Pedro Martínez

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.

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‐Hodgkins 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, p21and 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 (Fishers 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.

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.

Value of PCR detection of TCR gamma gene rearrangement in the diagnosis of cutaneous lymphocytic infiltrates.

In this study, we analyzed the reliability and usefulness of the polymerase chain reaction (PCR) for the detection of T-cell receptor (TCR)gamma gene monoclonal rearrangement. We first tested for the specificity and sensitivity of this strategy, against the classical criteria of Southern blot analysis (SBA). Of the 27 samples tested, results agreed in all but two. Broader analysis of these cases demonstrated the high specificity (absence of false positives) of the PCR strategy, together with its limited sensitivity (10% of false negatives). The usefulness of this PCR approach was then tested on a panel of 28 biopsy specimens of cutaneous lymphocytic infiltrates. Monoclonal TCR gamma rearrangement was detected in seven of eight cases of early stage mycosis fungoides (MF), one of two Sezary syndrome (SS) cases, two of two non-MF T-cell lymphoma, and two of three lymphomatoid papulosis. Monoclonality was not detected in any of the 11 benign cases (parapsoriasis and inflammatory dermatosis). Results obtained with this new molecular strategy provide additional support for the hypothesis of a monoclonal origin for most early stage T-cell MF. They also suggest the heterogeneous nature of some lymphomatoid papulosis lesions. Therefore, due to the difficulty in detecting T-cell monoclonality by immunohistochemical techniques, PCR can be a useful alternative strategy to SBA. It could also be used as a complementary technique in the routine diagnosis of T-cell cutaneous infiltrates.

The detection of B-cell monoclonal populations by polymerase chain reaction : accuracy of approach and application in gastric endoscopic biopsy specimens

The recently developed strategy for the detection of monoclonal B-cell populations, based on the selective amplification of predominant immunoglobulin heavy chain gene (IgH) rearrangement, is seen to be a suitable alternative to Southern blot analysis. The new technique uses a pair of consensus primers for variable (VH) and joining (JH) regions. We first tested the accuracy of this new approach on a broad series of 67 samples that had been well characterized by both Southern blot and immunohistochemical techniques before being subjected to blind testing. Our results show that this technique gave 100% specificity (absence of false-positive results) and approximately 70% sensitivity (30% false-negative results). The only exception was the presence of an IgH polymerase chain reaction ambiguous result in a case of Sezarys syndrome. The polymerase chain reaction technique was then applied to a panel of 27 frozen gastric endoscopy biopsy specimens following previous clinical suspicion of lymphoma. Monoclonality was detected in nine of 13 samples previously diagnosed as lymphomas and in one of two carcinomas. Further examination of the gastrectomy specimen in the latter case disclosed a B-cell lymphoma associated with the carcinoma. In spite of its limited sensitivity, the high specificity attained by this technique in the detection of monoclonality makes it a useful adjunct to routine morphologic criteria, as it is sometimes capable of detecting true positive cases that conventional morphology studies show to be negative.

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.

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 Hodgkins 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 p21was 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.

Evaluación de dos pautas de quimioprofilaxis tuberculosa en pacientes infectados por el virus de la inmunodeficiencia humana

Objetivo Evaluar la cumplimentacion, tolerancia y eficacia de una pauta corta de quimioprofilaxis para tuberculosis con isoniacida y rifampicina durante 3 meses frente a una pauta estandar de isoniacida durante 12 meses en pacientes con infeccion por el virus de la inmunodeficiencia humana (VIH). Pacientes Y Metodos Ensayo clinico prospectivo, comparativo, aleatorizado y abierto realizado en cuatro hospitales generales y un centro penitenciario de Castilla-La Mancha. La profilaxis se administro en pacientes PPD positivos y pacientes anergicos de acuerdo con las normas de los Centers for Diseases Control (CDC) de 1991. Los pacientes se distribuyeron de forma aleatoria en dos pautas: pauta de rifampicina a los que se les administraron 300 mg/dia de isoniacida y 600 mg/dia de rifampicina durante 3 meses, y pauta de isoniacida a los que se les administraron 300 mg/dia de isoniacida durante 12 meses. Resultados Se incluyeron 133 pacientes: 64 en la pauta isoniacida y 69 en la pauta rifampicina. Se tolero mejor la pauta de rifampicina, con un 28% de efectos adversos frente a un 55% en la pauta de isoniacida. La hepatotoxicidad fue mas frecuente en la pauta de isoniacida, con un riesgo relativo (RR) de 2,2 (intervalo de confianza [IC] del 95%, 1,23-4,01). La hepatotoxicidad grave, que obligo a suspender el tratamiento, se relaciono con el tiempo de administracion del farmaco, siendo mas frecuente en la pauta de 12 meses. Se cumplimento mejor la pauta corta, pero sin diferencias valorables. La incidencia de tuberculosis fue de 4,23 casos por 100 personasano para la pauta de isoniacida y 2,08 para la pauta de rifampicina, con un riesgo relativo para desarrollar tuberculosis con la pauta de rifampicina de 0,51 (IC del 95%, 0,09-2,8) frente a la pauta de isoniacida, no estadisticamente significativo. La estancia en prision se asocio con un riesgo significativo de tuberculosis, independientemente de la pauta de tratamiento (RR = 9,2; IC del 95%; 1,06-80,2). Conclusiones En pacientes con infeccion por el VIH con PPD positivo o anergicos, la pauta de rifampicina es al menos igual de eficaz para prevenir el desarrollo de tuberculosis que la pauta de isoniacida, y presenta menos efectos adversos.

From perturbation theory to Confinement: how the string tension is built up☆

Abstract We study the spatial volume dependence of electric flux energies for SU (2) Yang-Mills fields on the torus with twisted boundary conditions. The results approach smoothly the rotational invariant Confinement regime. The would-be string tension is very close to the infinite volume result already for volumes of (1.2 fm.) 3 . We speculate on the consequences of our result for the Confinement mechanism.

Loss of heterozygosity of p16 correlates with minimal residual disease at the end of the induction therapy in non-high risk childhood B-cell precursor acute lymphoblastic leukemia

We evaluated the incidence of MTS1/p16 deletions by loss of heterozygosity (LOH) analysis in 36 non-high risk B-cell precursor childhood acute lymphoblastic leukemia (BCP-ALL) and correlated these results with clinical features and with the presence of minimal residual disease (MRD) at the end of induction therapy. LOH was analyzed using three microsatellite markers flanking the p16 gene. MRD was studied by the polymerase chain reaction (PCR) for IgH and TCRdelta genes. All patients were classified and treated according to the BFM-86 protocol. A slower response to the induction treatment (MRD) was associated with LOH of p16 and worse clinical outcome. Thus, LOH of p16 may be a marker of chemotherapy resistance among the children classified as non-high risk BCP-ALL.