CD4+ tumor infiltrating lymphocytes are prognostic and independent of R-IPI in patients with DLBCL receiving R-CHOP chemo-immunotherapy

Despite the Revised International Prognostic Index's (R-IPI) undoubted utility in diffuse large B-cell lymphoma (DLBCL), significant clinical heterogeneity within R-IPI categories persists. Emerging evidence indicates that circulating host immunity is a robust and R-IPI independent prognosticator, most likely reflecting the immune status of the intratumoral microenvironment. We hypothesized that direct quantification of immunity within lymphomatous tissue would better permit stratification within R-IPI categories. We analyzed 122 newly diagnosed consecutive DLBCL patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) chemo-immunotherapy. Median follow-up was 4 years. As expected, the R-IPI was a significant predictor of outcome with 5-year overall survival (OS) 87% for very good, 87% for good, and 51% for poor-risk R-IPI scores (P < 0.001). Consistent with previous reports, systemic immunity also predicted outcome (86% OS for high lymphocyte to monocyte ratio [LMR], versus 63% with low LMR, P = 0.01). Multivariate analysis confirmed LMR as independently prognostic. Flow cytometry on fresh diagnostic lymphoma tissue, identified CD4+ T-cell infiltration as the most significant predictor of outcome with ≥23% infiltration dividing the cohort into high and low risk groups with regard to event-free survival (EFS, P = 0.007) and OS (P = 0.003). EFS and OS were independent of the R-IPI and LMR. Importantly, within very good/good R-IPI patients, CD4+ T-cells still distinguished patients with different 5 year OS (high 96% versus low 63%, P = 0.02). These results illustrate the importance of circulating and local intratumoral immunity in DLBCL treated with R-CHOP.

High-resolution loss of heterozygosity screening implicates PTPRJ as a potential tumor suppressor gene that affects susceptibility to non-Hodgkin's Lymphoma

We employed a Hidden-Markov-Model (HMM) algorithm in loss of heterozygosity (LOH) analysis of high-density single nucleotide polymorphism (SNP) array data from Non-Hodgkin’s lymphoma (NHL) entities, follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). This revealed a high frequency of LOH over the chromosomal region 11p11.2, containing the gene encoding the protein tyrosine phosphatase receptor type J (PTPRJ). Although PTPRJ regulates components of key survival pathways in B-cells (i.e., BCR, MAPK, and PI3K signaling), its role in B-cell development is poorly understood. LOH of PTPRJ has been described in several types of cancer but not in any hematological malignancy. Interestingly, FL cases with LOH exhibited down-regulation of PTPRJ, in contrast no significant variation of expression was shown in DLBCLs. In addition, sequence screening in Exons 5 and 13 of PTPRJ identified the G973A (rs2270993), T1054C (rs2270992), A1182C (rs1566734), and G2971C (rs4752904) coding SNPs (cSNPs). The A1182 allele was significantly more frequent in FLs and in NHLs with LOH. Significant over-representation of the C1054 (rs2270992) and the C2971 (rs4752904) alleles were also observed in LOH cases. A haplotype analysis also revealed a significant lower frequency of haplotype GTCG in NHL cases, but it was only detected in cases with retention. Conversely, haplotype GCAC was over-representated in cases with LOH. Altogether, these results indicate that the inactivation of PTPRJ may be a common lymphomagenic mechanism in these NHL subtypes and that haplotypes in PTPRJ gene may play a role in susceptibility to NHL, by affecting activation of PTPRJ in these B-cell lymphomas.

A new method to detect loss of heterozygosity using cohort heterozygosity comparisons

Background Loss of heterozygosity (LOH) is an important marker for one of the 'two-hits' required for tumor suppressor gene inactivation. Traditional methods for mapping LOH regions require the comparison of both tumor and patient-matched normal DNA samples. However, for many archival samples, patient-matched normal DNA is not available leading to the under-utilization of this important resource in LOH studies. Here we describe a new method for LOH analysis that relies on the genome-wide comparison of heterozygosity of single nucleotide polymorphisms (SNPs) between cohorts of cases and un-matched healthy control samples. Regions of LOH are defined by consistent decreases in heterozygosity across a genetic region in the case cohort compared to the control cohort. Methods DNA was collected from 20 Follicular Lymphoma (FL) tumor samples, 20 Diffuse Large B-cell Lymphoma (DLBCL) tumor samples, neoplastic B-cells of 10 B-cell Chronic Lymphocytic Leukemia (B-CLL) patients and Buccal cell samples matched to 4 of these B-CLL patients. The cohort heterozygosity comparison method was developed and validated using LOH derived in a small cohort of B-CLL by traditional comparisons of tumor and normal DNA samples, and compared to the only alternative method for LOH analysis without patient matched controls. LOH candidate regions were then generated for enlarged cohorts of B-CLL, FL and DLBCL samples using our cohort heterozygosity comparison method in order to evaluate potential LOH candidate regions in these non-Hodgkin's lymphoma tumor subtypes. Results Using a small cohort of B-CLL samples with patient-matched normal DNA we have validated the utility of this method and shown that it displays more accuracy and sensitivity in detecting LOH candidate regions compared to the only alternative method, the Hidden Markov Model (HMM) method. Subsequently, using B-CLL, FL and DLBCL tumor samples we have utilised cohort heterozygosity comparisons to localise LOH candidate regions in these subtypes of non-Hodgkin's lymphoma. Detected LOH regions included both previously described regions of LOH as well as novel genomic candidate regions. Conclusions We have proven the efficacy of the use of cohort heterozygosity comparisons for genome-wide mapping of LOH and shown it to be in many ways superior to the HMM method. Additionally, the use of this method to analyse SNP microarray data from 3 common forms of non-Hodgkin's lymphoma yielded interesting tumor suppressor gene candidates, including the ETV3 gene that was highlighted in both B-CLL and FL.

Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly expresses EBNA3A with conserved CD8+ T-cell epitopes

Post-transplantation lymphoproliferative disorders (PTLD) arise in the immunosuppressed and are frequently Epstein-Barr virus (EBV) associated. The most common PTLD histological sub-type is diffuse large B-cell lymphoma (EBV+DLBCL-PTLD). Restoration of EBV-specific T-cell immunity can induce EBV+DLBCL-PTLD regression. The most frequent B-cell lymphoma in the immunocompetent is also DLBCL. ‘EBV-positive DLBCL of the elderly’ (EBV+DLBCL) is a rare but well-recognized DLBCL entity that occurs in the overtly immunocompetent, that has an adverse outcome relative to EBV-negative DLBCL. Unlike PTLD (which is classified as viral latency III), literature suggests EBV+DLBCL is typically latency II, i.e. expression is limited to the immuno-subdominant EBNA1, LMP1 and LMP2 EBV-proteins. If correct, this would be a major impediment for T-cell immunotherapeutic strategies. Unexpectedly we observed EBV+DLBCL-PTLD and EBV+DLBCL both shared features consistent with type III EBV-latency, including expression of the immuno-dominant EBNA3A protein. Extensive analysis showed frequent polymorphisms in EBNA1 and LMP1 functionally defined CD8+ T-cell epitope encoding regions, whereas EBNA3A polymorphisms were very rare making this an attractive immunotherapy target. As with EBV+DLBCL-PTLD, the antigen presenting machinery within lymphomatous nodes was intact. EBV+DLBCL express EBNA3A suggesting it is amenable to immunotherapeutic strategies.

In silico analyses reveal common cellular pathways affected by loss of heterozygosity (LOH) events in the lymphomagenesis of Non-Hodgkin’s lymphoma (NHL)

Background The analysis of cellular networks and pathways involved in oncogenesis has increased our knowledge about the pathogenic mechanisms that underlie tumour biology and has unmasked new molecular targets that may lead to the design of better anti-cancer therapies. Recently, using a high resolution loss of heterozygosity (LOH) analysis, we identified a number of potential tumour suppressor genes (TSGs) within common LOH regions across cases suffering from two of the most common forms of Non-Hodgkin’s lymphoma (NHL), Follicular Lymphoma (FL) and Diffuse Large B-cell Lymphoma (DLBCL). From these studies LOH of the protein tyrosine phosphatase receptor type J (PTPRJ) gene was identified as a common event in the lymphomagenesis of these B-cell lymphomas. The present study aimed to determine the cellular pathways affected by the inactivation of these TSGs including PTPRJ in FL and DLBCL tumourigenesis. Results Pathway analytical approaches identified that candidate TSGs located within common LOH regions participate within cellular pathways, which may play a crucial role in FL and DLBCL lymphomagenesis (i.e., metabolic pathways). These analyses also identified genes within the interactome of PTPRJ (i.e. PTPN11 and B2M) that when inactivated in NHL may play an important role in tumourigenesis. We also detected genes that are differentially expressed in cases with and without LOH of PTPRJ, such as NFATC3 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 3). Moreover, upregulation of the VEGF, MAPK and ERBB signalling pathways was also observed in NHL cases with LOH of PTPRJ, indicating that LOH-driving events causing inactivation of PTPRJ, apart from possibly inducing a constitutive activation of these pathways by reduction or abrogation of its dephosphorylation activity, may also induce upregulation of these pathways when inactivated. This finding implicates these pathways in the lymphomagenesis and progression of FL and DLBCL. Conclusions The evidence obtained in this research supports findings suggesting that FL and DLBCL share common pathogenic mechanisms. Also, it indicates that PTPRJ can play a crucial role in the pathogenesis of these B-cell tumours and suggests that activation of PTPRJ might be an interesting novel chemotherapeutic target for the treatment of these B-cell tumours.

Ratios of T-cell immune-effectors with tumour associated macrophages and PD-1/PD-L1 axis immune-checkpoint molecules, as prognosticators in diffuse large B cell lymphoma: A population-based study

Background Risk-stratification of diffuse large B-cell lymphoma (DLBCL) requires identification of patients with disease that is not cured despite initial R-CHOP. Although the prognostic importance of the tumour microenvironment (TME) is established, the optimal strategy to quantify it is unknown. Methods The relationship between immune-effector and inhibitory (checkpoint) genes was assessed by NanoString™ in 252 paraffin-embedded DLBCL tissues. A model to quantify net anti-tumoural immunity as an outcome predictor was tested in 158 R-CHOP treated patients, and validated in tissue/blood from two independent R-CHOP treated cohorts of 233 and 140 patients respectively. Findings T and NK-cell immune-effector molecule expression correlated with tumour associated macrophage and PD-1/PD-L1 axis markers consistent with malignant B-cells triggering a dynamic checkpoint response to adapt to and evade immune-surveillance. A tree-based survival model was performed to test if immune-effector to checkpoint ratios were prognostic. The CD4*CD8:(CD163/CD68)*PD-L1 ratio was better able to stratify overall survival than any single or combination of immune markers, distinguishing groups with disparate 4-year survivals (92% versus 47%). The immune ratio was independent of and added to the revised international prognostic index (R-IPI) and cell-of-origin (COO). Tissue findings were validated in 233 DLBCL R-CHOP treated patients. Furthermore, within the blood of 140 R-CHOP treated patients immune-effector:checkpoint ratios were associated with differential interim-PET/CT+ve/-ve expression.

Prognostic molecular factors and algorithms in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas. As DLBCL is characterized by heterogeneous clinical and biological features, its prognosis varies. To date, the International Prognostic Index has been the strongest predictor of outcome for DLBCL patients. However, no biological characters of the disease are taken into account. Gene expression profiling studies have identified two major cell-of-origin phenotypes in DLBCL with different prognoses, the favourable germinal centre B-cell-like (GCB) and the unfavourable activated B-cell-like (ABC) phenotypes. However, results of the prognostic impact of the immunohistochemically defined GCB and non-GCB distinction are controversial. Furthermore, since the addition of the CD20 antibody rituximab to chemotherapy has been established as the standard treatment of DLBCL, all molecular markers need to be evaluated in the post-rituximab era. In this study, we aimed to evaluate the predictive value of immunohistochemically defined cell-of-origin classification in DLBCL patients. The GCB and non-GCB phenotypes were defined according to the Hans algorithm (CD10, BCL6 and MUM1/IRF4) among 90 immunochemotherapy- and 104 chemotherapy-treated DLBCL patients. In the chemotherapy group, we observed a significant difference in survival between GCB and non-GCB patients, with a good and a poor prognosis, respectively. However, in the rituximab group, no prognostic value of the GCB phenotype was observed. Likewise, among 29 high-risk de novo DLBCL patients receiving high-dose chemotherapy and autologous stem cell transplantation, the survival of non-GCB patients was improved, but no difference in outcome was seen between GCB and non-GCB subgroups. Since the results suggested that the Hans algorithm was not applicable in immunochemotherapy-treated DLBCL patients, we aimed to further focus on algorithms based on ABC markers. We examined the modified activated B-cell-like algorithm based (MUM1/IRF4 and FOXP1), as well as a previously reported Muris algorithm (BCL2, CD10 and MUM1/IRF4) among 88 DLBCL patients uniformly treated with immunochemotherapy. Both algorithms distinguished the unfavourable ABC-like subgroup with a significantly inferior failure-free survival relative to the GCB-like DLBCL patients. Similarly, the results of the individual predictive molecular markers transcription factor FOXP1 and anti-apoptotic protein BCL2 have been inconsistent and should be assessed in immunochemotherapy-treated DLBCL patients. The markers were evaluated in a cohort of 117 patients treated with rituximab and chemotherapy. FOXP1 expression could not distinguish between patients, with favourable and those with poor outcomes. In contrast, BCL2-negative DLBCL patients had significantly superior survival relative to BCL2-positive patients. Our results indicate that the immunohistochemically defined cell-of-origin classification in DLBCL has a prognostic impact in the immunochemotherapy era, when the identifying algorithms are based on ABC-associated markers. We also propose that BCL2 negativity is predictive of a favourable outcome. Further investigational efforts are, however, warranted to identify the molecular features of DLBCL that could enable individualized cancer therapy in routine patient care.

Nuevos marcadores pronósticos en el linfoma difuso de célula grande b gástrico : expresión de proteinas y metilación de promotores

203 p. : il. col.

Efficacy of Pharmacokinetics-Directed Busulfan, Cyclophosphamide, and Etoposide Conditioning and Autologous Stem Cell Transplantation for Lymphoma: Comparison of a Multicenter Phase II Study and CIBMTR Outcomes.

Busulfan, cyclophosphamide, and etoposide (BuCyE) is a commonly used conditioning regimen for autologous stem cell transplantation (ASCT). This multicenter, phase II study examined the safety and efficacy of BuCyE with individually adjusted busulfan based on preconditioning pharmacokinetics. The study initially enrolled Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) patients ages 18 to 80 years but was amended due to high early treatment-related mortality (TRM) in patients > 65 years. BuCyE outcomes were compared with contemporaneous recipients of carmustine, etoposide, cytarabine, and melphalan (BEAM) from the Center for International Blood and Marrow Transplant Research. Two hundred seven subjects with HL (n = 66) or NHL (n = 141) were enrolled from 32 centers in North America, and 203 underwent ASCT. Day 100 TRM for all subjects (n = 203), patients > 65 years (n = 17), and patients ≤ 65 years (n = 186) were 4.5%, 23.5%, and 2.7%, respectively. The estimated rates of 2-year progression-free survival (PFS) were 33% for HL and 58%, 77%, and 43% for diffuse large B cell lymphoma (DLBCL; n = 63), mantle cell lymphoma (MCL; n = 29), and follicular lymphoma (FL; n = 23), respectively. The estimated rates of 2-year overall survival (OS) were 76% for HL and 65%, 89%, and 89% for DLBCL, MCL, and FL, respectively. In the matched analysis rates of 2-year TRM were 3.3% for BuCyE and 3.9% for BEAM, and there were no differences in outcomes for NHL. Patients with HL had lower rates of 2-year PFS with BuCyE, 33% (95% CI, 21% to 46%), than with BEAM, 59% (95% CI, 52% to 66%), with no differences in TRM or OS. BuCyE provided adequate disease control and safety in B cell NHL patients ≤ 65 years but produced worse PFS in HL patients when compared with BEAM.

Genomic lesions associated with a different clinical outcome in diffuse large B-Cell lymphoma treated with R-CHOP-21

Despite recent therapeutic improvements, the clinical course of diffuse large B-cell lymphoma (DLBCL) still differs considerably among patients. We conducted this retrospective multi-centre study to evaluate the impact of genomic aberrations detected using a high-density genome wide-single nucleotide polymorphism-based array on clinical outcome in a population of DLBCL patients treated with R-CHOP-21 (rituximab, cyclophosphamide, doxorubicine, vincristine and prednisone repeated every 21_d). 166 DNA samples were analysed using the GeneChip Human Mapping 250K NspI. Genomic anomalies were analysed regarding their impact on the clinical course of 124 patients treated with R-CHOP-21. Unsupervised clustering was performed to identify genetically related subgroups of patients with different clinical outcomes. Twenty recurrent genetic lesions showed an impact on the clinical course. Loss of genomic material at 8p23.1 showed the strongest statistical significance and was associated with additional aberrations, such as 17p- and 15q-. Unsupervised clustering identified five DLBCL clusters with distinct genetic profiles, clinical characteristics and outcomes. Genetic features and clusters, associated with a different outcome in patients treated with R-CHOP, have been identified by arrayCGH.

Perfil imunoistoquímico dos linfomas difusos de grandes células B caninos utilizando-se o método de microarranjo de tecido (TMA)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Linfomas Double-Hit em casuística de Linfomas Não-Hodgkin B de alto grau: estudo clínico, morfológico, imunoistoquímico e citogenético

Pós-graduação em Patologia - FMB

Avaliação do C-MYC, BCL2, KI67 e índice mitótico como fatores prognósticos em cães com linfoma difuso de grandes células B

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)