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.

Molecular heterogeneity in chronic lymphocytic leukemia is dependent on BCR signaling: clinical correlation.

Chronic lymphocytic leukemia (CLL), the most frequent form of adult leukemia in Western countries, is characterized by a highly variable clinical course. Expression profiling of a series of 160 CLL patients allowed interrogating the genes presumably playing a role in pathogenesis, relating the expression of functionally relevant signatures with the time to treatment. First, we identified genes relevant to the biology and prognosis of CLL to build a CLL disease-specific oligonucleotide microarray. Second, we hybridized a training series on the CLL-specific chip, generating a biology-based predictive model. Finally, this model was validated in a new CLL series. Clinical variability in CLL is related with the expression of two gene clusters, associated with B-cell receptor (BCR) signaling and mitogen-activated protein kinase (MAPK) activation, including nuclear factor-kappaB1 (NF-kappaB1). The expression of these clusters identifies three risk-score groups with treatment-free survival probabilities at 5 years of 83, 50 and 17%. This molecular predictor can be applied to early clinical stages of CLL. This signature is related to immunoglobulin variable region somatic hypermutation and surrogate markers. There is a molecular heterogeneity in CLL, dependent on the expression of genes defining BCR and MAPK/NF-kappaB clusters, which can be used to predict time to treatment in early clinical stages.

Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

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.

Identification of genes involved in imatinib resistance in CML: a gene-expression profiling approach.

The use of the tyrosine kinase inhibitor imatinib, which blocks the enzymatic action of the BCR-ABL fusion protein, has represented a critical advance in chronic myeloid leukemia (CML) treatment. However, a subset of patients initially fails to respond to this treatment. Use of complementary DNA (cDNA) microarray expression profiling allows the identification of genes whose expression is associated with imatinib resistance. Thirty-two CML bone marrow samples, collected before imatinib treatment, were hybridized to a cDNA microarray containing 6500 cancer genes, and analyzed using bootstrap statistics. Patients refractory to interferon-alpha treatment were evaluated for cytogenetic and molecular responses for a minimum of 12 months. A set of 46 genes was differentially expressed in imatinib responders and non-responders. This set includes genes involved in cell adhesion (TNC and SCAM-1), drug metabolism (cyclooxygenase 1), protein tyrosine kinases and phosphatases (BTK and PTPN22). A six-gene prediction model was constructed, which was capable of distinguishing cytogenetic response with an accuracy of 80%. This study identifies a set of genes that may be involved in primary resistance to imatinib, suggesting BCR-ABL-independent mechanisms.

Adenovirus E1A orchestrates the urokinase-plasminogen activator system and upregulates PAI-2 expression, supporting a tumor suppressor effect.

Invasiveness and metastatic potential are the two most important properties defining malignancy. The adeno-virus E1A (Ad-E1A) gene has a dual effect as a proliferative gene and as a tumor-suppressor gene, decreasing tumor growth and the metastatic potential of malignant cells. In order to study genes related with the antimetastatic effect of Ad-E1A in human cells, we performed a microarray analysis using OncoChiptrade mark. In three independent experiments, NIH3T3, IMR90 and MDA MB 435 cells were infected with pLPC retroviruses carrying the adenovirus 12S E1A gene or the GFP gene. We analyzed cDNA expression by using the CNIO OncoChipTM, a cDNA microarray containing a total of 6386 genes represented by 7237 clones. uPA, uPAr, tPA, PAI-1 and PAI-2 were also studied at RNA and protein levels. Microarrays of cDNA expression, RT-PCR and Western blot performed in IMR90 E1A-expressing cells showed downregulation of uPA, uPAr, tPA, PAI-1 and upregulation of PAI-2. These results were confirmed in NIH3T3 and MDA MB 435 breast carcinoma cells, with PAI-2 upregulation by RT-PCR and Western blot. In addition, zymographic analysis demonstrated that E1A expression greatly reduced the gelatinase activity of the pro-MMP2 and -MMP9 proteins. We propose that adenovirus E1A may orchestrate the expression of most members of the urokinase-plasminogen activation system, downregulating potentially invasive genes and upregulating PAI-2, which is associated with a better prognosis in human tumors.

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.

Cutaneous follicular B-cell lymphoma: description of a series of 18 cases.

The lack of precise and homogeneous criteria for the recognition of primary cutaneous follicular lymphoma has hindered gaining data on the frequency and clinical and molecular features of this entity. In the course of a review of a series of primary cutaneous lymphoma from different Spanish hospitals, we collected a series of 18 cases of primary cutaneous follicular lymphoma and analyzed its clinical, morphologic, and biologic characteristics. In this review only cases with a follicular pattern of growth, germinal center cytology, and restriction to the skin in a minimum follow-up of 6 months have been included. Cases of primary cutaneous follicular lymphoma were characterized by the expression of classic markers of the germinal center, such as bcl6, CD10, and the presence of aggregates of follicular dendritic cells. They frequently express bcl2 protein, although classical t(14;18) was not found in any of the cases analyzed. Analysis of the bcl6 noncoding first exon showed somatic mutations in two of four cases analyzed, as would be expected in lymphoma deriving from the germinal center. Clinically, most cases showed initial involvement of the head and neck, with relapses in eight cases (involving the skin in five cases, both skin and lymph node in two cases, and lymph node in one case). No death attributable to the tumor was recorded. These data seem to imply that follicular lymphoma may present initially in the skin, lacking the characteristic t(14;18) and having a relatively indolent course. Recognition of these tumors and elucidation of their molecular alterations could lead to properly adapted staging and treatment protocols for these patients.

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.

p16(INK4a) gene alterations are frequent in lesions of mycosis fungoides.

Mycosis fungoides is usually an indolent disease that, after a variable period of time in a stable phase, evolves into a tumoral form with aggressive behavior. The molecular events that mark this progression remain essentially unknown to date, and this prompted us to investigate the hypothetical role of p16(INK4a) silencing in mycosis fungoides progression. We analyzed the three most frequent mechanisms of inactivation of the p16(INK4a) gene (deletion, promoter hypermethylation, and mutation) in nine cases with patch/plaque and tumoral lesions of mycosis fungoides. The existence of alterations was investigated by microsatellite analysis, methylation-specific polymerase chain reaction, and direct sequencing. Alterations of the p16(INK4a) gene were found in four of nine of the plaque lesions (hypermethylation in three samples and allelic loss in one sample) and seven of nine in the tumor lesions (hypermethylation in five samples and allelic loss in three samples). No case presented point mutations. Although a higher incidence of p16(INK4a) hypermethylation was observed in the cases that failed to achieve a complete remission, the limited size of our series prompted us to evaluate this finding cautiously. The results of this study therefore showed a common genetic alteration that is found more frequently in tumoral lesions than it is in plaque lesions of mycosis fungoides. It also offers data that could suggest a relationship between p16(INK4a) hypermethylation and unfavorable clinical outcome. Broader studies are needed to confirm both relationships.

Splenic marginal zone lymphoma with increased number of blasts: an aggressive variant?

Splenic marginal zone lymphoma (SMZL) is a recently described and distinctive type of splenic lymphoma and is characterized by an indolent clinical course. By analyzing a large series of SMZL cases, we recognized the existence of a subset of 6 cases characterized by an aggressive clinical course that led to death caused by the tumor in 5 of 6 cases, whereas the remaining patient showed signs of tumor progression. The morphological, immunohistological, and molecular study of these cases has allowed us to detect precise distinctive features of this SMZL variant. The cases included here were characterized by massive splenomegaly and a morphological picture showing a micronodular pattern of splenic involvement with follicle replacement, biphasic cytology, and marginal zone differentiation. Unlike classical SMZL cases, a conspicuous component of larger lymphocytes was distributed in the marginal zone ring, occasionally overrunning it, with isolated presence of the same cells within the central small cell component and also in the red pulp. The bone marrow and peripheral lymph nodes showed similar histological findings to those described for SMZL in these locations. The genetic and molecular study of these cases showed no alterations specific to other lymphoma types, such as t14;18 and t11;14. Instead of this, it showed 7q loss in 3 of 5 cases, p53 inactivation in 2 of 6 cases, cyclinD1 overexpression in 2 of 6 cases, and the presence of translocations involving the 1q32 region in 2 of 4 cases. The recognition of this aggressive variant, besides offering a prognostic indication, could lead to a more suitable form of clinical management of these patients. Further molecular studies would clarify the role of the different genetic alterations found.

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 protein expression associated with methylation of the p16INK4A gene is a frequent finding in Hodgkin's disease.

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.

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.

Cyclin-dependent kinase inhibitor p27KIP1 in lymphoid tissue: p27KIP1 expression is inversely proportional to the proliferative index.

Cell cycle progression is regulated by the combined action of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CDKIs). p27KIP1, which has a high degree of similarity with p21WAF1, is a general CDKI thought to be involved in G1 arrest in response to agents that inhibit cell cycle progression. The aims of this study were 1) to establish the pattern of expression of p27KIP1 protein in nontumor lymphoid tissue, 2) to determine whether p27KIP1 is involved in lymphomagenesis, and 3) to address the possible relationship between p27KIP1 and p21WAF1 expression in reactive and tumor lymphoid tissue. p27KIP1 protein was found to be mainly present in quiescent lymphocytes in reactive lymphoid tissue as well as in peripheral blood lymphocytes, with an inverse expression for p27KIP1 and Ki-67 proteins. The same p27KIP1 expression pattern was observed in lymphomas, independently of histological type; small resting cells were p27KIP1 positive, and large proliferating cells were p27KIP1 negative. Therefore, tumors with a low proliferative index were mostly positive, whereas tumors characterized by a higher growth fraction bad low p27KIP1 protein levels. An unexpected finding was the existence of a group of six cases of high-grade lymphomas (three diffuse large B-cell lymphomas and three Burkitt's lymphomas) with homogeneously strong staining for p27KIP1 protein. All 6 of these cases belong to a group of 28 cases characterized by blockage of the p53 tumor suppressor pathway, as determined by genetic (p53 mutation) or immunophenotypic studies (p53+/p21-). p27KIP1 expression was not seen in any case of aggressive non-Hodgkin's lymphoma with an intact p53 pathway. The results indicate that p27KIP1 is down-regulated in lymphomas with a high proliferative index, although it is highly expressed in high-grade lymphomas with defects in the p53 pathway.

p21WAF1/CIP1 and MDM2 expression in non-Hodgkin's lymphoma and their relationship to p53 status: a p53+, MDM2-, p21-immunophenotype associated with missense p53 mutations.

p53 is a tumour suppressor gene which is often found to be inactivated in most types of human cancer. p53 is a transcription factor, the inactivation of which may lead to significant variations in the levels of p53 downstream proteins, such as p21WAF1/CIP1 and MDM2. In view of the significance of p21WAF1/CIP1 and MDM2 as wild-type (wt) p53 targets, this study was undertaken to monitor the varying expression of these proteins in non-Hodgkin's lymphomas (NHLs) in relation to p53 gene status. A total of 57 cases of different histological types of NHL were included in this study. Proteins p53, p21WAF1/CIP1, and MDM2 were analysed by immunohistochemical techniques, taking the levels expressed in reactive lymphoid tissues as reference points. p53 gene point mutations (exons 5-8) were looked for using the PCR-SSCP technique and direct sequencing. Fifteen of the 57 cases studied showed 16 mutations at the p53 gene: 12 missense, one nonsense, two silent mutations, and one frameshift deletion. Most missense mutations were associated with high levels of p53 protein, while the nonsense mutations and frameshift deletion did not induce detectable levels of p53. All cases with mutation at the p53 gene (15) showed null or low levels of p21WAF1/CIP1 and MDM2 proteins, suggesting that null or missense mutations at this gene give rise to a protein that is unable to transactivate the p21WAF1/CIP1 and MDM2 genes. The association between missense p53 mutation and dissociate immunophenotype (p53+, MDM2-, p21-) was statistically significant (Fisher's exact test, P = 0.0024). This anomalous p53+, MDM2-, p21- phenotype was also found in a small group of five cases with wt p53; this could indicate that in these cases p53 transactivation capacity has been abrogated by a mechanism other than p53 mutation. Most cases with the wt p53 gene show simultaneous immunohistochemical expression of all three proteins and often display higher levels than those found in reactive lymphoid tissue. There is a tendency for EBV-positive cases to harbour high levels of p53+ and p21+, suggesting that EBV could be involved in the nuclear accumulation of p53 and p21WAF1/CIP1 in NHL.

MDM2 and p21WAF1/CIP1, wild-type p53-induced proteins, are regularly expressed by Sternberg-Reed cells in Hodgkin's disease.

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.

MDM2 expression in lymphoid cells and reactive and neoplastic lymphoid tissue. Comparative study with p53 expression.

MDM2 and p53 immunohistochemical protein expression was analysed in lymphocytes and in reactive and neoplastic lymphoid tissue. Phytohaemagglutinin (PHA)-stimulated lymphocytes displayed MDM2 and p53 co-expression. In 8 of 8 tonsils, 24 of 24 Hodgkin's disease (HD), and 10 of 24 high-grade non-Hodgkin's lymphoma (HG-NHL) specimens, MDM2 paralleled p53 nuclear expression in non-tumour and tumour cells. The number of positive cells was greater and the staining intensity was stronger for p53 than for MDM2. In another nine of the 24 HG-NHL cases studied, dissociated expression was observed, with high p53 expression and very low or absent MDM2 expression. In five cases, both MDM2 and p53 were negative. The eight low-grade NHL (LG-NHL) cases were also MDM2- and p53-negative. MDM2 and p53 expression in PHA-activated lymphocytes and reactive lymphoid tissue is probably an expression of opposing biological signals regulating cell proliferation. Parallel MDM2 and p53 expression in all HD and in 10 out of 24 HG/NHL cases may indicate that this growth suppressive pathway is maintained in those cases. However, dissociated MDM2/p53 expression (nine cases) and the absence of expression of both proteins (five cases) may represent examples of deregulation of this growth control pathway. These findings are in agreement with previous in vitro studies in cell lines regarding the role of MDM2/p53 lymphoid tissue, suggesting a possible role for MDM2 deregulation in lymphomagenesis.

p53 expression in non-Hodgkin's lymphomas: a marker of p53 inactivation?

The p53 gene located in the short arm of chromosome 17 at position 17p13, is involved in the negative regulation of cellular growth. p53 mutation seems to be the most frequent genetic alteration found in human cancer. Mutant conformation of the p53 gene is associated with cell proliferation and tumour progression, and in most cases implies p53 stabilization, which renders the p53 protein detectable through the use of immunohistochemical techniques. p53 expression is a frequent finding in high grade lymphomas of either B or T cell lineage, having been detected in 30% of cases in our series. The focal presence of p53+ cells was seen in a wide range of low and high grade lymphomas, including lymphadenitis and reactive tonsils. In 37.5% of cases this increased expression of p53 was secondary to mutation in highly conserved regions (exons 5-8). Unlike findings reported in other tumours, in lymphomas, p53 expression seems to be secondary to genetic alterations other than p53 mutation. Initial data suggest that the MDM2 protein could be involved in inactivating p53 protein in most of these cases. Finally, p53 expression has been found to be a poor prognostic marker in high grade B-cell lymphomas in a large series of cases. High p53 expression was associated with a short survival, this relation being stronger in cases with simultaneous bcl2 expression.