Sequential combination of bortezomib and WEE1 inhibitor, MK-1775, induced apoptosis in multiple myeloma cell lines.

INTRODUCTION: Multiple myeloma (MM) remains incurable due to high rates of relapse after various treatment regimens. WEE1 is a cell cycle related gene that regulates the G2/M checkpoint and promotes cell cycle suspension for consequent DNA repair. To date, there are clinical studies for the evaluation of WEE1 inhibitors in the treatment of solid tumors and studies on cell lines of non-MM hematological tumors. OBJECTIVES: To perform in vitro functional studies to verify the effect of the inhibition of WEE1 on MM cell lines viability and its potential as therapeutic target. MATERIAL AND METHODS: WEE1 expression was evaluated in 22 newly diagnosed MM patients and in four MM cell lines, RPMI-8226, U266 and SKO-007 and SK-MM2, by quantitative real-time PCR (qPCR). After treatment with the WEE1 inhibitor (MK-1775), with or without proteasome inhibitor (bortezomib) pretreatment, we assessed cell viability through Prestoblue functional test, microspheres formation in soft agar, and induction of apoptosis and cell cycle alterations by flow cytometry. RESULTS: All MM cell lines showed WEE1 expression by qPCR. RPMI-8226 and U266 showed a 50% reduction in cell viability after 24 h of incubation with MK-1775, at concentrations of 5 µM and 20 µM, respectively. SKO-007 showed dose and time dependence to this drug. Combination therapy with bortezomib and MK-1775 abolished the formation of soft agar microspheres in the RPMI-8226 cell line (also responsive to the use of both drugs) and U266, but SKO-007 was resistant to all drugs, isolated and combined. However, treatment of bortezomib followed by MK-1775 (sequential treatment) versus bortezomib alone showed statistically significant impact on cell lines total apoptosis: 88.8% vs 74.1% in RPMI-8222 (confirmed by cell cycle experiments); 92.5% vs 86.6% in U266; and 60.2% 30.9% on SKO-007 (p < 0.05). CONCLUSION: The sequential combination of bortezomib and WEE1 inhibitor, MK-1775, induced apoptosis in RPMI-8226, U266, and especially SKO-007 cell lines, more efficiently than the use of the same isolated drugs, highlighting its effect in inhibition of proliferation of tumor cells in MM cell lines. Our data suggest that WEE1 can figure as a MM target and that the sequential combination of bortezomib and MK-1775 may be explored in future clinical trials.

Targeting the polarization of tumor-associated macrophages and modulating mir-155 expression might be a new approach to treat diffuse large B-cell lymphoma of the elderly.

Aging immune deterioration and Epstein-Barr (EBV) intrinsic mechanisms play an essential role in EBV-positive diffuse large B-cell lymphoma (DLBCL) of the elderly (EBV + DLBCLe) pathogenesis, through the expression of viral proteins, interaction with host molecules and epigenetic regulation, such as miR-155, required for induction of M1 phenotype of macrophages. This study aims to evaluate the relationship between macrophage polarization pattern in the tumor microenvironment and relative expression of miR-155 in EBV + DLBCLe and EBV-negative DLBCL patients. We studied 28 EBV + DLBCLe and 65 EBV-negative DLBCL patients. Tumor-associated macrophages (TAM) were evaluated by expression of CD68, CD163 and CD163/CD68 ratio (degree of M2 polarization), using tissue microarray. RNA was extracted from paraffin-embedded tumor samples for miR-155 relative expression study. We found a significantly higher CD163/CD68 ratio in EBV + DLBCLe compared to EBV-negative DLBCL. In EBV-negative DLBCL, CD163/CD68 ratio was higher among advanced-staged/high-tumor burden disease and overexpression of miR-155 was associated with decreased polarization to the M2 phenotype of macrophages. The opposite was observed in EBV + DLBCLe patients: we found a positive association between miR-155 relative expression and CD163/CD68 ratio, which was not significant after outlier exclusion. We believe that the higher CD163/CD68 ratio in this group is probably due to the presence of the EBV since it directly affects macrophage polarization towards M2 phenotype through cytokine secretion in the tumor microenvironment. Therapeutic strategies modulating miR-155 expression or preventing immuno-regulatory and pro-tumor macrophage polarization could be adjuvants in EBV + DLBCLe therapy since this entity has a rich infiltration of M2 macrophages in its tumor microenvironment.

TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) stable silencing increases late apoptosis by upregulation of caspase 9 and APAF1 in RPMI8226 multiple myeloma cell line.

BACKGROUND: TP53 Regulated Inhibitor of Apoptosis 1 (TRIAP1) modulates apoptotic pathways preventing the formation of the apoptosome complex. Our group previous study showed that 90% of patients' multiple myeloma (MM) marrow-derived plasma cells present TRIAP1 overexpression as compared to normal plasma cells. Due to high prevalence and lack of information on TRIAP1's role in MM biology, we decided to explore the impact of TRAIP1 through stable gene silencing in MM cell lines and its effect on cell cycle and apoptosis. METHODS: TRIAP1 expression was examined in MM cell lines by quantitative real time PCR. Cell lines were submitted to transduction with lentiviral vector encoding a TRIAP1-specific short hairpin RNA (shRNA) and, as control, encoding a non-targeting shRNA (scramble). Apoptosis was assessed by flow cytometry with annexin V and propidium iodide staining (the later also used for cell cycle), APAF1 and Caspase 9 apoptosome related genes expression and Caspase 9 and Caspase 3/7 activity. RESULTS: RPMI8226 and U266 cell lines were chosen for transduction experiments since they present higher levels of TRIAP1 expression. Inhibition of TRIAP1 in RPMI8226 cells increased the percentage of apoptotic cells, accompanied by increased expression of APAF1 and Caspase 9, and Caspase 9 and Caspase 3/7 activity. Transduced U266 cell line did not show sustained inhibition of TRIAP1 expression nor apoptosis induction. CONCLUSION: Stable silencing of TRIAP1 induces late apoptosis through APAF1/Caspase 9 pathway at least in RPMI8226 cell line, suggesting that it could be exploited as a potential target at least for a subgroup of MM patients. GENERAL SIGNIFICANCE: In the present study, we demonstrated effects of TRIAP1 silencing on RPMI8226 MM cell line and established its mechanism mediated through APAF1 and Caspase 9. No relevant effect was found after gene silencing in U266 cell line.

Body Adiposity Index (BAI) as a predictor of body fat in an oldest old and independent Brazilian elderly cohort.

Background: Anthropometry and body mass index (BMI) do not assess body composition or its distribution in older adults; thus, individuals may have different fat percentages but similar BMI values. The body adiposity index (BAI) was recently proposed as a feasible and inexpensive method for estimating the percentage of body fat based on measurements of hip circumference and height. Objectives: The present study evaluated whether BAI and BMI are useful alternatives to dual-energy X-ray absorptiometry (DXA), which is rarely used in clinical practice, for predicting body fat in independent long-lived older adults. Methods: In this cross-sectional study, we used DXA to calculate the percentage of body fat, which was compared with BAI and BMI values. We performed Pearson correlation analyses and used Cronbach's alpha, described by Bland and Altman, to compare the reliability between the indexes. Results: Among 157 evaluated individuals (73.2% women, mean age 87 years, ± 3.57), men had a lower percentage of total fat, as assessed by DXA, and lower BAI indices than women. The correlation between BAI and DXA was moderate (r = 0.59 for men and r = 0.67 for women, p < 0.001). We confirmed the reliability based on Cronbach's alpha coefficients of 0.67 in men and 0.77 in women. We also observed that the BAI was strongly positively correlated with BMI in both men and women. Conclusion: The BAI, used in combination with BMI, can be an alternative to DXA for the assessment of body fat in the oldest old in clinical practice, mainly women, and can be used to add information to BMI.

MAGE-C1/CT7 and MAGE-C2/CT10 are frequently expressed in multiple myeloma and can be explored in combined immunotherapy for this malignancy.

The exact function of MAGE-C1/CT7 and MAGE-C2/CT10 is not yet understood in multiple myeloma (MM). However, the homologs MAGE-C1/CT7 and MAGE-C2/CT10 genes encode highly immunogeneic cancer/testis antigens (CTAs) and can be potential targets for T cell-based immunotherapy. MAGE-C1/CT7 and MAGE-C2/CT10 mRNA expression were investigated in MM patients, solitary plasmacytomas, monoclonal gammopathies of undetermined significance (MGUS) and bone marrow (BM) aspirates from healthy donors by RT-PCR. MAGE-C1/CT7 and MAGE-C1/CT10 were expressed in 67 and 59 % of the 46 MM analyzed patients. At least one of the genes was expressed in 76 % of MM cases. Solitary plasmacytoma also showed MAGE-C1/CT7 and MAGE-C2/CT10 expression. MAGE-C1/CT7 and MAGE-C2/CT10 were not expressed in normal BM samples, showing restricted expression of these CTA genes in MM, solitary plasmacytoma and MGUS. In the present study, we found high expression of the homologs MAGE-C1/CT7 and MAGE-C2/CT10 in monoclonal gammopathies and speculate whether these genes might represent a valuable therapeutic option for myeloma, in particular for combined immunotherapy.

Cancer/testis antigens expression and autologous serological response in a set of Brazilian non-Hodgkin's lymphoma patients.

BACKGROUND: Based on their tumor-associated expression pattern, cancer/testis antigens (CTAs) are considered potential targets for cancer immunotherapy. We aim to evaluate the expression of CTAs in non-Hodgkin's lymphoma (NHL) samples and the ability of these patients to elicit spontaneous humoral immune response against CTAs. METHODS: Expression of MAGE-A family, CT7/MAGE-C1, CT10/MAGE-C2, GAGE and NY-ESO-1 was analyzed by immunohistochemistry in a tissue microarray generated from 106 NHL archival cases. The humoral response against 19 CTAs was tested in 97 untreated NHL serum samples using ELISA technique. RESULTS: 11.3 % of NHL tumor samples expressed at least 1 CTA. MAGE-A family (6.6 %), GAGE (5.7 %) and NY-ESO-1(4.7 %) were the most frequently expressed antigens. We found no statistically significant correlation between CTA positivity and clinical parameters such as NHL histological subtype, Ann Arbor stage, international prognostic index score, response to treatment and overall survival. Humoral response against at least 1 CTA was observed in 16.5 % of NHL serum samples. However, overall seroreactivity was low, and strong titers (>1:1000) were observed in only two diffuse large B-cell lymphomas patients against CT45. CONCLUSION: Our findings are in agreement with most of published studies in this field to date and suggest an overall low expression of CTAs in NHL patients. However, as many new CTAs have been described recently and some of them are found to be highly expressed in NHL cell lines and tumor samples, further studies exploring the expression of different panels of CTAs are needed to evaluate their role as candidates for immunotherapy in NHL patients.

Cancer/Testis Antigen MAGE-C1/CT7: new target for multiple myeloma therapy.

Cancer/Testis Antigens (CTAs) are a promising class of tumor antigens that have a limited expression in somatic tissues (testis, ovary, fetal, and placental cells). Aberrant expression of CTAs in cancer cells may lead to abnormal chromosome segregation and aneuploidy. CTAs are regulated by epigenetic mechanisms (DNA methylation and acetylation of histones) and are attractive targets for immunotherapy in cancer because the gonads are immune privileged organs and anti-CTA immune response can be tumor-specific. Multiple myeloma (MM) is an incurable hematological malignancy, and several CTAs have been detected in many MM cell lines and patients. Among CTAs expressed in MM we must highlight the MAGE-C1/CT7 located on the X chromosome and expressed specificity in the malignant plasma cells. MAGE-C1/CT7 seems to be related to disease progression and functional studies suggests that this CTA might play a role in cell cycle and mainly in survival of malignant plasma cells, protecting myeloma cells against spontaneous as well as drug-induced apoptosis.

The role of regulatory T cells and TH17 cells in multiple myeloma.

The development of multiple myeloma (MM) involves a series of genetic alterations and changes in the bone marrow microenvironment, favoring the growth of the tumor and failure of local immune control. Quantitative and functional alterations in CD4(+) and CD8(+) T cells have been described in MM. The balance between T regulatory cells (Treg) and T helper (Th) 17 cells represents one essential prerequisite for maintaining anti-tumor immunity in MM. Tregs play an important role in the preservation of self-tolerance and modulation of overall immune responses against infections and tumor cells. In MM patients, Tregs seem to contribute to myeloma-related immune dysfunction and targeting them could, therefore, help to restore and enhance vital immune responses. Th17 cells protect against fungal and parasitic infections and participate in inflammatory reactions and autoimmunity. The interplay of TGF-ß and IL-6, expressed at high levels in the bone marrow of myeloma patients, may affect generation of Th17 cells both directly or via other pro-inflammatory cytokines and thereby modulate antitumor immune responses. A detailed analysis of the balance between Tregs and Th17 cells seems necessary in order to design more effective and less toxic modes of immunotherapy myeloma which still is an uncurable malignancy.

Evaluation of LAGE-1 and NY-ESO-1 expression in multiple myeloma patients to explore possible benefits of their homology for immunotherapy.

Due to the high homology between the LAGE-1 and NY-ESO-1 proteins, we hypothesized that an anti-NY-ESO-1 vaccine might elicit LAGE-1 immunity and hence may be effective in multiple myeloma (MM) patients with LAGE-1-positive/NY-ESO-1-negative tumors. Therefore, we set out to evaluate LAGE-1 and NY-ESO-1 mRNA and protein expression in MM patients in a bid to evaluate possible benefits of their homology for immunotherapy. LAGE-1 (a and b isoforms) and NY-ESO-1 mRNA expression was studied in 18 normal tissues and 50 bone marrow MM samples by RT-PCR. LAGE-1 and NY-ESO-1 protein expression was analyzed by immunohistochemistry (IHC) in 27 MM specimens using mAbs 219-510-23 and E978. Spontaneous serological immune response against both antigens was analyzed by ELISA in sera from 33 MM patients. LAGE-1 (a and b isoforms) was positive in 42% and NY-ESO-1 in 26% of the MM samples analyzed by RT-PCR. Both genes were found to be expressed in 18% of the cases, while at least one of the genes was found to be expressed in 50% of the cases. In LAGE-1 positive samples, 81% were positive for LAGE-1a and 19% were positive for both LAGE-1a and -1b. LAGE-1 and NY-ESO-1 protein expression could only be detected in two cases by IHC and there was a clear strong spontaneous antibody response to LAGE-1 and NY-ESO-1 in only one MM patient. In conclusion, LAGE-1a and NY-ESO-1 homology cannot be easily exploited in an anti-NY-ESO-1 vaccine given the low frequency of protein expression detected by IHC or serum analysis.

Structural profiles of TP53 gene mutations predict clinical outcome in diffuse large B-cell lymphoma: an international collaborative study.

The purpose of this study is to correlate the presence of TP53 gene mutations with the clinical outcome of a cohort of patients with diffuse large B-cell lymphoma (DLBCL) assembled from 12 medical centers. TP53 mutations were identified in 102 of 477 patients, and the overall survival (OS) of patients with TP53 mutations was significantly worse than those with wild-type TP53 (P < .001). However, subsets of TP53 mutations were found to have different effects on OS. Mutations in the TP53 DNA-binding domains were the strongest predictors of poor OS (P < .001). Mutations in the Loop-Sheet-Helix and Loop-L3 were associated with significantly decreased OS (P = .002), but OS was not significantly affected by mutations in Loop-L2. A subset of missense mutations (His158, His175, Ser245, Gln248, His273, Arg280, and Arg282) in the DNA-binding domains had the worst prognosis. Multivariate analysis confirmed that the International Prognostic Index and mutations in the DNA-binding domains were independent predictors of OS. TP53 mutations also stratified patients with germinal center B cell-like DLBCL, but not nongerminal center B cell-like DLBCL, into molecularly distinct subsets with different survivals. This study shows the prognostic importance of mutations in the TP53 DNA-binding domains in patients with DLBCL.

TGFbetaR2 aberrant methylation is a potential prognostic marker and therapeutic target in multiple myeloma.

Multiple myeloma (MM) is an incurable hematological malignancy. Different studies demonstrated the occurrence of genetic and epigenetic alterations in MM. The aberrant methylation is one of the most frequent epigenetic alterations in human genome. This study evaluated the aberrant methylation status of 20 genes in 51 MM samples by quantitative methylation-specific PCR (QMSP) and compared the methylation profile with clinicopathological characteristics of the patients. The QMSP analyses showed that PTGS2 (100.0%), SFN (100.0%), CDKN2B (90.2%), CDH1 (88.2%), ESR1 (72.5%), HIC1 (70.5%), CCND2 (62.7%), DCC (45.1%) and TGFbetaR2 (39.2%) are frequently hypermethylated in MM while aberrant methylation of RARbeta (16.6%), MGMT (12.5%), AIM1 (12.5%), CDKN2A (8.3%), SOCS1 (8.3%), CCNA1 (8.3%) and THBS1 (4.1%) are rare events. There was no methylation of GSTP1, MINT31, p14ARF and RB1 in the samples tested. Hypermethylation of ESR1 was correlated positively with isotype IgA, while aberrant methylation of THBS1 correlated negatively with isotype IgG. Furthermore, hypermethylation of DCC and TGFbetaR2 were correlated with poor survival. The multivariate analysis showed ISS and TGFbetaR2 hypermethylation strongly correlated with poor outcome. This study represents the first quantitative evaluation of promoter methylation in MM and our data provide evidence that TGFbetaR2 hypermethylation, besides ISS, may be useful as prognostic indicator in this disease.

Stew in its Own Juice: Protein Homeostasis Machinery Inhibition Reduces Cell Viability in Multiple Myeloma Cell Lines.

INTRODUCTION: Multiple myeloma (MM) cells accumulate in the bone marrow and produce enormous quantities of immunoglobulins, causing endoplasmatic reticulum stress and activation of protein handling machinery, such as heat shock protein response, autophagy and unfolded protein response (UPR). METHODS: We evaluated cell lines viability after treatment with bortezomib (B) in combination with HSP70 (VER-15508) and autophagy (SBI-0206965) or UPR (STF- 083010) inhibitors. RESULTS: For RPMI-8226, after 72 hours of treatment with B+VER+STF or B+VER+SBI, we observed 15% of viable cells, but treatment with B alone was better (90% of cell death). For U266, treatment with B+VER+STF or with B+VER+SBI for 72 hours resulted in 20% of cell viability and both treatments were better than treatment with B alone (40% of cell death). After both triplet combinations, RPMI-8226 and U266 presented the overexpression of XBP-1 UPR protein, suggesting that it is acting as a compensatory mechanism, in an attempt of the cell to handle the otherwise lethal large amount of immunoglobulin overload. CONCLUSION: Our in vitro results provide additional evidence that combinations of protein homeostasis inhibitors might be explored as treatment options for MM.

Prognostic impact of cancer/testis antigen expression in advanced stage multiple myeloma patients.

This study aims to analyze the expression of 14 cancer/testis (CT) antigens in multiple myeloma (MM) to identify possible prognostic markers and therapeutic targets. The expression of MAGEA1, MAGEA2, MAGEA3/6, MAGEA4, MAGEA10, MAGEA12, BAGE1, MAGEC1/CT7, the GAGE family, LAGE-1, PRAME, NY-ESO-1, SPA17 and SSX1 was studied by RT-PCR in 15 normal tissues, a pool of 10 normal bone marrow samples, 3 normal tonsils and bone marrow aspirates from 6 normal donors, 3 monoclonal gammopathies of undetermined significance (MGUS), 5 solitary plasmacytomas, 39 MM samples (95% advanced stage) and the MM cell line U266. MAGEC1/CT7 was expressed in bone marrow aspirates from one MGUS and one plasmacytoma. The frequencies at which CT antigen were found to be expressed in MM patients were MAGEC1/CT7 77%, LAGE-1 49%, MAGEA3/6 41%, MAGEA2 36%, GAGE family 33%, NY-ESO-1 33%, BAGE-1 28%, MAGEA1 26%, PRAME 23%, SSX-1 26%, MAGEA12 20.5%, MAGEA4 0%, and MAGEA10 0%. Cox's regression model showed that GAGE family expression and having >6 CT antigens expressed were independent prognostic factors when all patients were analyzed. However, MAGEC1/CT7 expression was the only independent prognostic factor when non-transplanted patients where analyzed. Based on our findings, MAGEC1/CT7, MAGEA3/6 and LAGE-1 are good candidates for immunotherapy, since together they cover 85% of our MM cases. Furthermore, expression of the GAGE family, >6 CT antigens and MAGEC1/CT7 seem to have impact on MM prognosis.

Is the follicular lymphoma international prognostic index better than the international prognostic index to identify high-risk follicular lymphoma patients?

The aims of this study are to validate follicular lymphoma international prognostic index (FLIPI) prognostic score and to compare it with the international prognostic index (IPI) in a cohort of 57 Brazilian patients. According to IPI, 24 patients (42%) were in the low-risk, 28 (49%) in the intermediate-risk, and 4 (7%) in the high-risk group. The distribution according to FLIPI was: 20 (35%) in the low-risk, 8 (14%) in the intermediate-risk, and 29 (51%) in the high-risk group. According to IPI score, median OS was not reached for the low-risk, it was 45 months for the intermediate-risk and 25 months for the high-risk group (p < 0.001). When FLIPI score was applied, median OS was not reached for the low and intermediate-risk, and was 42 months for the high-risk group (p = 0.0064). These findings suggest that: (1) FLIPI score could be validated in a Brazilian population; (2) FLIPI is more accurate than IPI to identify FL patients having worse prognosis (51%); (3) IPI seems to be a better tool for clinical decisions because it selected a smaller high-risk group (7%) having worse prognosis. In our opinion, IPI high-risk patients are the real candidates for more aggressive therapies, avoiding unnecessary over-treatment.

Anti-myeloma effects of ruxolitinib combined with bortezomib and lenalidomide: A rationale for JAK/STAT pathway inhibition in myeloma patients.

JAK proteins have been linked with survival and proliferation of multiple myeloma (MM) cells; therefore, JAK inhibition could be a therapeutic strategy for MM. We evaluated JAK1 and JAK2 expression in MM patients and the effects of JAK/STAT pathway inhibition on apoptosis, cell cycle, gene and protein expression in RPMI-8226 and U266 MM cell lines. 57% of patients presented overexpression of JAK2 and 27%, of JAK1. After treatment with ruxolitinib and bortezomib, RPMI-8226 and U266 presented 50% of cells in late apoptosis, reduction of anti-apoptotic genes expression and higher number of cells in SubG0 phase. Co-culture with stromal cells protected RPMI-8226 cells from apoptosis, which was reversed by lenalidomide addition. Combination of ruxolitinib, bortezomib and lenalidomide induced 72% of cell death, equivalent to bortezomib, lenalidomide and dexamethasone, combination used in clinical practice. Many JAK/STAT pathway genes, after treatment, had their expression reduced, mainly in RPMI-8226, with insignificant changes in U266. In this scenario, JAK/STAT pathway could pose as a new therapeutic target to be exploited, since it is constitutively active and contributes to survival of MM tumor cells.

Psychosocial adaptation and quality of life among Brazilian patients with different hematological malignancies.

This study aims to investigate the prevalence of posttraumatic stress disorder (PTSD) symptoms, anxiety, and depression in patients with hematological malignancies, and to investigate the possible relationship between these symptoms and variables such as demographic data, social support, and quality of life (QOL). We studied 107 patients: 54 with non-Hodgkin's lymphoma (NHL), 18 acute myelogenous leukaemia (AML), 10 acute lymphoblastic leukaemia (ALL), and 25 multiple myeloma (MM). Demographic data were collected, and three standardized instruments were applied to this group of patients: Hospital Anxiety and Depression Scale (HADS), Impact of Event Scale (IES), European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 questionnaire of QOL. The results showed a significant percentage of patients presenting with symptoms: 13% had high levels of intrusive thoughts, 20.5% had high levels of anxiety, and 16.8% had high levels of depression. Patients with MM had the lowest QOL scores in the EORTC physical functioning subscale. Patients under intravenous chemotherapy treatment had a higher level of anxiety than the monitoring patients. Patients with recent diagnosis had a level of intrusion symptoms (IES) relevantly higher than the others. The unemployed patients and those with lower social support had levels of stress, anxiety, and depression significantly higher than the others. Our results confirm the high incidence of intrusion, avoidance, anxiety, and depression in patients with hematological malignancies and highlight the importance of a multidisciplinary staff to complement the treatment of these patients, including psychosocial assistance.

Angiomirs expression profiling in diffuse large B-Cell lymphoma.

Despite advances in treatment, 30% of diffuse large B-cell lymphoma (DLBCL) cases are refractory or relapse after chemoimmunotherapy. Currently, the relationship between angiogenesis and angiomiRs in DLBCL is unknown. We classified 84 DLBCL cases according to stromal signatures and evaluated the expression of pro- and antiangiomiRs in paraffin embedded tissues of DLBCL and correlated them with microvascular density (MVD). 40% of cases were classified as stromal-1, 50% as stromal-2 and 10% were not classified. We observed increased expression of proangiomiRs Let-7f, miR-17, miR-18a, miR-19b, miR-126, miR-130a, miR-210, miR-296 and miR-378 in 14%, 57%, 30%, 45%, 12%, 12%, 56%, 58% and 48% of the cases, respectively. Among antiangiomiRs we found decreased expression of miR-16, miR-20b, miR-92a, miR-221 and miR-328 in, respectively, 27%, 71%, 2%, 44% and 11%. We found association between increased expression of proangiomiRs miR-126 and miR-130a and antiangiomiR miR-328 and the subtype non-GCB. We found higher levels of the antiangiomiRs miR-16, miR-221 and miR-328 in patients with low MVD and stromal-1 signature. IPI and CD34 confirmed independent impact on survival of the study group. None of the above angiomiRs showed significance as biomarker in an independent serum samples cohort of patients and controls. In conclusion, we confirmed association between antiangiomiRs miR-16, miR-221 and miR-328 and stromal-1 signature. Four angiomiRs emerged as potential therapeutic targets: proangiomiRs miR-17, miR-210 and miR-296 and antiangiomiR miR-20b. Although the four microRNAs seem to be important in DLBCL pathogenesis, they were not predictive of DLBCL onset or relapse in the serum independent cohort.

p16 gene methylation lacks correlation with angiogenesis and prognosis in multiple myeloma.

Methylation of p16 gene is a relatively frequent molecular finding in multiple myeloma (MM), but its clinical implication is disputable. Cell cycle regulators are now recognized as active in the control of angiogenesis, which is an integral component of pathogenesis and a target for new treatment modalities of this disease. On such background, we focused on determining whether loss of p16 function by methylation could be associated with increased angiogenesis and VEGF expression, and the prognostic relevance of p16 methylation in 50 untreated, newly diagnosed MM patients. Thirty-one percent (13/42) of 42 patients assessable for p16 gene methylation showed to be methylation-positive. High-angiogenesis was present in 73% of cases, but methylation of the p16 gene did not associate with angiogenesis or with VEGF expression. Also, p16 methylation did not show prognostic relevance or correlation with the clinical and laboratory parameters of prognostic significance in univariate analysis. P16 immunoexpression presented only a faint agreement with the molecular study. Therefore, p16 methylation seems to have no correlation with angiogenesis and VEGF expression, neither with overall and event-free survival in MM patients. Besides, P16 immunohistochemistry seems inadequate to substitute the molecular study of methylation in this type of tumor cells. Additional studies are needed to clarify the correspondence between the epigenetic alteration of the p16 gene and its protein immunexpression, and the clinical relevance of p16 methylation in MM patients.

Epstein-Barr viral load, interleukin-6 and interleukin-10 levels in post-transplant lymphoproliferative disease: a nested case-control study in a renal transplant cohort.

The possible correlation among Epstein-Barr virus (EBV) load, interleukin-6 (IL-6) and interleukin-10 (IL-10) levels has become an attractive issue and can provide a useful tool for diagnosis and monitoring of patients at risk for post-transplant lymphoproliferative disease (PTLD) development. At the time of diagnosis of PTLD, 11 patients were prospectively enrolled and 55 nested controls were selected from a 1800 renal transplant cohort. Real-time polymerase chain reaction (PCR) was used to quantify EBV load in peripheral blood mononuclear cells (PBMC). Serum IL-6 and IL-10 levels were determined using an enzyme-linked immunosorbent assay (ELISA). The median EBV load of PTLD cases was 17400 copies/10(6) PBMC, statistically different from controls (P=0.001). The median IL-6 level of PTLD cases was not different from controls (P=0.079). However, median IL-10 levels showed a significant difference in both groups (P < or = 0.001). The receiver-operating characteristic (ROC) curve analysis was applied to estimate the IL-10 cut-off value predictive of PTLD development. We found that 73.5 pg/ml has high sensitivity (1.00) and specificity (0.85). Also, Pearson's analysis showed a strong correlation between EBV load and serum IL-10 concentration (P < or = 0.001). This nested case-control study demonstrates that EBV load at diagnosis of PTLD correlates with IL-10 levels, and that monitoring of IL-10 can provide a less expensive and less time-consuming tool for PTLD diagnosis and close follow-up of patients at risk. Furthermore, we were able to define a cut-off value of IL-10 mostly predictive of PTLD development in this cohort. Our data suggest that serial measurements prior to PTLD development must be carried out to validate our hypothesis.

Molecular analysis of gastric washings in the diagnosis and monitoring of gastric lymphomas.

The monitoring of gastric lymphomas is often hampered by the inherently limited sampling provided by small endoscopic biopsy specimens. To investigate the feasibility of using gastric washing fluid for monitoring patients with known gastric lymphoma and for diagnosing gastric involvement in patients with extranodal nongastric lymphoma, we collected 49 gastric washings from 39 patients (29 patients with gastric lymphoma and 10 patients with nongastric extranodal lymphoma). Collection was done at the time of follow-up biopsy and when no endoscopic abnormalities were found. DNA was extracted from the washing fluid and analyzed for clonal IgH gene rearrangement by Southern blotting (J6 probe) and/or polymerase chain reaction (PCR) (using VH-FR3 and JH primers). Forty-one of 49 samples (84%) yielded sufficient DNA for molecular analysis. Sixteen of 41 analyzable gastric washing samples (39%) failed Southern blot analysis due to degraded or insufficient DNA. Concordance between the results of Southern blot analysis of the washing and histology of the simultaneous biopsy specimen was found in 20 (80%) of the remaining 25 samples. The IgH PCR result was concordant with biopsy histology in 33 out of 41 washing samples (80%). The overall concordance between molecular clonality studies of washings (Southern blotting and/or PCR) and biopsy histology was 83% (34 of 41). Of the 7 (18%) discrepant specimens, 2 were diagnosed histologically as lymphoma, but the simultaneous washings were negative by molecular studies. Five biopsy specimens were histologically benign, but the corresponding washings demonstrated clonal IgH gene rearrangement (3 cases by PCR and 2 cases by Southern blotting). This study demonstrates the diagnostic utility of molecular clonality analysis of gastric washings.