miRNA profile and disease severity in patients with sickle cell anemia.

Identification of biomarkers associated with severity in sickle cell anemia is desirable. Circulating serum microRNAs (miRNA) are targets studied as diagnostic or prognostic markers, but few studies have been conducted in sickle cell anemia. The purpose of this study is to identify specific signatures of miRNAs in plasma samples from sickle cell anemia patients according to severity indexes. Screening of the miRNAs expression was performed in 8 patients, classified by tricuspid regurgitation velocity (TRV) measure: 4 with TRV ≥ 2.5 m/s and 4 with TRV < 2.5 m/s. The samples were analyzed by real-time PCR using Megaplex RT Human Pool A and Pool B comprising 667 distinct miRNAs. Seventeen miRNAs were differentially expressed between the two groups (p < 0.05). Five differentially expressed miRNAs (miR15b, miR502, miR510, miR544, miR629) were selected for validation in a cohort of 52 patient samples, 26 with TRV ≥ 2.5 m/s. Another two severity scores were also used: organ injury score (OIS) and Bayesian score (BS). Univariate binary logistic regressions were performed to analyze the data. Five out of 17 differentially expressed miRNAs were selected for validation in 52 patient samples: miR15b, miR502, miR510, miR544, and miR629. Two miRNAs (miR510 and miR629) were significantly decreased in cases of greater severity. Whereas miR510 expression discriminated the patients according to TRV and OIS, miR629 expression did it according to BS. This is the first study investigating plasma miRNAs as possible biomarkers for SCA severity. Our data suggest that low levels of miR510 and miR629 expression are associated with greater SCA disease severity. Further studies are still necessary to elucidate mechanism of these miRNAs and their related proteins.

Single nucleotide variants in immune-response genes and the tumor microenvironment composition predict progression of mantle cell lymphoma.

BACKGROUND: There is evidence to consider that the tumor microenvironment (TME) composition associates with antitumor immune response, and may predict the outcome of various non-Hodgkin lymphoma subtypes. However, in the case of mantle cell lymphoma (MCL), a rare and aggressive disease, there is lacking a detailed study of the TME components, as well as an integrative approach among them in patients' samples. Also, from the genetic point of view, it is known that single nucleotide variants (SNVs) in immune-response genes are among important regulators of immunity. At present, it is uncertain whether SNVs in candidate immune-response genes and the TME composition are able to alter the prognosis in MCL. METHODS: We assessed a detailed TME composition in 88 MCL biopsies using immunohistochemistry, which was automatically analyzed by pixel counting (Aperio system). We also genotyped SNVs located in candidate immune-response genes (IL12A, IL2, IL10, TGFB1, TGFBR1, TGFBR2, IL17A, IL17F) in 95 MCL patients. We tested whether the SNVs could modulate the respective protein expression and TME composition in the tumor compartment. Finally, we proposed survival models in rituximab-treated patients, considering immunohistochemical and SNV models. RESULTS: High FOXP3/CD3 ratios (p = 0.001), high IL17A levels (p = 0.003) and low IL2 levels (p = 0.03) were individual immunohistochemical predictors of poorer survival. A principal component, comprising high quantities of macrophages and high Ki-67 index, also worsened outcome (p = 0.02). In the SNV model, the CC haplotype of IL10 (p < 0.01), the GG genotype of IL2 rs2069762 (p = 0.02) and the AA+AG genotypes of TGFBR2 rs3087465 (p < 0.01) were independent predictors of outcome. Finally, the GG genotype of TGFB1 rs6957 associated with lower tumor TGFß levels (p = 0.03) and less CD163+ macrophages (p = 0.01), but did not modulate patients' survival. CONCLUSIONS: Our results indicate that the TME composition has relevant biological roles in MCL. In this setting, immunohistochemical detection of T-reg cells, IL17A and IL2, coupled with SNV genotyping in IL10, TGFBR2 and IL2, may represent novel prognostic factors in this disease, following future validations.

May critical molecular cross-talk between indoleamine 2,3-dioxygenase (IDO) and arginase during human aging be targets for immunosenescence control?

BACKGROUND: This study aimed to identify novel plasma metabolic signatures with possible clinical relevance during the aging process. A biochemical quantitative phenotyping platform, based on targeted electrospray ionization tandem mass spectrometry technology, was used for the identification of any eventual perturbed biochemical pathway by the aging process in prospectively collected peripheral blood plasma from 166 individuals representing the population of São Paulo city, Brazil. RESULTS: Indoleamine 2,3-dioxygenase (IDO) activity (Kyn/Trp) was significantly elevated with age, and among metabolites most associated with elevations in IDO, one of the strongest correlations was with arginase (Orn/Arg), which could also facilitate the senescence process of the immune system. Hyperactivity of IDO was also found to correlate with increased blood concentrations of medium-chain acylcarnitines, suggesting that deficiencies in beta-oxidation may also be involved in the immunosenescence process. Finally, our study provided evidence that the systemic methylation status was significantly increased and positively correlated to IDO activity. CONCLUSIONS: In the present article, besides identifying elevated IDO activity exhibiting striking parallel association with the aging process, we additionally identified increased arginase activity as an underlying biochemical disturbance closely following elevations in IDO. Our findings support interventions to reduce IDO or arginase activities in an attempt to preserve the functionality of the immune system, including modulation of myeloid-derived suppressor cells (MDSCs), T cells, macrophages, and dendritic cells' function, in old individuals/patients.

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.

International, evidence-based consensus diagnostic criteria for HHV-8-negative/idiopathic multicentric Castleman disease.

Human herpesvirus-8 (HHV-8)-negative, idiopathic multicentric Castleman disease (iMCD) is a rare and life-threatening disorder involving systemic inflammatory symptoms, polyclonal lymphoproliferation, cytopenias, and multiple organ system dysfunction caused by a cytokine storm often including interleukin-6. iMCD accounts for one third to one half of all cases of MCD and can occur in individuals of any age. Accurate diagnosis is challenging, because no standard diagnostic criteria or diagnostic biomarkers currently exist, and there is significant overlap with malignant, autoimmune, and infectious disorders. An international working group comprising 34 pediatric and adult pathology and clinical experts in iMCD and related disorders from 8 countries, including 2 physicians that are also iMCD patients, was convened to establish iMCD diagnostic criteria. The working group reviewed data from 244 cases, met twice, and refined criteria over 15 months (June 2015 to September 2016). The proposed consensus criteria require both Major Criteria (characteristic lymph node histopathology and multicentric lymphadenopathy), at least 2 of 11 Minor Criteria with at least 1 laboratory abnormality, and exclusion of infectious, malignant, and autoimmune disorders that can mimic iMCD. Characteristic histopathologic features may include a constellation of regressed or hyperplastic germinal centers, follicular dendritic cell prominence, hypervascularization, and polytypic plasmacytosis. Laboratory and clinical Minor Criteria include elevated C-reactive protein or erythrocyte sedimentation rate, anemia, thrombocytopenia or thrombocytosis, hypoalbuminemia, renal dysfunction or proteinuria, polyclonal hypergammaglobulinemia, constitutional symptoms, hepatosplenomegaly, effusions or edema, eruptive cherry hemangiomatosis or violaceous papules, and lymphocytic interstitial pneumonitis. iMCD consensus diagnostic criteria will facilitate consistent diagnosis, appropriate treatment, and collaborative research.

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.

Observational study of multiple myeloma in Latin America.

Relatively little is known about the outcomes of multiple myeloma in Latin America, a world region where incorporation of novel agents is generally slow. In the current retrospective-prospective study, we aimed to describe the patterns of care and treatment results in five Latin American countries. Between April 2007 and October 2009, patients who had been diagnosed from January 2005 to December 2007 were registered at 23 institutions from Argentina, Brazil, Chile, Mexico, and Peru. We divided patients into two cohorts, according to transplantation eligibility, and analyzed them with regard to first-line treatment and overall survival (OS). We analyzed a total of 852 patients, 46.9 % of whom were female. The median follow-up was 62 months. Among transplantation-ineligible patients (N = 461), the mean age was 67.4 years, approximately one third of patients received a thalidomide-based treatment in the first line, and the median OS was 43.0 months. Transplantation-eligible patients (N = 391) had a mean age of 54.7 years and a median OS of 73.6 months. Autologous transplantation was performed in 58.6 % of the patients for whom this procedure was initially planned and in only 26.9 % of the overall patients. Our long-term results reflect the contemporary literature for patients with multiple myeloma treated with autologous transplantation and thalidomide-based regimens in clinical trials and observational studies. However, further efforts are needed to approve and incorporate novel agents in Latin American countries, as well as to increase access to transplantation, in order to achieve the expected improvements in patient outcomes.

FOXP3 and CTLA4 overexpression in multiple myeloma bone marrow as a sign of accumulation of CD4(+) T regulatory cells.

INTRODUCTION: Multiple myeloma (MM) development involves a series of genetic abnormalities and changes in the bone marrow (BM) microenvironment, favoring the growth of the tumor and failure of local immune control. T regulatory (Treg) cells play an important role in dampening anti-tumor immune responses while T-helper-17 (Th17) cells seem to be critical for the eradication of malignant cells. The aim of our study was to characterize the expression of Treg- and Th17-related genes in total myeloma BM samples to assess their role as biomarkers, prognostic factors, and possible therapeutic targets in this incurable disease. METHODS: Expression of markers for Treg (FOXP3, CTLA4) and Th17 cells (RORγt) was determined by quantitative real-time PCR in BM aspirates of 46 MM patients, four patients with monoclonal gammopathy of undetermined significance, five solitary plasmacytomas, and five healthy BM donors. Gene expression was evaluated regarding an influence on the patients' overall survival (OS). RESULTS: FOXP3 and CTLA4 presented a sixfold (p = 0.02) and 30-fold higher expression (p = 0.03), respectively, in MM patients than in controls. RORγt expression was similar in MM patients and controls. Median OS of MM patients was 16.8 (range 4.5-29.1) months, and international staging system was the only independent prognostic factor for patients survival. CONCLUSIONS: Overexpression of FOXP3 and CTLA4 in total BM samples suggests a local accumulation of immunosuppressive Tregs, the MM tumor environment, possibly dampening anti-tumor host immune responses. Therapeutic approaches targeting Treg cells and restoring local anti-tumor immunity may provide new treatment strategies for this incurable malignancy.

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.

Immune reconstitution inflammatory syndrome-associated lymphoma: A retrospective Brazilian cohort.

After initiating combined antiretroviral therapy (cART), individuals with human immunodeficiency virus (HIV) may develop Hodgkin/non-Hodgkin lymphoma due to immune reconstitution inflammatory syndrome (IRIS). This retrospective cohort study evaluated the incidence, clinical features and prognosis of IRIS-associated lymphomas in Brazilian patients. Incidence in 2000-2019 was 9.8% (27/276 patients with HIV and lymphoma; viral load drop >1 log). Time between HIV diagnosis and cART initiation was <1 year in 70.3% of cases. Time between cART initiation and lymphoma diagnosis was <3 months in 11 cases and 3-6 months in 16 cases. Overall and progression-free survival rates were similar between cases of non-IRIS-associated lymphoma and IRIS-associated lymphoma.

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.

Factors associated with survival in patients with lymphoma and HIV.

OBJECTIVE: To analyze the factors associated with survival in the largest cohort of individuals with HIV and lymphoma so far described in Brazil. DESIGN: A retrospective, observational, multicenter study involving five institutions in São Paulo, Brazil. METHODS: The medical records of consecutive patients with HIV diagnosed with lymphoma between January 2000 and December 2019 were screened. Inclusion criteria consisted of age over 17 years and a biopsy-confirmed diagnosis of lymphoma. The data collected included age, sex, staging (Ann Arbor system), duration of HIV infection, CD4 + lymphocyte count, HIV viral load, lactate dehydrogenase, erythrocyte sedimentation rate and serum beta-2-microglobulin levels, treatment and outcome. RESULTS: Overall, 276 patients were included. Median age was 42 years. Most patients were male (74.3%) and with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 (28.6% and 46.4%, respectively). Most had non-Hodgkin lymphomas (89.2%, n  = 246), particularly diffuse large B-cell lymphoma (40.9%) and Burkitt lymphoma (26.4%). Hodgkin lymphoma accounted for 9.4%. Advanced stages III/IV were predominant (86.8%). HIV viral load at the moment of lymphoma diagnosis was detectable in 52.9% of patients. A CD4 + cell count of <200 cells/µl was recorded for 53% of the patients. Most patients (62.4%) were on combination antiretroviral therapy. The factors that significantly affected survival were: the ECOG performance status, lymphoma subtype, staging, beta-2-microglobulin level, central nervous system (CNS) infiltration, site of CNS infiltration, relapsed/refractory lymphoma and International Prognostic Index score. CONCLUSIONS: HIV status, CD4 + -lymphocyte count and relapsed/refractory disease affected survival. Rituximab did not appear to improve outcome in HIV-related lymphomas.

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.

An overview of cancer/testis antigens expression in classical Hodgkin's lymphoma (cHL) identifies MAGE-A family and MAGE-C1 as the most frequently expressed antigens in a set of Brazilian cHL patients.

ABSTRACT: BACKGROUND: Cancer/testis antigens are considered potential targets for immunotherapy due to their tumor-associated expression pattern. Although recent studies have demonstrated high expression of CT45 in classical Hodgkin's lymphomas (cHL), less is known about the expression pattern of other families of CTAs in cHL. We aim to evaluate the expression of MAGE-A family, MAGE-C1/CT7, MAGE-C2/CT10, NY-ESO1 and GAGE family in cHL and to correlate their expression with clinical and prognostic factors in cHL. METHODS: Tissue microarray was generated from 38 cHL archival cases from Pathology Department of Universidade Federal de Sao Paulo. Immunohistochemistry (IHC) was done using the following panel of antibodies: MAGE-A family (MA454, M3H67, 57B and 6C1), GAGE (#26), NY-ESO-1 (E978), MAGE-C1/CT7 (CT7-33) and MAGE-C2/CT10 (CT10#5). RESULTS: We found CTA expression in 21.1% of our cHL series. Among the tested CTAs, only MAGE-A family 7/38 (18.4%) and MAGE-C1/CT7 5/38 (13.2%) were positive in our cHL samples. We found higher CTA positivity in advanced stage (28.6%) compared to early stage (11.8%) disease, but this difference was not statistically significant. Analysis of other clinicopathological subgroups of cHL including histological subtypes, EBV status and response to treatment also did not demonstrate statistical significant differences in CTA expression. CONCLUSION: We found CTA expression in 21.1% of cHL samples using our panel. Our preliminary findings suggest that from all CTAs included in this study, MAGE-A family and MAGE-C1/CT7 are the most interesting ones to be explored in further studies.

Tumor Microenvironment Proteomics: Lessons From Multiple Myeloma.

Although the "seed and soil" hypothesis was proposed by Stephen Paget at the end of the 19th century, where he postulated that tumor cells (seeds) need a propitious medium (soil) to be able to establish metastases, only recently the tumor microenvironment started to be more studied in the field of Oncology. Multiple myeloma (MM), a malignancy of plasma cells, can be considered one of the types of cancers where there is more evidence in the literature of the central role that the bone marrow (BM) microenvironment plays, contributing to proliferation, survival, migration, and drug resistance of tumor cells. Despite all advances in the therapeutic arsenal for MM treatment in the last years, the disease remains incurable. Thus, studies aiming a better understanding of the pathophysiology of the disease, as well as searching for new therapeutic targets are necessary and welcome. Therefore, the present study aimed to evaluate the protein expression profiling of mononuclear cells derived from BM of MM patients in comparison with these same cell types derived from healthy individuals, in order to fill this gap in MM treatment. Proteomic analysis was performed using the mass spectrometry technique and further analyses were done using bioinformatics tools, to identify dysregulated biological pathways and/or processes in the BM microenvironment of patients with MM as a result of the disease. Among the pathways identified in this study, we can highlight an upregulation of proteins related to protein biosynthesis, especially chaperone proteins, in patients with MM. Additionally, we also found an upregulation of several proteins involved in energy metabolism, which is one of the cancer hallmarks. Finally, with regard to the downregulated proteins, we can highlight mainly those involved in different pathways of the immune response, corroborating the data that has demonstrated that the immune system of MM is impaired and, therefore, the immunotherapies that have been studied recently for the treatment of the disease are extremely necessary in the search for a control and a cure for these patients who live with the disease.

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.