profile and overall survival.

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Abstract: PURPOSE: Current therapies fail to cure a significant proportion of patients with Hodgkin's lymphoma (HL). Predictive systems for stratification of the disease and selection of treatment based on sets of clinical variables, such as the international prognostic score (IPS), are of relatively small practical value. The predictive use of biologic parameters has so far provided limited and inconsistent results. Here we explore the influence of a set of molecular markers on the outcome of HL. PATIENTS AND METHODS: Forty molecular markers involved in B-cell differentiation and activation, signal transduction, cell cycle, and apoptosis control were analyzed in 259 classic HL patient cases by using tissue microarrays. Univariate analysis was performed to evaluate the influence of markers on favorable outcome (complete remission of > 12 months). Significant variables were included in a multivariate logistic regression analysis, and the probability of favorable outcome was estimated. RESULTS: Univariate analysis revealed four molecular markers that predicted outcome, and the multivariate analysis showed p53, Bcl-X(L), and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) to have independent significance. The combination of these factors determined two groups of patients (group I, zero to one factor; group II, two to three factors) with a probability of a favorable outcome of.948 and.687, respectively. A multivariate Cox's model shows that these biologic risk groups have special predictive power in low-IPS patients. CONCLUSION: The data from this exploratory study suggest that the accumulation of molecular events seems to influence the outcome of HL, particularly in the low-IPS group.

Abstract: Diffuse large B-cell lymphoma (DLBCL) patients are treated using relatively homogeneous protocols, irrespective of their biological and clinical variability. Here we have developed a protein-expression-based outcome predictor for DLBCL. Using tissue microarrays (TMAs), we have analyzed the expression of 52 selected molecules in a series of 152 DLBCLs. The study yielded relevant information concerning key biological aspects of this tumor, such as cell-cycle control and apoptosis. A biological predictor was built with a training group of 103 patients, and was validated with a blind set of 49 patients. The predictive model with 8 markers can identify the probability of failure for a given patient with 78% accuracy. After stratifying patients according to the predicted response under the logistic model, 92.3% patients below the 25 percentile were accurately predicted by this biological score as "failure-free" while 96.2% of those above the 75 percentile were correctly predicted as belonging to the "fatal or refractory disease" group. Combining this biological score and the International Prognostic Index (IPI) improves the capacity for predicting failure and survival. This predictor was then validated in the independent group. The protein-expression-based score complements the information obtained from the use of the IPI, allowing patients to be assigned to different risk categories.

Abstract: p18INK4c is a cyclin-dependent kinase (CDK) inhibitor that interferes with the Rb-kinase activity of CDK6/CDK4. Disruption of p18INK4c in mice impairs B-cell terminal differentiation and confers increased susceptibility to tumor development; however, alterations of p18INK4c in human tumors have rarely been described. We used a tissue-microarray approach to analyze p18INK4c expression in 316 Hodgkin lymphomas (HLs). Nearly half of the HL cases showed absence of p18INK4c protein expression by Reed-Sternberg (RS) cells, in contrast with the regular expression of p18INK4c in normal germinal center cells. To investigate the cause of p18INK4c repression in RS cells, the methylation status of the p18INK4c promoter was analyzed by methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing. Hypermethylation of the p18INK4c promoter was detected in 2 of 4 HL-derived cell lines, but in none of 7 non-Hodgkin lymphoma (NHL)-derived cell lines. We also detected p18INK4c hypermethylation, associated with absence of protein expression, in 5 of 26 HL tumors. The correlation of p18INK4c immunostaining with the follow-up of the patients showed shorter overall survival in negative cases, independent of the International Prognostic Score. These findings suggest that p18INK4c may function as a tumor suppressor gene in HL, and its inactivation may contribute to the cell cycle deregulation and defective terminal differentiation characteristic of the RS cells.

Abstract: Tumoral cells in Hodgkin lymphoma (HL) display an increased growth fraction and diminished apoptosis, implying a profound disturbance of the cell cycle and apoptosis regulation. However, limitations of molecular techniques have prevented the analysis of the tumor suppressor pathways and cell-cycle checkpoints. Tissue microarray (TMA) is a powerful tool for analyzing a large number of molecular variables in a large series of tumors, although the feasibility of this technique has not yet been demonstrated in heterogeneous tumors. The expression of 29 genes regulating the cell cycle and apoptosis were analyzed by immunohistochemistry and in situ hybridization in 288 HL biopsies using TMA. The sensitivity of the technique was validated by comparing the results with those obtained in standard tissue sections. The results revealed multiple alterations in different pathways and checkpoints, including G1/S and G2/M transition and apoptosis. Striking findings were the overexpression of cyclin E, CDK2, CDK6, STAT3, Hdm2, Bcl2, Bcl-X(L), survivin, and NF-kappaB proteins. A multiparametric analysis identified proteins associated with increased growth fraction (Hdm2, p53, p21, Rb, cyclins A, B1, D3, and E, CDK2, CDK6, SKP2, Bcl-X(L), survivin, STAT1, and STAT3), and proteins associated with apoptosis (NF-kappaB, STAT1, and RB). The analysis also demonstrated that Epstein-Barr virus (EBV)-positive cases displayed a characteristic profile, confirming the pathogenic role of EBV in HL. Survival probability depends on multiple biologic factors, including overexpression of Bcl2, p53, Bax, Bcl-X(L), MIB1, and apoptotic index. In conclusion, Hodgkin and Reed-Sternberg cells harbor concurrent and overlapping alterations in the major tumor suppressor pathways and cell-cycle checkpoints. This appears to determine the viability of the tumoral cells and the clinical outcome.

Abstract: Mycosis fungoides (MF) is the most frequent type of cutaneous T-cell lymphoma, whose diagnosis and study is hampered by its morphologic similarity to inflammatory dermatoses (ID) and the low proportion of tumoral cells, which often account for only 5% to 10% of the total tissue cells. cDNA microarray studies using the CNIO OncoChip of 29 MF and 11 ID cases revealed a signature of 27 genes implicated in the tumorigenesis of MF, including tumor necrosis factor receptor (TNFR)-dependent apoptosis regulators, STAT4, CD40L, and other oncogenes and apoptosis inhibitors. Subsequently a 6-gene prediction model was constructed that is capable of distinguishing MF and ID cases with unprecedented accuracy. This model correctly predicted the class of 97% of cases in a blind test validation using 24 MF patients with low clinical stages. Unsupervised hierarchic clustering has revealed 2 major subclasses of MF, one of which tends to include more aggressive-type MF cases including tumoral MF forms. Furthermore, signatures associated with abnormal immunophenotype (11 genes) and tumor stage disease (5 genes) were identified.

Abstract: 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 IgV(H) 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.

Abstract:

Abstract: Association of Hodgkin lymphoma and non-Hodgkin lymphoma is rare and, specifically, the combination of Hodgkin lymphoma and mantle cell lymphoma has not been previously described. Here we describe composite mantle cell lymphoma and Hodgkin lymphoma affecting the spleen in one case and the eyelid and cervical lymph nodes in a second. In both, nodules of classical Hodgkin lymphoma were intermixed with diffuse or nodular areas of typical mantle cell lymphoma. Immunohistochemical and molecular analyses confirmed cyclin D1 overexpression secondary to the translocation t(11;14) in the small mantle cell lymphoma component; with CD30, CD15, and EBV expression in the Hodgkin and Reed-Sternberg cells. Finally, clonal analysis of rearranged immunoglobulin genes performed on microdissected Hodgkin and Reed-Sternberg and mantle cell lymphoma cells provided definite evidence of separate clonal origins of the two tumors in the patients. These EBV-positive, clonally unrelated tumors seem to represent true composite neoplasms, in contrast to cases showing merely clonal progression.

Abstract: p14(ARF), the alternative product from the human INK4a/ARF locus, antagonizes Hdm2 and mediates p53 activation in response to oncogenic stimuli. An immunohistochemical study of p14(ARF) expression in 74 samples of aggressive B-cell lymphomas was performed, demonstrating an array of different abnormalities. A distinct nucleolar expression pattern was detected in nontumoral tissue and a subset of lymphomas (50/74). In contrast, a group of cases (8/74) showed absence of p14(ARF) expression, dependent either on promoter hypermethylation or gene loss. Additionally, 16 out of 74 cases displayed an abnormal nuclear p14(ARF) overexpression not confined to the nucleoli, as confirmed by confocal microscopy, and that was associated with high levels of p53 and Hdm2. A genetic study of these cases failed to show any alteration in the p14(ARF) gene, but revealed the presence of p53 mutations in over 50% of these cases. An increased growth fraction and a more aggressive clinical course, with a shortened survival time, also characterized the group of tumors with p14(ARF) nuclear overexpression. Moreover, this p14(ARF) expression pattern was more frequent in tumors displaying accumulated alterations in the p53, p16(INK4a), and p27(KIP1) tumor supressors. These observations, together with the consideration of the central role of p14(ARF) in cell cycle control, suggest that p14(ARF) abnormal nuclear overexpression is a sensor of malfunction of the major cell cycle regulatory pathways, and consequently a marker of a high tumor aggressivity.

Abstract: The development of human cancers is frequently associated with the silencing of the two major tumor suppressor pathways represented by retinoblastoma protein and p53. As the incidence of p53 mutations is significantly lower in Hodgkin's lymphoma than in other neoplasias, we investigated whether the malfunction of other proteins in this pathway could be responsible for its inactivation. Because the existence of nucleolar complexes between p14(ARF) and Hdm2 has been described as having a critical effect on p53 function by inhibiting its degradation, we analyzed the expression and subcellular localization of these proteins in 52 cases and in Hodgkin's cell lines. Two of four cell lines revealed loss of p14(ARF) expression secondary to gene promoter methylation, this being mutually exclusive with p53 mutations (1 of 4), illustrating the existence of selective pressure to inactivate the p53 pathway. The majority of Hodgkin's samples showed a strong nucleolar expression of p14(ARF) that was not associated with Hdm2. They also showed the existence of Hdm2/p53 complexes, and the absence of complexes containing either p14(ARF)/Hdm2 or p14(ARF)/p53. The different localization of Hdm2 (nucleoplasm) and p14(ARF) (nucleoli) observed in Hodgkin's tumors and cell lines is associated with the presence of short alternatively spliced transcripts of Hdm2 lacking the ARF-binding region and the nuclear export signal. The absence of these p14(ARF)/Hdm2 nucleolar complexes could be sufficient to inactivate the pathway and may explain the low frequency of p53 mutations in this tumor.

Abstract: Oct1 and Oct2 are transcription factors of the POU homeo-domain family that bind to the Ig gene octamer sites, regulating B-cell-specific genes. The function of these transcription factors is dependent on the activity of B-cell-restricted coactivators such as BOB.1/OBF.1. Independent studies of the expression of these proteins in non-Hodgkin's lymphoma have been restricted to single markers, and most lack data concerning immunohistochemical expression. Thus, we have investigated the expression of Oct1, Oct2, and BOB.1/OBF.1 in human reactive lymphoid tissue and in a series of 140 Hodgkin and non-Hodgkin's lymphomas. None of these proteins was found to be restricted to B cells, although only B cells expressed high levels of all three markers. Additionally, germinal center B cells showed stronger Oct2 and BOB.1/OBF.1 staining. Consequently, most B-cell lymphomas showed reactivity for all three antibodies. Oct2 expression was significantly higher in germinal center-derived lymphomas, although other B-cell lymphomas also displayed a high level of Oct2 expression. Although T-cell lymphomas and Hodgkin's lymphomas expressed some of these proteins, they commonly exhibited less reactivity than B-cell lymphomas. Despite not being entirely cell-specific, the strong nuclear expression of Oct2 and BOB.1/OBF.1 by germinal center- derived lymphomas makes these antibodies a potentially useful tool in lymphoma diagnosis.

Abstract: Monocytoid B cells (MBCs) are a subset of B cells that may be recognized in several reactive and tumoral lymph node conditions, including toxoplasmic lymphadenitis, infectious mononucleosis, and Hodgkin's lymphoma. Although this is a commonly observed cell population, which has even given its name to a type of lymphoma, MBC lymphoma, scarcely any information is available about the function and characteristics of this cell type. A relationship with marginal zone (MZ) B lymphocytes has been claimed for MBCs, but this has not yet been fully proven. Indeed, specific markers for MBCs are still lacking, which has made it difficult to analyze their relationship with other B cell subpopulations and confirm the existence of tumors deriving from this B cell subset. We used a panel of cell cycle markers to explore the characteristics of MBCs and their relationship with MZ B cells, nodal MZ lymphoma, and splenic MZ lymphoma. We therefore compared the phenotypic profile of MBCs in different conditions with normal MZ B cells within the spleen and mesenteric lymph nodes, with a group of seven cases of nodal MZ/MBC lymphoma and another group of five cases of splenic MZ lymphoma. MBCs were mainly in the G(0) to G(1) phases, as deduced from the presence of a proportion of between 10 and 35% Ki67-positive cells, whereas very low expression was observed with cyclin A and cyclin B staining. Nests of MBCs were clearly labeled by the expression of p21(WAF1), a cyclin-dependent kinase inhibitor (CKI), rarely detectable in benign lymphocytes, and by cyclin E. Basically all MBCs were bcl-2-negative, and high cyclin D2 and cyclin D3 were also detected in these cells, at proportions and intensities above expected levels, when the percentage of proliferating cells was taken into account. p27(KIP1) expression was characterized by homogeneous reactivity, higher than that observed in other B cell populations with a relatively high-growth fraction. Immunoglobulin staining showed undetectable light and heavy chains. However, splenic MZ cells, nodal MZ lymphoma, and splenic MZ lymphoma showed a distinct expression of IgM and bcl-2, with high p27 (KIP1) nuclear expression and undetectable or low levels of cyclin A, B, E, or D, or p21(WAF1) expression. The data from this study show an unexpected immunophenotype in MBCs, different from the one observed in splenic and lymph node MZ B cells. This suggests that either MBCs are a unique B cell population from a distinct cell lineage, or if related to MZ cells, they would represent a definite differentiation stage characterized by a distinctive immunophenotype. They also show so-called MZ/MBC lymphoma to be more closely related to lymph node and splenic MZ B cells, as they do not share the most distinctive features of MBCs.

Abstract: The description of primary cutaneous follicular lymphoma has raised interest in the differential diagnosis of this versus disseminated follicular lymphoma involving the skin. We report here on four cases of Stage IV follicular lymphoma, diagnosed in skin biopsy, in which cutaneous lesion was the most noticeable feature of clinical presentation. In all cases, the morphological features were superimposed over typical nodal follicular lymphoma. Apart from classic B-cell markers, they were characterized by CD10 and bcl6 positivity, markers of follicle germinal center cells; and bcl2 expression, with a corresponding t(14;18) translocation in three of three cases examined. In all four cases, bone marrow study and clinical staging revealed disease that had disseminated since diagnosis. Follow-up showed relapsing cutaneous and nodal disease in two cases. The only difference observed with a control group of 10 cases of primary cutaneous follicular lymphoma was the absence in this group of t(14; 18). Disseminated classical follicular lymphoma has to be considered in the differential diagnosis of follicular lymphoma presenting in the skin. This series of cases suggests that the presence of t(14;18) could imply the existence of disease that has disseminated beyond the skin and that cases harboring this translocation could be candidates for systemic polychemotherapy.

Abstract: Here we have addressed the role that zetaPKC plays in NF-kappaB activation using mice in which this kinase was inactivated by homologous recombination. These mice, although grossly normal, showed phenotypic alterations in secondary lymphoid organs reminiscent of those of the TNF receptor-1 and of the lymphotoxin-beta receptor gene-deficient mice. The lack of zetaPKC in embryonic fibroblasts (EFs) severely impairs kappaB-dependent transcriptional activity as well as cytokine-induced phosphorylation of p65. Also, a cytokine-inducible interaction of zetaPKC with p65 was detected which requires the previous degradation of IkappaB. Although in zetaPKC-/- EFs this kinase is not necessary for IKK activation, in lung, which abundantly expresses zetaPKC, IKK activation is inhibited.

Abstract: Many human tumors harbor mutations that result in deregulation of Cdk4 activity. Most of these mutations involve overexpression of D-type cyclins and inactivation of INK4 inhibitors. In addition, a mutation in the Cdk4 protein has been described in patients with familial melanoma (Wolfel, T., Hauer, M., Schneider, J., Serrano, M., Wolfel, C., et al. (1995) Science 269, 1281-1284; Zuo, L., Weger, J., Yang, Q., Goldstein, A. M., Tucker, M. A., et al. (1996) Nat. Genet. 12, 97-99). This mutation, R24C, renders the Cdk4 protein insensitive to inhibition by INK4 proteins including p16(INK4a), a major candidate for the melanoma susceptibility locus. Here we show that knock-in mice expressing a Cdk4 R24C allele are highly susceptible to melanoma development after specific carcinogenic treatments. These tumors do not have mutations in the p19(ARF)/p53 pathway, suggesting a specific involvement of the p16(INK4a)/Cdk4/Rb pathway in melanoma development. Moreover, by using targeted mice deficient for other INK4 inhibitors, we show that deletion of p18(INK4c) but not of p15(INK4b) confers proliferative advantage to melanocytic tumor growth. These results provide an experimental scenario to study the role of Cdk4 regulation in melanoma and to develop novel therapeutic approaches to control melanoma progression.

Abstract: An 86-year-old woman presented with a 3-year history of an erythematous axillary lesion, which was histologically confirmed to be extramammary Paget's disease (EMPD) confined to the epidermis and adnexa. Surprisingly, spontaneous clinical regression occurred in the lesion, but Paget's cells persisted within the epidermis and adnexa on histologic examination. One year of intermittent topical chemotherapy with 5-fluorouracil resulted in ulcers that were interpreted as EMPD and completely excised. Histologic examination showed a complete absence of Paget's cells. To our knowledge, only one previous report investigated apparent spontaneous clinical resolution with histologic persistence of EMPD. We emphasize that topical 5-fluorouracil cannot be considered a safe treatment modality for EMPD, but it may be useful in certain cases in which the extent of the lesions, or the general condition of the patient, advise against surgery or radiotherapy.

Abstract: p16 protein binds and inactivates cyclin D-CDK4/6 complexes, stopping the cell cycle at the G1/S boundary. Loss of p16 expression is found frequently in human cancer tissues, often resulting from allelic loss or promoter region hypermethylation in non-Hodgkin's lymphomas. Hodgkin's disease has been shown to be a monoclonal neoplasm of B-cells in which a majority of cells are cycling. In the attempt to identify hypothetical CDK inhibitor inactivation that could explain the accumulation of proliferating cells, we decided to focus on the p16INK4A gene. To determine whether inactivation of this gene is implicated in the development of Hodgkin's disease, we immunostained 40 cases with a monoclonal antibody for the p16 protein. At the same time, we used a methylation-specific PCR technique to determine the methylation status of exon 1 of the p16INK4A gene in 23 cases in this series. Loss of p16 expression was found in 30 of 37 cases (absence of expression in most Hodgkin's/Reed-Sternberg cells, with a normal scattered pattern of p16 expression in the reactive background). Only seven samples showed nuclear p16 expression in a significant proportion of large tumoral cells. In agreement with this finding, hypermethylation of p16INK4A gene was found in 14 of 23 cases by PCR. All the p16 cases found positive by immunohistochemistry also showed unmethylated DNA. These results show that loss of p16 protein expression is usually observed in Hodgkin's/Reed-Sternberg cells in Hodgkin's disease, frequently associated with p16INK4A gene hypermethylation. The high frequency of abnormal methylation found in this study suggests that this genetic event may play an important role in the pathogenesis of the disease.

Abstract: Cell cycle progression is regulated by the combined action of cyclins, cyclin-dependent kinases (CDKs), and CDK-inhibitors (CDKi), which are negative cell cycle regulators. p27KIP1 is a CDKi key in cell cycle regulation, whose degradation is required for G1/S transition. In spite of the absence of p27KIP1 expression in proliferating lymphocytes, some aggressive B-cell lymphomas have been reported to show an anomalous p27KIP1 staining. We analysed p27KIP1 expression in a series of Diffuse Large B-cell Lymphoma (DLBCL), correlating it with the proliferative index and clinical outcome, to characterize the implications of this anomalous staining in lymphomagenesis in greater depth. For the above mentioned purposes, an immunohistochemical technique in paraffin-embedded tissues was employed, using commercially available antibodies, in a series of 133 patients with known clinical outcomes. Statistical analysis was performed in order to ascertain which clinical and molecular variables may influence outcome, in terms of disease-free survival (DFS) and overall survival (OS). The relationships between p27KIP1 and MIB-1 (Ki-67) were also tested. An abnormally high expression of p27KIP1 was found in lymphomas of this type. The overall correlation between p27KIP1 and MIB-1 showed there to be no significant relationship between these two parameters, this differing from observations in reactive lymphoid and other tissues. Analysis of the clinical relevance of these findings showed that a high level of p27KIP1 expression in this type of tumour is an adverse prognostic marker, in both univariate and multivariate analysis. These results show that there is abnormal p27KIP1 expression in DLBCL, with adverse clinical significance, suggesting that this anomalous p27KIP1 protein may be rendered non-functional through interaction with other cell cycle regulator proteins.

Abstract: Type-1 neurofibromatosis (NF-1) or Von Recklinghausen disease is an autosomal dominant hereditary condition that may affect the gastrointestinal tract in 25% of cases and which takes three main forms: ganglioneuromatosis, stromal tumors, and tumors in the duodenum and periampullar region. Not infrequently, these patients present with gastrointestinal bleeding. We present the case of a 48-year-old patient diagnosed as having NF-1, with relapsing episodes of gastrointestinal hemorrhage, in which we discovered the simultaneous presence of a stromal tumor in the jejunum together with polypoid and diffuse ganglioneuromatosis in the colon.

Abstract: p27 cyclin-dependent kinase inhibitor downregulation is essential for transition to the S phase of the cell cycle. Thus, proliferating cells in reactive lymphoid tissue show no detectable p27 expression. Nevertheless, anomalous high p27 expression has been shown to be present in a group of aggressive B-cell lymphomas with high proliferation index and adverse clinical outcome. This suggests that abnormally accumulated p27 protein has been rendered functionally inactive. We analyzed the causes of this anomalous presence of p27 in a group of aggressive B-cell lymphomas, including 54 cases of diffuse large B-cell lymphomas and 20 Burkitt's lymphomas. We simultaneously studied them for p27, cyclin D3, cyclin D2, cyclin D1, and cyclin E expression, because it has been stated that high levels of expression of cyclin D1 or E lead to increased p27 levels in some cell types. A statistically significant association between p27 and cyclin D3 expression was found for the group as a whole. Additionally, when dividing the cases according to the level of expression of cyclin D3 by reactive germinal centers, it was observed that cases with stronger cyclin D3 expression also show higher p27 expression. The relationship between both proteins was also shown at a subcellular level by laser confocal studies, showing that in cases with high expression of both proteins there was a marked colocalization. Additional evidence in favor of p27 sequestration by cyclin D3 was provided by coimmunoprecipitation studies in a Burkitt's cell line (Raji) showing the existence of cyclin D3/p27 complexes and the absence of CDK2/p27 complexes. These results could support the hypothesis that there are cyclin D3/p27 complexes in a subset of aggressive B-cell lymphomas in which p27 lacks the inhibitory activity found when it is bound to cyclin E/CDK2 complexes. This interaction between both proteins could lead to an abnormal nuclear accumulation, detectable by immunohistochemical techniques.

Abstract: AIMS: Crystal-storing histiocytosis is a rare disorder described in patients with lymphoproliferative diseases, mainly in cases of multiple myeloma but also in lymphoplasmacytic lymphoma (immunocytoma). Most cases involve one single organ which, in the majority, is related directly to the presence of tumour. We describe a 44-year-old man with a clinical picture of multifocal fibrosclerosis (with mesenteric panniculitis, peritoneal, mediastinal and orbital fibrosis) in which the autopsy showed a systemic infiltrate of crystal-storing histiocytes and functional alteration of the organs involved, associated with IgG-kappa type immunocytoma. METHODS AND RESULTS: Histology showed a systemic infiltration, with a predilection for adipose tissue, by a diffuse cellular infiltrate composed of small lymphocytes, plasmacytoid lymphocytes and plasma cells, admixed with large number of crystal-storing histiocytes. Intracytoplasmic crystals were not identified either in the plasma cells or plasmacytoid lymphocytes. The neoplastic cells and the crystalline inclusions displayed reactivity with antibodies for IgG and the kappa light chain. A polymerase chain reaction study for the IgH gene showed a monoclonal rearrangement. Ultrastructural studies showed needle-shaped crystals surrounded by a single unit membrane. CONCLUSION: This case is, to the authors' knowledge, the first to be described in which crystal-storing histiocytosis is associated with a clinical picture of multifocal fibrosclerosis, which suggests that lymphoproliferative processes should be considered in the differential diagnosis of the various conditions associated with multifocal fibrosclerosis.

Abstract: PURPOSE: The goal of this work was to perform a comprehensive exploration of the relationship between the clinical outcome of diffuse large B-cell lymphoma (DLBCL) and the expression of a panel of tumor suppressor and oncogenic proteins, which includes some cell-cycle regulator proteins involved in the p53 pathway. PATIENTS AND METHODS: To this end, we collected the clinical data of 141 patients with DLBCL and immunohistochemically analyzed diagnostic tumoral tissue from each patient for the presence of Ki67 (MIB1, Immuno-tech, Marseille, France), bcl2, p53, p21/WAF1, MDM2, and retinoblastoma (Rb) proteins. RESULTS: The results show that several proteins are associated with some of the clinical traits analyzed. Multivariate analysis showed that an extended overall survival (OS) time was associated with low growth fraction, high Rb protein, and low MDM2 expression, as well as with known clinical parameters. The probability of inducing a complete remission (CR) was only associated with clinical parameters, although univariate study showed that a low growth fraction was associated with a higher probability of inducing a CR. Univariate study of disease-free survival (DFS) showed that tumors with high bcl2 expression and nodal origin have a shorter DFS time, although multivariate study only confirmed the adverse effect of bcl2 expression. CONCLUSION: Taking all these results into consideration, it seems that although the overall outcome for patients with DLBCL is decided by a combination of different clinical and biologic variables, the expression of some of these cell-cycle regulator proteins appears to be specifically associated with the different clinical features of tumors.

Abstract: The CDKN2A gene located on chromosome region 9p21 encodes the cyclin-dependent kinase-4 inhibitor p16/INK4A, a negative cell cycle regulator. We analyzed p16/INK4A expression in different types of non-Hodgkin's lymphoma to determine whether the absence of this protein is involved in lymphomagenesis, while also trying to characterize the genetic events underlying this p16/INK4A loss. To this end, we investigated the levels of p16/INK4A protein using immunohistochemical techniques in 153 cases of non-Hodgkin's lymphoma, using as reference the levels found in reactive lymphoid tissue. The existence of gene mutation, CpG island methylation, and allelic loss were investigated in a subset of 26 cases, using single-strand conformational polymorphism and direct sequencing, Southern Blot, polymerase chain reaction, and microsatellite analysis, respectively. Loss of p16/INK4A expression was detected in 41 of the 112 non-Hodgkin's lymphomas studied (37%), all of which corresponded to high-grade tumors. This loss of p16/INK4A was found more frequently in cases showing tumor progression from mucosa-associated lymphoid tissue low-grade lymphomas (31 of 37) or follicular lymphomas (4 of 4) into diffuse large B-cell lymphomas. Analysis of the status of the p16/INK4A gene showed different genetic alterations (methylation of the 5'-CpG island of the p16/INK4A gene, 6 of 23 cases; allelic loss at 9p21, 3 of 16 cases; and nonsense mutation, 1 of 26 cases). In all cases, these events were associated with loss of the p16/INK4A protein. No case that preserved protein expression contained any genetic change. Our results demonstrate that p16/INK4A loss of expression contributes to tumor progression in lymphomas. The most frequent genetic alterations found were 5'-CpG island methylation and allelic loss.

Abstract: The p53 tumour suppressor gene is a cell cycle regulator, able to induce cell cycle arrest to allow DNA repair or apoptosis. The molecular mechanisms underlying p53 action imply transactivation of p53 dependent genes such as WAF1 (for wild type p53 associated fragment 1) and the murine double minute (MDM2) gene. In some cases, inactivation of the p53 gene results from p53 gene mutations leading to p53 protein accumulation, but in others it may results from mechanisms other than mutation, such as interaction with viral or cellular proteins. The expression of p53 protein and p53 transactivated gene proteins p21/WAF1 and MDM2, combined with in situ detection of apoptosis, was studied in specimens of CMV-infected patients as an in vivo model of p53 alteration not due to point mutation. p53 positivity was found in CMV + cells in different tissues, in cells with typical inclusion bodies, and in in situ hybridization and immunohistochemistry CMV + cells without inclusions (hidden infection). Although this p53 reactivity was accompanied by the expression of MDM2 and p21/WAF1 proteins, the patterns of MDM2 and p21/WAF1 protein expression were mutually exclusive, and were associated with the presence or absence of inclusion bodies. Nuclei bearing inclusion bodies were usually MDM2+, p21/ WAF1-, while hidden infected cells were usually MDM2-, p21/WAF1+. Apoptosis was not detected in any tissue section from CMV-infected patients. Two alternative patterns were found in CMV-infected tissues: p53+, p21/WAF1+, MDM2-, or p53+, p21/WAF1-. MDM2+ protein expression. These may represent examples of p53 dependent alternative effects in the course of CMV infection. Early stages are represented by CMV + cells without inclusion bodies, which display p53 and p21/ WAF1 expression, suggesting that p53 could be acting as a growth suppressor protein. Late CMV infection is represented by cells harbouring inclusion bodies. These cells showed a p53+, p21/WAF1-, MDM2+ profile, consistent with MDM2 mediated p53 inactivation. The absence of p21/WAF1 expression and lack of apoptosis suggest that the p53 protein expressed by MDM2+ cells could be functionally inactivated in CMV-infected cells with inclusion bodies. Previous studies have suggested that p53 inactivation by MDM2 over-expression occurs in sarcomas and lymphomas. Our observations seem to indicate that this mechanism of MDM2 mediated p53 inactivation may play a role in the late phase of CMV infection.

Abstract: Mutations in the p53 tumour suppressor gene are the most common genetic alteration found in human cancers. Most of them are accompanied by stabilization of the protein, which renders it detectable through immunohistochemical techniques. Although p53 expression is a very common finding in Hodgkin's disease (HD), the status of the p53 gene is scarcely known, due to the difficulty in sequencing this gene in a lesion in which tumour cells are thought to constitute a very minor subpopulation, diluted in a background of supposedly benign cells. The pattern of expression of two downstream p53 proteins (MDM2 and p21 WAF1/CIP1, was studied as an indirect way of assessing p53 gene status. MDM2 is a wild-p53 inducible protein which may form a complex with p53, abrogating its function, as has been found in human sarcomas and other malignancies. p21WAF1/CIP1 is another protein inducible by wild-type p53, involved in inhibiting cell-cycle progression, through binding to cyclin/cyclin-dependent-kinase complexes. MDM2 and p21WAF1/CIP1 immunostaining was detected in all the cases analysed, independently of histological type, and were mainly present in Sternberg-Reed and Hodgkin (H & SR) cells. These immunohistochemical results were confirmed by Western blotting. To study the cause of MDM2 protein accumulation, MDM2 mRNA expression was also investigated by reverse transcription polymerase chain reaction (RT-PCR). The results show the presence of MDM2 transcripts in all cases of HD, albeit at lower levels than those found in reactive lymphoid tissue. These results seem to support the hypothesis that p53 is transcriptionally active in at least some of the H & SR cells in HD, and is able to induce MDM2 and p21WAF1/CIP1 protein expression.

Abstract: Retinoblastoma (Rb) tumour-suppressor protein plays a critical role in cell cycle control. Rb inactivation is a frequent phenomenon in tumours of different cell lineages, in which the absence of Rb protein has been considered to be a marker of Rb disregulation. We used modern immunohistochemical techniques to study the expression of Rb protein in a large series of 130 patients with Hodgkin's disease. Simultaneously, Western blot was used to analyse a more restricted group (12 patients) to confirm the immunohistochemical results and to clarify the phosphorylation status of Rb protein. As the level of Rb expression varied according to cell cycle stage, we also performed immunostaining for Ki67, a protein present in proliferating cells. To make comparison possible, we first characterised the amount and phosphorylation status of Rb protein in reactive lymphoid tissue and phytohaemagglutinin (PHA)-stimulated lymphocytes. The presence of p53 in Sternberg-Reed cells was also included in the study, as both proteins (p53 and Rb) have been found to be closely associated in cell cycle control. PHA-stimulated peripheral blood lymphocytes showed a parallel increase in Rb and cell cycle progression, together with progressive Rb phosphorylation. In reactive lymphoid tissue there was also a clear correlation between Rb expression and the Ki67 proliferation index (R = 0.96, P = 0.038). When analysing Hodgkin's disease samples, a clear difference emerges between cases of nodular lymphocyte predominance, which preserve the relationship between Rb and Ki67 expression (r = 0.8727, P = 0.000), and classical forms of Hodgkin's disease (nodular sclerosis and mixed cellularity), which display a strong deviation from this pattern. Two main anomalies were found: (1) One group of 21/130 cases with partial or total loss of Rb protein expression, which could reflect the existence of genetic alterations, or an altered transcriptional or translational regulation of Rb gene. (2) Another group with an abnormally high Rb/Ki67 ratio, which could support conflicting interpretations: (i) excess Rb protein for controlling cell cycle progression; or (ii) adhesion of Rb protein to other cellular or viral proteins, such as p53 and MDM2. The results of this study indicate an anomalous pattern of expression of Rb in classical forms of Hodgkin's disease, and suggest the possibility of undertaking functional studies (E1A adhesion, p16 expression) with the aim of better characterising the status of Rb protein, and correlating these findings with clinical course in Hodgkin's disease patients.

Abstract: