Clinical Characteristics, Treatment and Evolution of Splenic and Nodal Marginal Zone Lymphomas-Retrospective and Multicentric Analysis of Portuguese Centers.

INTRODUCTION: Treatment of Splenic (SMZL) and Nodal (NMZL) Marginal Zone Lymphoma is not consensual. Histologic transformation (HT) to aggressive lymphoma is a poorly understood event, with an unfavorable outcome. OBJECTIVES: Describe the clinical characteristics, treatment, outcomes and incidence of HT. METHODS: Characteristics of patients with SMZL and NMZL consecutively diagnosed in 8 Portuguese centers were retrospectively reviewed. Endpoints were overall survival (OS), time to first systemic treatment (TTFST), frequency of HT and time to transformation (TTT). RESULTS: This study included 122 SMZL and 68 NMZL, most of them received systemic treatment: 55.4% and 76.5%, respectively. Splenectomy was performed in 58.7% of patients with SMZL. Different treatment protocols were used. OS or TTFST did not differ significantly according to treatments. Given the small sample size, no conclusion can be made concerning the role of Rituximab in the treatment of NMZL and SMZL based in these results. HT was documented in 18 patients, mainly in SMZL, with a cumulative incidence at 5 years of 4.2%. We confirmed that age is a prognostic factor. CONCLUSION: Randomized prospective trials are needed to standardize treatment in MZL. Patients with HT did appear to have shorter OS in comparison with those who did not experience HT (OS 5 years of 68.4% vs. 80.4%), but the number of HT was too small to reach statistical significance.

Clinical risk scores do not accurately identify a very high risk population with diffuse large B cell lymphoma-an analysis of 386 Portuguese patients.

The identification of high-risk patients deserving alternative first-line treatments to R-CHOP is a research priority in diffuse large B cell lymphoma (DLBCL). Despite the increasing recognition of biological features underlying aggressive behavior, clinical scores remain the basis for prognostic evaluation and treatment stratification in DLBCL. We performed a retrospective analysis of patients with DLBCL uniformly treated with immunochemotherapy with the aim of assessing the discriminative power of the NCCN international prognostic index (IPI) and the GELTAMO-IPI scores in risk group stratification and compared them with the IPI. Additionally, we investigated if bulky disease, gender, beta-2 microglobulin (ß2m), body mass index, and B-symptoms have independent prognostic impact. We confirmed the discriminative ability of the three prognostic scores in terms of progression-free survival and overall survival and found that the NCCN-IPI performs better in the identification of a high-risk population compared to the IPI and the GELTAMO scores. In an attempt to improve the prognostic power of the NCCN-IPI we analyzed additional clinical variables. Bulky disease and elevated ß2m were found to be independent predictors of prognosis when controlling for the NCCN-IPI risk groups. However, they seem to bring no incremental power to the latter in the identification of poor outcome patients. We support the use of the NCCN-IPI for the clinical identification of high-risk patients in DLBCL. Future studies to unravel the biological heterogeneity within NCCN-IPI groups are needed to improve risk prediction and design targeted therapies for poor prognosis patients.

Revisiting the immune microenvironment of diffuse large B-cell lymphoma using a tissue microarray and immunohistochemistry: robust semi-automated analysis reveals CD3 and FoxP3 as potential predictors of response to R-CHOP.

Gene expression studies have identified the microenvironment as a prognostic player in diffuse large B-cell lymphoma. However, there is a lack of simple immune biomarkers that can be applied in the clinical setting and could be helpful in stratifying patients. Immunohistochemistry has been used for this purpose but the results are inconsistent. We decided to reinvestigate the immune microenvironment and its impact using immunohistochemistry, with two systems of image analysis, in a large set of patients with diffuse large B-cell lymphoma. Diagnostic tissue from 309 patients was arrayed onto tissue microarrays. Results from 161 chemoimmunotherapy-treated patients were used for outcome prediction. Positive cells, percentage stained area and numbers of pixels/area were quantified and results were compared with the purpose of inferring consistency between the two semi-automated systems. Measurement cutpoints were assessed using a recursive partitioning algorithm classifying results according to survival. Kaplan-Meier estimators and Fisher exact tests were evaluated to check for significant differences between measurement classes, and for dependence between pairs of measurements, respectively. Results were validated by multivariate analysis incorporating the International Prognostic Index. The concordance between the two systems of image analysis was surprisingly high, supporting their applicability for immunohistochemistry studies. Patients with a high density of CD3 and FoxP3 by both methods had a better outcome. Automated analysis should be the preferred method for immunohistochemistry studies. Following the use of two methods of semi-automated analysis we suggest that CD3 and FoxP3 play a role in predicting response to chemoimmunotherapy in diffuse large B-cell lymphoma.

Dysregulation of autophagy in human follicular lymphoma is independent of overexpression of BCL-2.

Overexpression of the anti-apoptotic protein BCL-2 is characteristic of human follicular lymphoma (FL) and some cases of diffuse large B cell lymphoma (DLBCL). We aimed to determine autophagy status in primary FL and DLBCL samples and the BCL-2+/BCL-2- lymphoma cell lines using both autophagy PCR array and tissue microarray (TMA). A greater number of autophagy machinery genes were up-regulated in the BCL-2+ Su-DHL4 cell line compared with BCL-2- Su-DHL8 cells, at both the basal level and in response to autophagic stress. The autophagy-related gene expression profiles were determined in purified and unpurified malignant human lymph node biopsies. Seven autophagy machinery genes were up-regulated in purified FL B-cells compared with reactive B-cells. Only 2 autophagy machinery genes were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. Expression of autophagy substrates p62 and LC3 were determined by TMAs. FL samples showed significantly decreased levels of both p62 and LC3 compared with reactive and DLBCL, indicative of an increased autophagy activity in FL. In summary, these results demonstrate that FL showed increased basal autophagy activity, regardless of overexpression of BCL-2 in this disease.

Invasive validation of the Complior Analyse in the assessment of central artery pressure curves: a methodological study.

INTRODUCTION: Arteries are the target, the place, and the common denominator of cardiovascular diseases; hence, study of arterial function is of greatest importance in clinical practice. The aim of this study was to evaluate the accuracy of carotid pulse wave analysis using the new version of the Complior device--the Complior Analyse. METHODS AND RESULTS: This was a cross-sectional study that included 15 patients (seven women), mean age 62.07±10.59 years, referenced for cardiac catheterization. Pressure curves were obtained simultaneously in the ascending aorta (invasively) and in the right common carotid artery (using the Complior Analyse). Mean central arterial pressures, augmentation indexes, and wave morphology obtained using both methods were compared. A good concordance between methods was obtained for all the parameters measured, with intraclass correlation coefficients above 0.9. Bland-Altman analysis also indicated a good accuracy profile of the Complior device, with small mean differences observed for all parameters and most values confined within 2 SD of the mean difference. This was further confirmed by the strong Pearson correlation coefficients, with r² coefficients above 0.92 for all the variables studied. The correlations observed were independent of sex, age, arterial pressure, and BMI. CONCLUSION: The results presented and available research clearly indicate that the Complior Analyse device measures carotid pressure waves accurately; therefore, it is a simple and reliable noninvasive alternative for pressure wave analysis.

Reproducibility of aortic pulse wave velocity as assessed with the new Complior Analyse.

INTRODUCTION: The aim of this study was to assess the interobserver and intraobserver reproducibility, as well as the temporal variability of the new Complior Analyse assessing aortic pulse wave velocity (PWV). METHODS: Eighty-seven participants (60% men), mean age 34.26 ± 16.58 years, were enrolled in a cross-sectional study. All patients were subjected to sequential measures of aortic PWV by two experienced operators. In a group of 27 participants, PWV was also determined 1 month after the first moment evaluation to assess the temporal stability of the PWV estimations with the device. RESULTS: The analysis of concordance showed a very good agreement for paired PWV values in terms of both the intraobserver and the interobserver variability and also the temporal variability. The intraclass correlation coefficients were above 0.98 for the three conditions (P<0.0001), indicating an excellent strength of agreement. Further evidences in favor of a good overall performance of the device were determined from the Bland-Altman analysis, with small mean differences for intrareproducibility, interreproducibility, and temporal reproducibility (respectively, 0.02 ± 0.38, 0.10 ± 0.45, and 0.07 ± 0.51 m/s), and with differences mainly between 2 SDs of the mean difference. The correlations observed were independent of sex, age, arterial pressure, heart rate, and BMI. CONCLUSION: The data showed an excellent reproducibility of the Complior Analyse for the assessment of aortic PWV when used in ideal conditions and by experienced observers. The technical profile presented shows that this device meets the requirements of quality for its inclusion in integrated clinical follow-up programs.

HIV status does not impair the outcome of patients diagnosed with diffuse large B-cell lymphoma treated with R-CHOP in the cART era.

OBJECTIVE: To compare the outcome of patients diagnosed with HIV infection and diffuse large B-cell lymphoma (DLBCL) treated with R-CHOP in the cART era with that of a HIV-negative control group. METHODS: From 2003 to 2011, 305 patients (97 HIV-positive) were diagnosed with DLBCL and treated with R-CHOP. Clinical features were compared using chi-square or Fisher's exact test. Survival analysis was performed using the Kaplan-Meier method and log-rank test. Multivariate analysis was performed using the Cox regression proportional hazards model. RESULTS: HIV-positive patients had more B symptoms and extranodal sites of disease at diagnosis, but the proportion of patients with high-intermediate/high-risk disease according to the international prognostic index (IPI) was similar between groups. Response rate was 73%, both for patients with and without HIV infection. After a median follow-up of 48 months, 30 patients relapsed after achieving a complete remission, including four HIV-positive patients. Ninety-six patients have died (19 HIV-positive), 73 of them due to DLBCL. Three patients (one HIV-positive) died due to treatment toxicity. Patients with HIV infection had a significantly longer disease-free survival (DFS) (5-year: 94 vs. 77%; P = 0.03) and overall survival (OS) (78 and 64% for HIV-positive and HIV-negative patients, respectively; P = 0.03). These results were confirmed on multivariate analysis when controlled for other potential prognostic confounders. CONCLUSION: HIV-positive patients diagnosed with DLBCL in the cART era have an excellent outcome when treated with standard immunochemotherapy. Therefore, the choice of chemotherapy in patients with lymphoma should not be influenced by HIV status.

Poor concordance among nine immunohistochemistry classifiers of cell-of-origin for diffuse large B-cell lymphoma: implications for therapeutic strategies.

PURPOSE: The opportunity to improve therapeutic choices on the basis of molecular features of the tumor cells is on the horizon in diffuse large B-cell lymphoma (DLBCL). Agents such as bortezomib exhibit selective activity against the poor outcome activated B-cell type (ABC) DLBCL. In order for targeted therapies to succeed in this disease, robust strategies that segregate patients into molecular groups with high reliability are needed. Although molecular studies are considered gold standard, several immunohistochemistry (IHC) algorithms have been published that claim to be able to stratify patients according to their cell-of-origin and to be relevant for patient outcome. However, results are poorly reproducible by independent groups. EXPERIMENTAL DESIGN: We investigated nine IHC algorithms for molecular classification in a dataset of DLBCL diagnostic biopsies, incorporating immunostaining for CD10, BCL6, BCL2, MUM1, FOXP1, GCET1, and LMO2. IHC profiles were assessed and agreed among three expert observers. A consensus matrix based on all scoring combinations and the number of subjects for each combination allowed us to assess reliability. The survival impact of individual markers and classifiers was evaluated using Kaplan-Meier curves and the log-rank test. RESULTS: The concordance in patient's classification across the different algorithms was low. Only 4% of the tumors have been classified as germinal center B-cell type (GCB) and 21% as ABC/non-GCB by all methods. None of the algorithms provided prognostic information in the R-CHOP (rituximab plus cyclophosphamide-adriamycin-vincristine-prednisone)-treated cohort. CONCLUSION: Further work is required to standardize IHC algorithms for DLBCL cell-of-origin classification for these to be considered reliable alternatives to molecular-based methods to be used for clinical decisions.

The microenvironment of AIDS-related diffuse large B-cell lymphoma provides insight into the pathophysiology and indicates possible therapeutic strategies.

Despite the use of highly active antiretroviral therapy (HAART), AIDS-related lymphoma remains common. We investigated the tumor, microenvironment, and viral components in 41 AIDS-related diffuse large B-cell lymphomas (AR-DLBCLs) in the pre- and post-HAART era. The outcome has improved and the frequency of the prognostically unfavorable immunoblastic histology has decreased after HAART. Compared with sporadic cases, AR-DLBCL demonstrated increased hyperproliferation (P < .001) and c-Myc rearrangements, reduced CD4(+) (P < .001) and FOXP3(+) T cells (P < .001), increased activated cytotoxic cells (P < .001), but no difference in tumor-associated macrophages. Our analysis showed that AR-DLBCL is highly angiogenic with higher blood-vessel density than sporadic cases (P < .001) and highlighted the role of Epstein-Barr virus in angiogenesis. We recognized viral profiles and as a second step examined the reactive cytotoxic cell infiltrates. Our observation of markedly higher numbers of cytotoxic cells in AR-DLBCL with LMP1 and/or p24 compared with cases lacking viral antigens (P < .001) has important clinical implications, implicitly linked to the immunosurveillance theory. Whereas early initiation of HAART should improve immunosurveillance and reduce the incidence of LMP1-positive AR-DLBCL, cases without viral antigens appear able to avoid immunologic reaction and likely require additional strategies to improve surveillance.

Expression of FOXP3, CD68, and CD20 at diagnosis in the microenvironment of classical Hodgkin lymphoma is predictive of outcome.

PURPOSE: The immune microenvironment is key to the pathophysiology of classical Hodgkin lymphoma (CHL). Twenty percent of patients experience failure of their initial treatment, and others receive excessively toxic treatment. Prognostic scores and biomarkers have yet to influence outcomes significantly. Previous biomarker studies have been limited by the extent of tissue analyzed, statistical inconsistencies, and failure to validate findings. We aimed to overcome these limitations by validating recently identified microenvironment biomarkers (CD68, FOXP3, and CD20) in a new patient cohort with a greater extent of tissue and by using rigorous statistical methodology. PATIENTS AND METHODS: Diagnostic tissue from 122 patients with CHL was microarrayed and stained, and positive cells were counted across 10 to 20 high-powered fields per patient by using an automated system. Two statistical analyses were performed: a categorical analysis with test/validation set-defined cut points and Kaplan-Meier estimated outcome measures of 5-year overall survival (OS), disease-specific survival (DSS), and freedom from first-line treatment failure (FFTF) and an independent multivariate analysis of absolute uncategorized counts. RESULTS: Increased CD20 expression confers superior OS. Increased FOXP3 expression confers superior OS, and increased CD68 confers inferior FFTF and OS. FOXP3 varies independently of CD68 expression and retains significance when analyzed as a continuous variable in multivariate analysis. A simple score combining FOXP3 and CD68 discriminates three groups: FFTF 93%, 62%, and 47% (P < .001), DSS 93%, 82%, and 63% (P = .03), and OS 93%, 82%, and 59% (P = .002). CONCLUSION: We have independently validated CD68, FOXP3, and CD20 as prognostic biomarkers in CHL, and we demonstrate, to the best of our knowledge for the first time, that combining FOXP3 and CD68 may further improve prognostic stratification.

EBNA3B-deficient EBV promotes B cell lymphomagenesis in humanized mice and is found in human tumors.

Epstein-Barr virus (EBV) persistently infects more than 90% of the human population and is etiologically linked to several B cell malignancies, including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B cell lymphoma (DLBCL). Despite its growth transforming properties, most immune-competent individuals control EBV infection throughout their lives. EBV encodes various oncogenes, and of the 6 latency-associated EBV-encoded nuclear antigens, only EBNA3B is completely dispensable for B cell transformation in vitro. Here, we report that infection with EBV lacking EBNA3B leads to aggressive, immune-evading monomorphic DLBCL-like tumors in NOD/SCID/γc-/- mice with reconstituted human immune system components. Infection with EBNA3B-knockout EBV (EBNA3BKO) induced expansion of EBV-specific T cells that failed to infiltrate the tumors. EBNA3BKO-infected B cells expanded more rapidly and secreted less T cell-chemoattractant CXCL10, reducing T cell recruitment in vitro and T cell-mediated killing in vivo. B cell lines from 2 EBV-positive human lymphomas encoding truncated EBNA3B exhibited gene expression profiles and phenotypic characteristics similar to those of tumor-derived lines from the humanized mice, including reduced CXCL10 secretion. Screening EBV-positive DLBCL, HL, and BL human samples identified additional EBNA3B mutations. Thus, EBNA3B is a virus-encoded tumor suppressor whose inactivation promotes immune evasion and virus-driven lymphomagenesis.

Do we need to do fluorescence in situ hybridization analysis in myelodysplastic syndromes as often as we do?

Although conventional cytogenetics is considered the gold standard to detect chromosomal abnormalities in myelodysplastic syndromes (MDS), fluorescence in situ hybridization (FISH) is being increasingly used additionally. However, the real contribution of FISH analysis in the cytogenetic diagnosis of MDS has not been well defined. The aim of this study was to evaluate whether FISH studies are able to reveal chromosomal abnormalities in MDS patients undetected by conventional cytogenetics. One hundred seventy-four FISH studies were performed on bone marrow samples of 60 patients with MDS. The number of FISH studies in each patient was variable (1-5). FISH studies confirmed the G-banding cytogenetic findings in 99.4% (153/154) of samples and detected cytogenetic abnormalities in 25% (5/20) of cases in which the conventional cytognetic study failed. These results indicate that FISH studies provide relevant information in MSD in which the conventional cytogenetic analysis was unsuccessful but add little value to a normal katyotype in conventional cytogenetic analysis.