NETs detection and quantification in paraffin embedded samples using confocal microscopy.

Detection and quantification of Neutrophil Extracellular Traps (NETs) in tissue samples has become a topic of great interest to understand their pathological role in various diseases. We describe a semi-automatic method of visualization and quantification of NETs in paraffin-embedded intracoronary thrombus aspirate samples. This study is based on colocalization of myeloperoxidase (MPO) and citrullinated histone 3 (H3Cit) as hallmark of the presence of NETs. For the analysis we used the confocal immunofluorescence microscopy technology to quantify the number of fields and the total area (in µm2) containing NETs in each thrombus sample. This observer-independent quantification method could be a useful tool to standardize the study of NETs in paraffin-embedded tissues, enabling comparison of results among different laboratories.

Polar Marine Microorganisms and Climate Change.

The large diversity of marine microorganisms harboured by oceans plays an important role in planet sustainability by driving globally important biogeochemical cycles; all primary and most secondary production in the oceans is performed by microorganisms. The largest part of the planet is covered by cold environments; consequently, cold-adapted microorganisms have crucial functional roles in globally important environmental processes and biogeochemical cycles cold-adapted extremophiles are a remarkable model to shed light on the molecular basis of survival at low temperature. The indigenous populations of Antarctic and Arctic microorganisms are endowed with genetic and physiological traits that allow them to live and effectively compete at the temperatures prevailing in polar regions. Some genes, e.g. glycosyltransferases and glycosylsynthetases involved in the architecture of the cell wall, may have been acquired/retained during evolution of polar strains or lost in tropical strains. This present work focusses on temperature and its role in shaping microbial adaptations; however, in assessing the impacts of climate changes on microbial diversity and biogeochemical cycles in polar oceans, it should not be forgotten that physiological studies need to include the interaction of temperature with other abiotic and biotic factors.

Long-term persistence of molecular disease after histological remission in low-grade gastric MALT lymphoma treated with H. pylori eradication. Lack of association with translocation t(11;18): a 10-year updated follow-up of a prospective study.

BACKGROUND: Localized low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma can regress after Helicobacter pylori eradication, but IgV(H) gene monoclonality may persist. We studied the long-term histological and molecular follow-up of 24 patients and the possible association of t(11;18) with the persistent monoclonality. PATIENTS AND METHODS: From January 1994, 24 untreated patients with stage I low-grade gastric MALT lymphoma associated with H. pylori were prospectively studied. They all received eradication treatment and were sequentially followed-up with endoscopies for histological and molecular studies. Rearrangement of the IgV(H) gene was studied by PCR analysis. MALT1 locus alterations were studied by FISH. RESULTS: Twenty-two of the 24 patients (91%) achieved disappearance of the lymphoma. Eighteen (82%) of the 22 histologically cured patients and 16 of the 19 (84%) with long follow-up had monoclonality. Three patterns of development of IgV(H) gene rearrangements were observed: four patients (21%) had polyclonal rearrangements; eight (58%) had maintained/intermittent monoclonality and four (21%) had occasional monoclonality, mostly after H. pylori reinfection. Only one patient (6%) with persistent monoclonality relapsed. The remaining 18 patients maintained the remission, despite the persistent monoclonality in 15, for a median of 66 months (range 20-113). t(11;18) was not found in any of the patients with persistent monoclonality. Time and the number of endoscopies performed were not related with the occurrence of monoclonality. CONCLUSIONS: In stage I low-grade gastric MALT lymphoma eradication of H. pylori achieves prolonged histological remission in 90% of patients, but molecular remission is not accomplished in most cases. Molecular disease persists for years, but is not associated with t(11;18).

Richter's transformation of chronic lymphocytic leukemia. The possible role of fludarabine and the Epstein-Barr virus in its pathogenesis.

Transformation of CLL into a large cell lymphoma has an incidence of 3-5%. We have studied 101 cases of CLL treated with fludarabine over a 10-year period (1990-2000) and observed a 12% incidence of transformation. In six of 12 patients, transformation was documented within 4 months following treatment with fludarabine. Pathological material, available in nine cases, was investigated for latent EBV by staining for LMP-1 by immunohistochemistry and EBERs-1 and 2 by in situ hybridisation. LMP-1 and EBERs were demonstrated in three of the nine samples. In two cases there was a different pattern of immunoglobulin gene rearrangement in the transformed cells assessed by PCR (FR3 fragment) compared to the original CLL clone. One of these two cases showed evidence of latent EBV. The other seven cases, of which two were EBV positive, showed identical pattern of Ig gene rearrangement in both the CLL and the transformed cells. We suggest that the relatively high incidence of transformation in this series may be due to immunosuppression mainly related to fludarabine, although other agents and prior therapies may have also contributed.

Risk of malignant lymphoma associated with human herpesvirus-8: a case-control study in Spain.

No overall increased risk of lymphoma associated with antibodies to human herpesvirus-8 was found in 526 lymphomas and 599 controls (odds ratio (OR)=1.04, 95% confidence interval (CI)=0.62-1.75); significant increases were noted for 19 lymphoplasmacytic lymphomas (OR=4.47, 95% CI=1.34-14.85) and nine low-grade lymphoma/lymphoma B-cell NOS (OR=5.82, 95% CI=1.07-31.73).

Neural cell adhesion molecule expression in plasma cells in bone marrow biopsies and aspirates allows discrimination between multiple myeloma, monoclonal gammopathy of uncertain significance and polyclonal plasmacytosis.

AIMS: Differential diagnosis between multiple myeloma (MM), monoclonal gammopathy of uncertain significance (MGUS), and polyclonal plasmacytosis may be difficult in cases with not much bone marrow infiltration. Normal plasma cells express the antigens CD138, CD38, CD19, CD10 and D-related human leucocyte antigen (HLA-DR). Myelomatous plasma cells lack B lymphoid-associated markers and may express cell surface antigens associated with other haematopoietic lineages such as NCAM/CD56 (neural cell adhesion molecule). Recently, a monoclonal antibody, anti-CD56, has become available that can be used in fixed tissues embedded in paraffin, and it has been reported that osteoblastic cells of trabecular bone strongly express NCAM/CD56. METHODS AND RESULTS: We analysed NCAM molecule expression in 35 samples from patients with plasma cell disorders: 14 cases of MM, 16 cases of MGUS, and five cases of polyclonal plasmacytosis using immunohistochemistry in parallel in bone marrow core biopsies processed routinely and in bone marrow smears from the same patients. Of the MM samples 78% were CD56+ in smears and 92% positive in biopsies. We did not find strong CD56 expression in MGUS samples. One of five samples of polyclonal plasmacytosis was CD56+. A case was considered to be positive for CD56 expression if >50% of the CD138+ plasma cells expressed NCAM with an intensity on a par with that of the osteoblasts. CONCLUSION: We conclude that CD56 antibody is a very useful marker in the study of plasma cell proliferation in bone marrow biopsies and in bone marrow aspirates and is a great help in discriminating between MM, MGUS, and polyclonal plasmacytosis, especially in those cases with low infiltration.

[Diagnostic evaluation of nasopharyngeal carcinoma: role of Epstein-Barr virus]. / Evaluación diagnóstica del carcinoma nasofaríngeo: papel del virus de Epstein-Barr.

We present a retrospective series of 27 nasopharyngeal carcinomas, selected from those attended at Ramón y Cajal Hospital between 1977 and 1996, with the aim of review the role of the study of Epstein-Barr virus in the diagnostic process of nasopharyngeal carcinoma. Twenty-seven patients, ranging from 14 to 81 years, with an average age of 50 years were selected. Male/female ratio was 1,7. All but one case were Caucasian. A neck mass was the first symptom in 40% of cases, with a mean diagnostic delay of 17 months. Only 8 cases (23%) did not exhibit neck nodes at the moment of diagnosis. CT and MRI were essential to establish staging: 5 stage I, 7 stage II and 15 stage IV, due to regional extension and/or bone erosion. Radiotherapy was employed in all cases, helped by chemotherapy in 20% of them. With a mean follow-up of 62 months, 5-years survival was 32% (IC 14,06-52,09). Of 27 cases of nasopharyngeal carcinoma 4 were differentiated (type I), 2 moderately differentiated (type II) and 22 undifferentiated (type III). While LMP-1 was only expressed by 41% of cases, PCR detected Epstein-Barr virus genome in 26 cases (96%) and in situ hybridization for EBERs was positive in all cases. Thus, all nasopharyngeal carcinomas were related to Epstein-Barr virus. Expression of LMP-1 seemed to worse the prognosis of nasopharyngeal carcinoma.

Regression of gastric high grade mucosa associated lymphoid tissue (MALT) lymphoma after Helicobacter pylori eradication.

BACKGROUND: Most low grade gastric lymphomas arising from the mucosa associated lymphoid tissue (MALT) are related to Helicobacter pylori colonisation. Cases with disease limited to the stomach can be cured after H pylori eradication and remain in remission for years. In contrast, high grade lymphomas of the stomach, although also related to H pylori, do not usually respond to eradication treatment. CASE REPORT: A 36 year old patient was referred from another hospital with a diagnosis of a low grade gastric MALT lymphoma associated with H pylori. The patient was in stage I and while waiting for the biopsies to be reviewed H pylori eradication therapy was given as the first step of treatment. Review of the biopsies showed a high grade immunoblastic lymphoma with areas of low grade gastric MALT lymphoma (high grade gastric MALT lymphoma or diffuse large B cell lymphoma with areas of MALT type lymphoma of the WHO classification). The patient received no further treatment but has been closely followed up for 32 months with sequential endoscopies to obtain biopsies for histological studies, H pylori cultures, and polymerase chain reaction analysis of the IgH gene. RESULTS: After H pylori eradication the patient had a complete histological response that has been maintained for 32 months. Monoclonal IgH gene rearrangement persisted for 32 months. CONCLUSION: The response of this patient indicates the possibility that some cases of high grade gastric MALT lymphoma (possibly patients in stage I with a superficial or limited disease) may still be responsive to H pylori antigenic drive and may be cured with eradication therapy. Prospective studies should be performed to identify patients with high grade gastric MALT lymphomas that may respond to eradication therapy and be spared of other more aggressive treatments.

Thyroid-infiltrating B lymphocytes in Graves' disease are related to marginal zone and memory B cell compartments.

B lymphocytes that infiltrate the thyroid (Thy-B cells) in Graves' patients appear to be implicated in the pathophysiology of this disorder. The goal of the present study was to examine the nature of these Thy-B cells. To this end, Thy-B lymphocytes were isolated from surgical thyroidal samples, and their phenotype was determined by using mouse monoclonal antibodies (mAb) directed against a wide variety of surface markers, followed by flow cytometry multicolor analysis. The results show that most Thy-B cells (approximately 60%) exhibited IgM(+) IgD(low to -) surface immunoglobulin (Ig) profile, whereas the minor cell fraction (approximately 30%) consisted of switched IgG(+) memory B lymphocytes. Thy-B cells expressed low levels of CD5, CD23, and CD62L, which distinguished them from the resting B-cell pool, the major B-cell subset in the blood. In addition, they lacked CD38, CD10, and CD71, characteristic molecules for the germinal center B lymphocytes. In addition, Thy-B lymphocytes showed peculiar patterns both of adhesion molecules (CD62L(-), CD44(intermediate)), and of activation molecules (CD69(+), CD80(+), and, in part, CD95(+)). Taken together, these results suggest that the Thy-B lymphocyte subset consists of a combination of IgM(+) B cells resembling marginal zone B lymphocytes, and isotype-switched memory B cells.

Deletions within the epstein-barr virus latent membrane protein-1 oncogene in adult ordinary, HIV-associated and paediatric Hodgkin's disease.

The aims of this study were the following: a) to perform Epstein-Barr virus (EBV) strain type assignment in three groups of Hodgkin's disease(HD): adult ordinary (39 cases), paediatric (24 cases), and HIV-associated (30 cases) and to compare the prevalence of type 1 and type 2 in each of the groups with that existing in two reference populations made up of 50 adults and 39 children; b) to assess the frequency of latent membrane protein-1 (LMP-1) 30-base pair (bp) deletions in the HD groups and in the healthy controls; and c) to relate the presence of LMP-1 deletions with EBV type. Type 2 EBV was observed in 12.8% of ordinary HD, in 26.7% of HIV-associated HD, in 25% of paediatric HD, in 4% of adult controls, and in none of the healthy children. The existence of double infections by type 1 and 2 EBV was also observed in 5.1% of ordinary HD, in 6.7% of HIV-associated HD, and in 10% of adult controls. The 30-bp deletion was identified overall in 33.3% of ordinary HD, in 83.3% of HIV-positive HD, 79.2% of paediatric HD, 34.7% of adult controls, and 36.4% of healthy children. Statistical analysis showed a significant association of the deleted strains with HD occurring in HIV-positive patients (P= 0.00003) and childhood HD (P= 0.006). On the other hand, the prevalence of the 30-bp deletion in the adult ordinary HD group reflects the prevalence of the deletion in the general population. Co-infections by deleted and non-deleted EBV strains were detected in 12.8% of ordinary HD, in 33.3% of HIV-associated HD, in 50% of paediatric HD, in 26.5% of adult controls, and in 27.3% of healthy children. Concerning the relationship between the deletion and the EBV typing, 26% of type 1 specimens carried the 30-bp deletion in an isolated manner compared with 64.7% of type 2. The statistical analysis showed that the deletion was associated with type 2 strains when coinfections were excluded and only the cases in which the deletion appeared alone were considered (P=0.003).

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.

Treatment of low grade gastric mucosa-associated lymphoid tissue lymphoma in stage I with Helicobacter pylori eradication. Long-term results after sequential histologic and molecular follow-up.

BACKGROUND AND OBJECTIVES: Most cases of gastric low-grade mucosa-associated lymphoid tissue (MALT) lymphoma are associated with H. pylori. In localized disease (stage I), eradication of H. pylori can result in histologic regression of the lymphoma in 50% to 100% of the patients. Moreover, in half of the apparently cured patients a monoclonal rearrangement of the IgH gene can be demonstrated. However, data on the long-term outcome of the patients are scarce. We report the evolution of a series of patients followed-up since 1994 in order to evaluate the long-term outcome of the apparently cured lymphoma. DESIGN AND METHODS: From January 1994 to July 2000, 19 consecutive patients with stage I gastric low grade MALT lymphoma were sequentially studied in our hospital. They had all been diagnosed by endoscopy and had had a complete staging (including CT-scan, contrast X-ray of the small bowel, bone marrow biopsies, immunophenotyping of bone marrow and peripheral blood and, in the later years, endoscopic ultrasonography). Diagnosis required established histologic criteria for low grade MALT lymphoma in the samples obtained by endoscopy. The investigation of H. pylori status included histologic search, serology and breath test urea-(13)C. Only patients in stage I disease associated with H. pylori were included in the study. Patients received standard triple therapy for eradication of H.pylori and after treatment were sequentially followed-up with endoscopies performed every 2-3 months in the first year, every 6 months in the second year and then yearly. Post-treatment biopsies were obtained by endoscopy for histologic studies, H. pylori cultures and molecular studies. The criteria of Wotherspoon et al. were used for the histological evaluation. Molecular studies were performed with a polymerase chain reaction analysis of the IgH gene using semi-nested procedures with consensus primers for the V(H) (Fr3A/Fr2A) and J(H) (LJH and VLJH) regions. RESULTS: After the eradication treatment, 18 of the 19 patients (94.7%) achieved histologic regression of the MALT lymphoma that occurred after a mean of 4.6 months (range 2-19). In 11 of the 18 histologically cured patients (61%) a monoclonal rearrangement of the IgH gene was demonstrated. In 2 patients the monoclonality disappeared completely, but 9 of the 11 patients (82%) had either persistent (3 patients) or intermittently persistent (5 patients) monoclonality for as long as 64 months. None of the patients who achieved a histologic remission (either with or without monoclonality) relapsed after a mean follow-up of 37 months (range 2-78). Two patients were lost to follow-up and another patient died of a gastric carcinoma; the remaining 15 patients are still in histologic remission after a mean period of 43 months (range 5-78). Ten patients studied between 1994 and the end of 1996 are in remission after a mean of 59 months (range 33-78). INTERPRETATIONS AND CONCLUSIONS: In most cases of gastric low-grade MALT lymphoma in stage I eradication of H. pylori can produce histologic regression of the lymphoma and this regression can be maintained for years. However, IgH gene monoclonality can be detected and persists in most cases. Although this persistent monoclonality seems to indicate the presence of a latent lymphoma population, over a period of 6 years it has not so far influenced the outcome. These findings indicate that in cases of localized gastric low-grade MALT lymphoma associated with H. pylori, the first step of treatment should be eradication of the H. pylori; however, a close and long follow-up is essential to determine the ultimate outcome of these patients and the possible significance of the persistent monoclonality.

Utility of flow cytometric analysis of mast cells in the diagnosis and classification of adult mastocytosis.

The diagnosis of bone marrow (BM) involvement in mastocytosis has mainly been based on conventional histology. Nevertheless, in recent years, three major methodological advances have been made: the measurement of serum tryptase levels, the immunohistochemical assessment of mast cell (MC) tryptase, and the immunophenotypical characterization of BMMC using flow cytometry (FCM). The most characteristic immunophenotypic feature in mastocytosis is the coexpression of CD2 and CD25 antigens, which are never present in normal BMMC and constitute a phenotypic hallmark of BMMC in adult mastocytosis. Such observations would support the need to include the immunophenotypic analysis of MC in the diagnosis of mastocytosis.

Lymphoepithelioma-like carcinoma of the uterine cervix: a case report studied by in situ hybridization and polymerase chain reaction for Epstein-Barr virus.

Lymphoepithelioma-like carcinomas have been reported outside the nasopharynx in many sites, including the uterine cervix. The association with the Epstein-Barr virus in the latter site is still controversial. To date, Epstein-Barr virus genome has only been demonstrated in Asian patients. We report a case of lymphoepithelioma-like carcinoma of the uterine cervix in a white woman in whom the Epstein-Barr virus infection was tested for by in situ hybridization and polymerase chain reaction. The results of both techniques were negative. Our case and a review of the literature support the contention that cervical lymphoepithelioma-like carcinoma is not associated with Epstein-Barr virus infection in non-Asian patients.

Expression of high amounts of the CD117 molecule in a case of B-cell non-Hodgkin's lymphoma carrying the t(14:18) translocation.

The c-kit proto-oncogen (CD117) has been described to be present in normal and neoplastic hemopoietic cells including both myeloid and lymphoid lineages. Among the normal lymphoid cells CD117 expression would be restricted to a small subset of NK-cells, and to early T-cell precursors and it is not expressed by normal B-cells. Regarding chronic lymphoproliferative disorders the only data provided up to now suggests that CD117 expression is restricted to cases of Hodgkin's disease and anaplastic large-cell lymphoma. In the present paper we describe a case of a B-cell chronic lymphoproliferative disorder carrying the t(14:18) translocation as demonstrated by molecular studies, in which the flow cytometric immunophenotypic analysis of both peripheral blood and bone marrow samples revealed the expression of high amounts of the CD117 antigen in the surface of the clonal B-cell population. Further studies are necessary to explore both the functional role of c-kit expression in the neoplastic B-cells from this patient and its potential utility for the diagnosis and follow-up of patients with B-cell non-Hodgkin's lymphoma.

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.