Action and resistance of monoclonal CD20 antibodies therapy in B-cell Non-Hodgkin Lymphomas.

Anti-CD20 monoclonal antibodies (mAbs) have improved patient's survival with Non-Hodgkin Lymphoma, when combined with chemotherapy. Several mechanisms of action have been reported, including antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and induction of apoptosis. Despite the large amount of studies and published data, the role each mechanism played in vivo is not fully understood. Furthermore, the reason why a significant percentage of patients are refractory or resistant remains unknown. Several activated intracellular signaling pathways have been implicated in the mechanisms of resistance of rituximab. In the present manuscript, we review those mechanisms and new anti-CD20 mAbs, as well as the efforts being accomplished to overcome it, focusing on new drugs targeting pathways implicated in resistance to rituximab.

MicroRNA signatures in B-cell lymphomas.

Accurate lymphoma diagnosis, prognosis and therapy still require additional markers. We explore the potential relevance of microRNA (miRNA) expression in a large series that included all major B-cell non-Hodgkin lymphoma (NHL) types. The data generated were also used to identify miRNAs differentially expressed in Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) samples. A series of 147 NHL samples and 15 controls were hybridized on a human miRNA one-color platform containing probes for 470 human miRNAs. Each lymphoma type was compared against the entire set of NHLs. BL was also directly compared with DLBCL, and 43 preselected miRNAs were analyzed in a new series of routinely processed samples of 28 BLs and 43 DLBCLs using quantitative reverse transcription-polymerase chain reaction. A signature of 128 miRNAs enabled the characterization of lymphoma neoplasms, reflecting the lymphoma type, cell of origin and/or discrete oncogene alterations. Comparative analysis of BL and DLBCL yielded 19 differentially expressed miRNAs, which were confirmed in a second confirmation series of 71 paraffin-embedded samples. The set of differentially expressed miRNAs found here expands the range of potential diagnostic markers for lymphoma diagnosis, especially when differential diagnosis of BL and DLBCL is required.

Mantle cell lymphoma: transcriptional regulation by microRNAs.

Mantle cell lymphoma (MCL) pathogenesis is still partially unexplained. We investigate the importance of microRNA (miRNA) expression as an additional feature that influences MCL pathway deregulation and may be useful for predicting patient outcome. Twenty-three MCL samples, eight cell lines and appropriate controls were screened for their miRNAs and gene expression profiles and DNA copy-number changes. MCL patients exhibit a characteristic signature that includes 117 miRNA (false discovery rate <0.05). Combined analysis of miRNAs and the gene expression profile, paired with bioinformatics target prediction (miRBase and TargetScan), revealed a series of genes and pathways potentially targeted by a small number of miRNAs, including essential pathways for lymphoma survival such as CD40, mitogen-activated protein kinase and NF-kappaB. Functional validation in MCL cell lines demonstrated NF-kappaB subunit nuclear translocation to be regulated by the expression of miR-26a. The expression of 12 selected miRNAs was studied by quantitative PCR in an additional series of 54 MCL cases. Univariate analysis identified a single miRNA, miR-20b, whose lack of expression distinguished cases with a survival probability of 56% at 60 months. In summary, using a novel bioinformatics approach, this study identified miRNA changes that contribute to MCL pathogenesis and markers of potential utility in MCL diagnosis and clinical prognostication.

Transcriptomal profiling of the cellular response to DNA damage mediated by Slug (Snai2).

Snai2-deficient cells are radiosensitive to DNA damage. The function of Snai2 in response to DNA damage seems to be critical for its function in normal development and cancer. Here, we applied a functional genomics approach that combined gene-expression profiling and computational molecular network analysis to obtain global dissection of the Snai2-dependent transcriptional response to DNA damage in primary mouse embryonic fibroblasts (MEFs), which undergo p53-dependent growth arrest in response to DNA damage. Although examination of the response showed that overall expression of p53 target gene expression patterns was similarly altered in both control and Snai2-deficient cells, we have identified and validated candidate Snai2 target genes linked to Snai2 gene function in response to DNA damage. This work defines for the first time the effect of Snai2 on p53 target genes in cells undergoing growth arrest, elucidates the Snai2-dependent molecular network induced by DNA damage, points to novel putative Snai2 targets, and suggest a mechanistic model, which has implications for cancer management.

Hodgkin's lymphoma cells express alternatively spliced forms of HDM2 with multiple effects on cell cycle control.

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.

Splenic small B-cell lymphoma with predominant red pulp involvement: a diffuse variant of splenic marginal zone lymphoma?

AIMS: Splenic marginal zone lymphoma (SMZL) has been characterized by a micronodular pattern of infiltration, biphasic cytology, follicular replacement and the presence of marginal zone differentiation. Here we describe four cases with some distinctive features, such as diffuse splenic infiltration, lack of micronodules, marginal zone cytology, p53 inactivation and cutaneous involvement. METHODS AND RESULTS: In the course of a review of cases of SMZL, we recognized the existence of a subset of four cases of splenic B-cell lymphoma, with predominantly red pulp involvement, absence of follicular replacement, and a monomorphous population of tumoral cells resembling marginal zone B-cells, with scattered nucleolated blast cells. The immunophenotype (bcl2+, CD5-, CD10-, CD43-, CD23-, cyclin D1-, IgD- (3/4)) was consistent with SMZL. Bone marrow infiltration (4/4) and peripheral blood involvement (2/4) showed similar findings to those described for SMZL in these locations. However, unlike classical SMZL, 2/4 had cutaneous involvement, and 4/4 cases showed either p53 mutation or anomalous p53 staining (p53+, p21-). CONCLUSIONS; In spite of a diffuse pattern of splenic infiltration, cutaneous involvement and p53 alterations, these cases have findings that overlap with those corresponding to classic SMZL (symptomatology, morphology of bone marrow, lymph nodes, peripheral blood involvement, and immunophenotype). We suggest that these cases be considered a putative variant of SMZL.

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

Splenic marginal zone lymphoma (SMZL) is considered to be an indolent extranodal B-cell lymphoma. Despite its low aggressivity, histologic progression has been described in sporadic reports, although the frequency, characteristics, and underlying molecular abnormalities of this phenomenon are largely unknown. We review here the clinical, morphologic, immunohistochemical, and molecular features of a series of 12 SMZL cases that showed progression to large B-cell lymphoma (LBCL). The most frequent location of secondary LBCL was in peripheral lymph node. This occurred between 12 and 85 months after diagnosis of SMZL. However, in two cases LBCL was diagnosed at the initial stage of the disease (one spleen tumoral nodule and one hilar lymph node). The histologic and immunophenotypic features of these cases were similar to those of transformed LBCL at other sites. In four cases the immunoglobulin heavy chain gene polymerase chain study revealed the same rearrangement pattern in both primary and secondary tumors, thereby confirming their identity and excluding the possibility of a second malignancy. As is the case with other low-grade lymphoproliferative disorders, SMZL may undergo high-grade transformation. These 12 cases represent 13% of our series of SMZL with adequate follow-up. The incidence of large cell transformation in SMZL seems to be lower than in follicular lymphoma (25-60%) and mantle cell lymphoma (11-39%), although it is similar to the frequency of transformation in B-chronic lymphocytic lymphoma/small lymphocytic lymphoma (1-10%). The mean proliferative index (MIB1 staining) in initial SMZL specimens of cases with LBCL transformation was 28.6%, higher than that of MIB1 staining in the overall SMZL series (21.8%), although not statistically significantly so. p53 or p16INK4a inactivation in this series was observed in only one case, in contrast with the situation observed in chronic lymphocytic leukemia, follicular lymphoma, and mantle cell lymphoma. It seems that progression in SMZL is mainly independent of p53 or p16INK4a inactivation. The frequency of the 7q deletion in this series was 3 of 7 (42%). 7q loss may play an alternative role in the inactivation of the p53 and p16INK4a pathway, thereby favoring tumoral progression.

Overall survival in aggressive B-cell lymphomas is dependent on the accumulation of alterations in p53, p16, and p27.

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

Molecular heterogeneity of splenic marginal zone lymphomas: analysis of mutations in the 5' non-coding region of the bcl-6 gene.

Splenic marginal zone lymphoma (SMZL) has been recognized as a distinctive type of small B cell lymphoma, and defined on the basis of its morphological, phenotypic, clinical and molecular characteristics. In spite of this, the borders of the entity, the homogeneity of the cases and the presumably cell origin of SMZL remain controversial issues. The frequency of mutation in the 5' non-coding region of the bcl-6 gene has been used as a marker of germinal center derivation, which may be used to establish the molecular heterogeneity of different non-Hodgkin lymphoma (NHL) types. This roughly parallels the characteristics and frequency of the somatic hypermutations found in the immunoglobulin heavy chain variable region (IgVH) genes. This study analyzed mutations of bcl-6 in the 5' non-coding region in 22 SMZL cases and, for the purpose of comparison with different B cell subsets, in microdissected germinal centers, mantle zones and marginal zone subpopulations from reactive splenic lymphoid follicles. A majority of the SMZL cases studied, 19/22 (87%), bear unmutated bcl-6 gene, while mutation was only observed in 3/22 (13%) cases. Analysis of normal B cell subpopulations showed bcl-6 hypermutation in 3/10 (30%) germinal center clones, 5/14 (35%) marginal zone clones; and unmutated sequences in all clones derived from mantle cells. The frequency of these mutations in normal spleen confirms previous findings on the hypermutation IgVH process in normal B cell populations. The data presented here support the existence of molecular heterogeneity in this entity, and give additional results in favor of the hypothesis that, in spite of initial morphological observations, a significant proportion of SMZL cases could derive from an unmutated naive precursor, different from the marginal zone, and possibly located in the mantle zone of splenic lymphoid follicles. Thus the marginal zone differentiation of these tumors could be related more with the splenic microenvironment than it is to the histogenetic characteristics of the tumor.

Unique phenotypic profile of monocytoid B cells: differences in comparison with the phenotypic profile observed in marginal zone B cells and so-called monocytoid B cell lymphoma.

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.

Epstein-Barr virus-latent membrane protein 1 expression has a favorable influence in the outcome of patients with Hodgkin's Disease treated with chemotherapy.

The effect of molecular factors in the outcome of Hodgkin's Disease (HD) is being currently studied. In a previous series of HD, including patients treated only with radiotherapy and patients treated with chemotherapy (with or without radiotherapy), we found that a high proliferation index had an adverse influence in overall survival (OS) and in the achievement of a complete remission (CR). Loss of Rb expression also had an adverse prognostic influence in achievement of CR. On the other hand LMP1-EBV expression had a favorable influence for OS. The expression of other molecular factors, p53, bcl2 and CD15 did not show prognostic influence. In the present paper we have studied the effect of these molecular variables in 110 patients, of the previous series who had been treated with chemotherapy. A retrospective study was performed in these 110 patients with HD treated with chemotherapy (ABVD or variants, 62%, or regimes not containing adriamycin, 38%) with or without adjutant radiotherapy, collected at the 11 centers belonging to the Spanish Collaborative Group for the Study of Hodgkin's Disease. The prognostic value of clinical variables and the expression of p53, bcl2, CD15, Rb, LMP 1-EBV and proliferative fraction demonstrated with sensitive immunohistochemical methods were studied. Cox's multivariate analysis was performed to assess their influence in failure-free survival (FFS) and OS. A multivariate logistic regression analysis was performed for studying the effect of the variables in the achievement of a CR. Of the clinical variables, only advanced stage (III/IV) had a significant independent adverse influence in FFS, in OS and in the achievement of CR and advanced age in OS. Of the molecular variables, LMP1-EBV had an independent and strong favorable influence in FFS, in OS and in the achievement of CR. Rb expression had a modest favorable influence in CR. The rest of the molecular variables had no independent influence on the outcome of the disease. In conclusion these results confirm the favorable prognostic value of LMP1-EBV expression in the subset of patients with HD treated with chemotherapy.

p27KIP1 is abnormally expressed in Diffuse Large B-cell Lymphomas and is associated with an adverse clinical outcome.

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.

Anomalous high p27/KIP1 expression in a subset of aggressive B-cell lymphomas is associated with cyclin D3 overexpression. p27/KIP1-cyclin D3 colocalization in tumor cells.

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.

7q31-32 allelic loss is a frequent finding in splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL) has been recognized as an entity defined on the basis of its morphological, phenotypic, and clinical characteristic features. Nevertheless, no characteristic genetic alterations have been described to date for this entity, thus making an exact diagnosis of SMZL difficult in some cases. As initial studies showed that chromosome region 7q22-32 is deleted in some of these cases, we analyzed a larger group of SMZL and other lymphoproliferative disorders that may partially overlap with it. To better define the frequency of 7q deletion in SMZL and further identify the deleted region, polymerase chain reaction analysis of 13 microsatellite loci spanning 7q21-7q36 was performed on 20 SMZL and 26 non-SMZL tissue samples. The frequency of allelic loss in SMZL (8/20; 40%) was higher than that observed in other B-cell lymphoproliferative syndromes (2/26; 7.7%). This difference was statistically significant (P < 0.05). The most frequently deleted microsatellite was D7S487 (5/11; 45% of informative cases). Surrounding this microsatellite the smallest common deleted region of 5cM has been identified, defined between D7S685 and D7S514. By comparative multiplex polymerase chain reaction analysis, we detected a homozygous deletion in the D7S685 (7q31.3) marker in one case. These results suggest that 7q31-q32 loss may be used as a genetic marker of this neoplasia, in conjunction with other morphologic, phenotypic, and clinical features. A correlation between 7q allelic loss and tumoral progression (death secondary to the tumor or large cell transformation) in SMZL showed a borderline statistical significance. The observation of a homozygous deletion in this chromosomal region may indicate that there is a tumor suppressor gene involved in the pathogenesis of this lymphoproliferative neoplasia.

Loss of p16/INK4A protein expression in non-Hodgkin's lymphomas is a frequent finding associated with tumor progression.

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.

Analysis of the frequency of microsatellite instability and p53 gene mutation in splenic marginal zone and MALT lymphomas.

AIMS: Studies of the genetic characteristics of splenic marginal zone lymphoma (SMZL) have failed to identify genetic changes specific to this tumour. Microsatellite instability is a type of genomic instability associated with different types of human cancer. Although microsatellite instability is rare in B cell non-Hodgkin's lymphomas, it has been found in some specific subsets, such as marginal zone lymphomas arising in mucosa associated lymphoid tissue (MALT), where an association with p53 mutation has been described. Because it has been proposed that SMZL and MALT are close in histogenetic terms, this study investigated the comparative frequency of microsatellite instability and p53 mutation in patients with SMZL and MALT lymphomas. METHODS: Microsatellite instability was investigated using seven microsatellite marker loci in 14 patients with SMZL and 20 patients with MALT lymphomas. In an attempt to clarify the role of p53 gene mutation in the pathogenesis of SMZL, exons 5-8 were also investigated by polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) and direct sequencing in a total of 20 patients with SMZL and 22 patients with MALT lymphomas. RESULTS: Microsatellite instability was not detected in patients with SMZL, although five of 20 patients with MALT lymphomas had microsatellite instability. The frequency of p53 mutation was low in both series (two of 20 patients with SMZL and one of 22 patients with MALT lymphomas). No significant association was found between p53 mutation and microsatellite instability. CONCLUSIONS: These results indicate that microsatellite instability is not associated with the molecular pathogenesis of SMZL, confirming the relatively increased frequency of microsatellite instability in MALT lymphomas, and perhaps suggesting that MALT and SMZL have different mechanisms of tumorigenesis.

Adverse clinical outcome in Hodgkin's disease is associated with loss of retinoblastoma protein expression, high Ki67 proliferation index, and absence of Epstein-Barr virus-latent membrane protein 1 expression.

Previous studies have shown that in non-Hodgkin's lymphomas and others neoplasms, tumoral progression, treatment response, and outcome are related to the expression of different oncogenic and tumor suppressor proteins. This study aimed to determine the prognostic significance of the expression of p53, bcl2, retinoblastoma protein (Rb), Ki67, CD15, and latent membrane protein 1-Epstein-Barr Virus (LMP1-EBV) proteins in Hodgkin's disease. A retrospective study was performed on 140 patients collected at the 11 participating centers belonging to the Spanish Collaborative Group for the Study of Hodgkin's Disease. A highly sensitive immunohistochemical method with previous microwave-induced antigen retrieval technique was used for the demonstration of the above-mentioned proteins. A Cox's multivariate analysis was performed to evaluate the impact of the variables in the overall survival, together with a logistic regression model for the achievement of complete remission. Univariate statistical analysis confirmed the prognostic significance of the alredy known clinical parameters: stage, age over 60 years, and B symptoms. High proliferation index (Ki67) and loss of Rb expression were also found to be adverse prognostic factors influencing respectively lower overall survival and failure to achieve complete remission. Multivariate analysis confirmed the independent significance of these two parameters and additionally identifies LMP1-EBV expression as a favorable prognostic marker, in relation with overall survival. Histopathological type, p53, bcl2, and CD15 expression lack significant influence on the outcome of this series. The progression of the disease or the response to treatment in HD patients is the consequence of the interrelationship of different factors, among which LMP1 expression, loss of Rb, and high growth fraction seems to play a more relevant role.

Expression of p21WAF1/CIP1 in fetal and adult tissues: simultaneous analysis with Ki67 and p53.

AIMS: To determine the expression of p21WAF1/CIP1 in relation to the expression of Ki67 and p53 in various normal adult and fetal tissues, and to investigate its distribution throughout the cell cycle. METHODS: The expression of p21WAF1/CIP1 in relation to Ki67 and p53 was analysed in adult and fetal tissues using immunohistochemical techniques. Heat induced epitope retrieval techniques were used to characterise the presence of p21WAF1/CIP1 in different tissues, as well as to detect its distribution throughout the cell cycle. In addition, flow cytometry and western blotting were used to test whether the level of p21WAF1/CIP1 expression varied at different phases of the cell cycle in phytohaemagglutinin (PHA) stimulated lymphocytes. RESULTS: p21WAF1/CIP1 expression varied from one tissue to another, and it was restricted mainly to the squamous and glandular epithelium, where it appeared in association with p53. Human tissues in which p21WAF1/CIP1 was found showed a mutually exclusive topographical sequential expression between p21WAF1/CIP1 and Ki67. This was confirmed by double labelling studies, which showed that p21WAF1/CIP1 positive cells were in the G0 phase. Unlike these findings of a decline in p21WAF1/CIP1 expression after the G0 phase, PHA stimulated lymphocytes showed a level of p21WAF1/CIP1 expression that rose as the cell progressed through the cell cycle. CONCLUSIONS: The analysis of p21WAF1/CIP1 expression in relation to the status of p53 should take into account the existence of variable p21WAF1/CIP1 expression in different tissues. This could provide an explanation for the varying frequency of p53 mutations in tumours of different cellular origin. In tissues characterised by regular p21WAF1/CIP1 expression, it appears in a pattern that is consistent with the proposed role of this inhibitor of cyclin dependent kinases in cell cycle arrest-that of inducing cell differentiation. The conflicting results of in vivo and in vitro studies could support the hypothesis that microenvironmental conditions may influence the location of p21WAF1/CIP1 in different phases of the cell cycle.